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International audience; Rabies virus (RABV) is a neurotropic virus that causes fatal encephalitis in humans and animals and still kills up to 59,000 people worldwide every year. To date, only preventive or post-exposure vaccination protects against the disease but therapeutics are missing. After screening a library of 80 kinases inhibitors, we identified two compounds as potent inhibitors of RABV infection: Tyrphostin 9 and Rottlerin. Mechanism of action studies show that both inhibitors interfere with an early step of viral cycle and can prevent viral replication. In presence of Tyrphostin 9, the viral entry through endocytosis is disturbed leading to improper delivery of viral particles in cytoplasm, whereas Rottlerin is inhibiting the transcription, most likely by decreasing intracellular ATP concentrations, and therefore the replication of the viral genome.

Transient receptor potential mucolipin 2 and 3 (TRPML2 and TRPML3), as key channels in the endosomal-lysosomal system, are associated with many different cellular processes, including ion release, membrane trafficking and autophagy. In particular, they can also facilitate viral entry into host cells and enhance viral infection. We previously identified that two selective TRPML agonists, ML-SA1 and SN-2, that showed antiviral activities against dengue virus type 2 (DENV2) and Zika virus (ZIKV) in vitro, but their antiviral mechanisms are still elusive. Here, we reported that ML-SA1 could inhibit DENV2 replication by downregulating the expression of both TRPML2 and TRPML3, while the other TRPML activator, SN-2, suppressed DENV2 infection by reducing only TRPML3 expression. Consistently, the channel activities of both TRPML2 and TRPML3 were also found to be associated with the antiviral activity of ML-SA1 on DENV2 and ZIKV, but SN-2 relied only on TRPML3 channel activity. Further mechanistic experiments revealed that ML-SA1 and SN-2 decreased the expression of the late endosomal marker Rab7, dependent on TRPML2 and TRPML3, indicating that these two compounds likely inhibit viral infection by promoting vesicular trafficking from late endosomes to lysosomes and then accelerating lysosomal degradation of the virus. As expected, neither ML-SA1 nor SN-2 inhibited herpes simplex virus type I (HSV-1), whose entry is independent of the endolysosomal network. Together, our work reveals the antiviral mechanisms of ML-SA1 and SN-2 in targeting TRPML channels, possibly leading to the discovery of new drug candidates to inhibit endocytosed viruses.

Severe fever with thrombocytopenia syndrome (SFTS) is an acute infectious disease caused by a novel phlebovirus (SFTSV), characterized by fever, thrombocytopenia and leukocytopenia which lead to multiple organ failure with high mortality in severe cases. The SFTSV has spread rapidly in recent years and posed a serious threat to public health in endemic areas. However, specific antiviral therapeutics for SFTSV infection are rare. In this study, we demonstrated that two peptides, SGc1 and SGc8, derived from a hydrophobic region of the SFTSV glycoprotein Gc, could potently inhibit SFTSV replication in a dose-dependent manner without apparent cytotoxicity in various cell lines and with low immunogenicity and good stability. The IC50 (50% inhibition concentration) values for both peptides to inhibit 2 MOI of SFTSV infection were below 10 μM in L02, Vero and BHK21 cells. Mechanistically, SGc1 and SGc8 mainly inhibited viral entry at the early stage of the viral infection. Inhibition of SFTSV replication was specific by both peptides because no inhibitory effect was shown against other viruses including Zika virus and Enterovirus A71. Taken together, our results suggested that viral glycoprotein-derived SGc1 and SGc8 peptides have antiviral potential and warrant further assessment as an SFTSV-specific therapeutic.

During virus entry, members of the Polyomaviridae transit the endolysosomal network en route to the endoplasmic reticulum (ER), from which degraded capsids escape into the cytoplasm and enter the nucleus. Emerging evidence suggests that viruses require both endosomal acidification and the correct ionic balance of K+ and Ca2+ ions in endosomes for correct virus trafficking and genome release. Here, using two polyomaviruses with different capsid architectures, namely Simian virus 40 (SV40) and Merkel cell polyomavirus (MCPyV), we describe methods to rapidly quantify virus infection using IncuCyte ZOOM imaging analysis, and use this system to investigate the role of both K+ and Ca2+ channels during the early stages of virus entry. Using broad spectrum blockers of both K+ and Ca2+ channels to specifically target host cell ion channel functionality, we show that MCPyV, but not SV40 can be inhibited by K+ channel modulators, whilst both viruses are restricted by the broad spectrum Ca2+ channel inhibitor verapamil. Using a panel of more specific Ca2+ blockers, we show that both MCPyV and SV40 are dependent on the activity of two-pore Ca2+ channels (TPCs), as the TPC-specific blocker tetrandrine could prevent the capsid disassembly and nuclear transport required for virus entry. We therefore reveal a novel target to restrict the entry of polyomaviruses, which given the known role of TPCs during endolysosomal-ER fusion, is likely to be applicable to other viruses that transit this pathway., Highlights • We describe novel high-throughput assays to study SV40 and MCPyV infection. • MCPyV, but not SV40, is sensitive to K+ channel inhibition. • Verapamil inhibits MCPyV and SV40 infection. • Tetrandrine is a potent inhibitor of MCPyV and SV40 infection. • Two-pore channel 1/2 activity is essential for polyomavirus entry.

Primary peripheral blood monocytes are responsible for the hematogenous dissemination of human cytomegalovirus (HCMV) following a primary infection. In order to facilitate viral spread, HCMV extends the naturally short 48-h lifespan of monocytes by stimulating a non-canonical activation of Akt during viral entry, which leads to the increased expression of a specific subset of antiapoptotic proteins. In this study, global analysis of the Akt signaling network showed HCMV induced a more robust activation of the entire network when compared to normal myeloid growth factors. Furthermore, we found a unique interplay between HCMV-activated Akt and the stress response transcription heat shock factor 1 (HSF1) that allowed for the synthesis of both cap- and internal ribosome entry site (IRES)-containing antiapoptotic mRNAs such as myeloid cell leukemia-1 (Mcl-1) and X-linked inhibitor of apoptosis (XIAP), respectively. As generally a switch from cap-dependent to IRES-mediated translation occurs during cellular stress, the ability of HCMV to concurrently drive both types of translation produces a distinct milieu of prosurvival proteins needed for the viability of infected monocytes. Indeed, we found inhibition of XIAP led to death of ∼99% of HCMV-infected monocytes while having minimal effect on the viability of uninfected cells. Taken together, these data indicate that the aberrant activation of the Akt network by HCMV induces the upregulation of a unique subset of antiapoptotic proteins specifically required for the survival of infected monocytes. Consequently, our study highlights the possibility of exploiting these virus-induced changes to prevent viral spread in immunocompromised patients at high-risk for HCMV exposure.

Dengue virus (DENV) infection has become a severe public health problem worldwide. However, there is no specific antiviral drug available yet. In this study, we found that DENV serotype 2 (DENV2) infection enhanced the expression of β3 integrin on human umbilical vein endothelial cells (HUVECs) and that DENV2 antigens co-localized with β3 integrin. DENV2 envelope protein (E) directly interacted with β3 integrin, and their interacting sites were located at domain III of E protein (EDIII). Several synthetic peptides were designed based on the amino acid sequence of EDIII, and peptides P4 and P7 could inhibit DENV2 entry into HUVECs in a dose-dependent manner. The inhibitory concentration (IC50) of the two peptides was 19.08 ± 2.52 μM for P4 and 12.86 ± 5.96 μM for P7. Moreover, P7 containing an FG-loop, but not P4, could also inhibit DENV1 entry into HUVECs. Our results suggest a novel mechanism in which interaction between β3 integrin and EDIII is involved in DENV entry. The findings on the inhibitory effect of the peptides on viral entry have significance for anti-DENV drug design.

Ebola virus (EBOV) causes fatal hemorrhagic fever with high death rates in human. Currently, there are no available clinically-approved prophylactic or therapeutic treatments. The recently solved crystal structure of cleavage-primed EBOV glycoprotein (GPcl) in complex with the C domain of endosomal protein Niemann-Pick C1 (NPC1) provides a new target for the development of EBOV entry inhibitors. In this work, a computational approach using docking and molecular dynamic simulations is carried out for the rational design of peptide inhibitors. A novel cyclo-peptide (Pep-3.3) was identified to target at the late stage of EBOV entry and exhibit specific inhibitory activity against EBOV-GP pseudotyped viruses, with 50% inhibitory concentration (IC50) of 5.1 μM. In vitro binding assay and molecular simulations revealed that Pep-3.3 binds to GPcl with a KD value of 69.7 μM, through interacting with predicted residues in the hydrophobic binding pocket of GPcl. Mutation of predicted residues T83 caused resistance to Pep-3.3 inhibition in viral infectivity, providing preliminary support for the model of the peptide binding to GPcl. This study demonstrates the feasibility of inhibiting EBOV entry by targeting GPcl with peptides.

RSV continues to be a high priority for vaccine and antiviral drug development. Unfortunately, no safe and effective RSV vaccine is available and treatment options are limited. Over the past decade, several studies have focused on the role of RSV G protein on viral entry, viral neutralization, and RSV-mediated pathology. Anti-G murine monoclonal antibody (mAb) 131-2G treatment has been previously shown to reduce weight loss, bronchoalveolar lavage (BAL) cell number, airway reactivity, and Th2-type cytokine production in RSV-infected mice more rapidly than a commercial humanized monoclonal antibody (mAb) against RSV F protein (Palivizumab). In this study, we have tested two human anti-RSV G mAbs, 2B11 and 3D3, by both prophylactic and therapeutic treatment for RSV in the BALB/c mouse model. Both anti-G mAbs reduced viral load, leukocyte infiltration and IFN-γ and IL-4 expression in cell-free BAL supernatants emphasizing the potential of anti-G mAbs as anti-inflammatory and antiviral strategies.

The past decade has seen the re-emergence of Chikungunya virus (CHIKV) as a major global health threat, affecting millions around the world. Although fatal infections are rare among infected patients, the occurrence of long-lasting polyarthralgia has a significant impact on patients' quality of lives and ability to work. These issues were the stimuli for this study to determine the potential of baicalin, a bioflavonoid, as the novel antiviral compound against CHIKV. It was found that baicalin was well tolerated by Vero, BHK-21 and HEK 293T cells with maximal nontoxic doses >600 μM, ≈ 350 μM and ≈110 μM, respectively. Antiviral assays indicated that baicalin was the most effective inhibitor when tested for its direct virucidal activity with EC 50 ≈ 7 μM, followed by inhibition of virus entry into the host cell, attachment of virus particle to cellular receptors and finally intracellular replication of viral RNA genome. In silico analysis using molecular docking demonstrated close interactions between baicalin and CHIKV envelope protein with considerably strong binding affinity of −9.7 kcal/mol. qRT-PCR analysis revealed that baicalin had the greatest effect on the synthesis of viral negative stand RNA with EC 50 ≈ 0.4 μM followed by the inhibition of synthesis of positive-strand genomic (EC 50 ≈ 13 μM) and subgenomic RNAs (EC 50 ≈ 14 μM). These readings indicate that the compound efficiently inhibits replicase complexes formation but is a less potent inhibitor of existing replicase complexes. Coherent with this hypothesis, the use of recombinant CHIKV replicons harboring Renilla luciferase marker showed that replication of corresponding replicon RNAs was only slightly downregulated at higher doses of baicalin, with EC 50 > 100 μM. Immunofluorescence and western blotting experiments demonstrated dose-dependent inhibition of expression of different viral proteins. It was also observed that levels of important protein markers for cellular autophagy (LC3) and apoptosis (Bax) were reduced in baicalin treatment groups as compared with untreated virus infected controls. In summary, given its low toxicity and high efficacy against CHIKV, baicalin has great potential to be developed as the novel antiviral compound for CHIKV. In vivo studies to evaluate its activity in a more complexed system represent a necessary step for future analysis.

Members of the family Filoviridae cause severe, often fatal disease in humans, for which there are no approved vaccines and only a few experimental drugs tested in animal models. Retro-2, a small molecule that inhibits retrograde trafficking of bacterial and plant toxins inside host cells, has been demonstrated to be effective against a range of bacterial and virus pathogens, both in vitro and in animal models. Here, we demonstrated that Retro-2 and its derivatives, Retro-2.1 and compound 25, blocked infection by Ebola virus and Marburg virus in vitro. We show that the derivatives were more potent inhibitors of infection as compared to the parent compound. Pseudotyped virus assays indicated that the compounds affected virus entry into cells while virus particle localization to Niemann-Pick C1-positive compartments showed that they acted at a late step in virus entry. Our work demonstrates a potential for Retro-type drugs to be developed into anti-filoviral therapeutics.

Antiviral targeting of virus envelope proteins is an effective strategy for therapeutic intervention of viral infections. Here, we took a computer-guided approach with the aim of identifying new antivirals against the envelope protein E2 of bovine viral diarrhea virus (BVDV). BVDV is an enveloped virus with an RNA genome responsible for major economic losses of the cattle industry worldwide. Based on the crystal structure of the envelope protein E2, we defined a binding site at the interface of the two most distal domains from the virus membrane and pursued a hierarchical docking-based virtual screening search to identify small-molecule ligands of E2. Phenyl thiophene carboxamide derivative 12 (PTC12) emerged as a specific inhibitor of BVDV replication from in vitro antiviral activity screening of candidate molecules, displaying an IC 50 of 0.30 μM against the reference NADL strain of the virus. Using reverse genetics we constructed a recombinant BVDV expressing GFP that served as a sensitive reporter for the study of the mechanism of action of antiviral compounds. Time of drug addition assays showed that PTC12 inhibited an early step of infection. The mechanism of action was further dissected to find that the compound specifically acted at the internalization step of virus entry. Interestingly, we demonstrated that similar to PTC12, the benzimidazole derivative 03 (BI03) selected in the virtual screen also inhibited internalization of BVDV. Furthermore, docking analysis of PTC12 and BI03 into the binding site revealed common interactions with amino acid residues in E2 suggesting that both compounds could share the same molecular target. In conclusion, starting from a targeted design strategy of antivirals against E2 we identified PTC12 as a potent inhibitor of BVDV entry. The compound can be valuable in the design of antiviral strategies in combination with already well-characterized polymerase inhibitors of BVDV. Fil: Pascual, María José. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas; Argentina Fil: Merwaiss, Fernando. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas; Argentina Fil: Leal, Emilse Soledad. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Centro de Investigaciones en Bionanociencias "Elizabeth Jares Erijman"; Argentina Fil: Quintana, Maria Eugenia. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Virología; Argentina Fil: Capozzo, Alejandra Victoria. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Virología; Argentina Fil: Cavasotto, Claudio Norberto. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigación en Biomedicina de Buenos Aires - Instituto Partner de la Sociedad Max Planck; Argentina Fil: Bollini, Mariela. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Centro de Investigaciones en Bionanociencias "Elizabeth Jares Erijman"; Argentina Fil: Alvarez, Diego Ezequiel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas; Argentina

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) has infected over 200 million people throughout the world as of August 2021. There are currently no approved treatments providing high chance of recovery from a severe case of coronavirus disease 2019 (COVID-19) caused by SARS-CoV-2, and the beneficial effect of Remdesivir and passive immunization therapies may only be seen when administered early on disease onset. The emergence of variants is also raising concerns regarding the efficacy of antibody therapies, antivirals, and vaccines. Therefore, there is still a need to develop new antivirals. Here, we investigated the suitability of primary human epithelial cells from the trachea/bronchia (NHBE) and small airway (SAEC) as lung models of SARS-CoV-2 infection to determine, whether the microbicide polyphenylene carboxymethylene (PPCM) has antiviral activity against SARS-CoV-2. Both NHBE and SAEC expressed proteins required for virus entry in lung epithelial cells. However, these cells were only low to moderately permissive to SARS-CoV-2 as titers increased at best by 2.5 log10 during an 8-day kinetic. Levels of replication in SAEC, unlike in NHBE, were consistent with data from other studies using human normal tissues or air-liquid interface cultures, suggesting that SAEC may be more relevant to use than NHBE for drug screening. PPCM EC50 against SARS-CoV-2 was between 32 and 132 μg/ml with a selectivity index between 12 and 41, depending on the cell type and the infective dose used. PPCM doses were consistent with those previously showing effect against other human viruses. Finally, PPCM antiviral effect observed in SAEC was in line with reduction of inflammatory markers observed overly expressed in severe COVID-19 patients. Altogether, our data support the fact that PPCM should be further evaluated in vivo for toxicity and antiviral activity against SARS-CoV-2.

Many natural lectins have been reported to have antiviral activity. As some of these have been put forward as potential development candidates for preventing or treating viral infections, we have set out in this review to survey the literature on antiviral lectins. The review groups lectins by structural class and class of source organism we also detail their carbohydrate specificity and their reported antiviral activities. The review concludes with a brief discussion of several of the pertinent hurdles that heterologous proteins must clear to be useful clinical candidates and cites examples where such studies have been reported for antiviral lectins. Though the clearest path currently being followed is the use of antiviral lectins as anti-HIV microbicides via topical mucosal administration, some investigators have also found systemic efficacy against acute infections following subcutaneous administration.

Viral infection induces translocation of the nucleolar protein GLTSCR2 from the nucleus to the cytoplasm, resulting in attenuation of the type I interferon IFN-β. Addressing the role of GLTSCR2 in viral replication, we detect that knocking down GLTSCR2 by shRNAs results in significant suppression of viral replication in mammalian and chicken cells. Injection of chicken embryo with the GLTSCR2-specific shRNA-1370 simultaneously or 24 h prior to infection with Newcastle disease virus (NDV) substantially reduces viral replication in chicken embryo fibroblasts. Injection of shRNA-1370 into chicken embryo also reduces the replication of avian influenza virus (AIV). In contrast, GLTSCR2-derived protein G4-T, forming α-helical dimers, increases replication of seven various DNA and RNA viruses in cells. Our studies reveal that alteration of the function of cellular GLTSCR2 plays a role in supporting viral replication. GLTSCR2 should be seriously considered as a therapeutic target for developing broad spectrum antiviral agents to effectively control viral infection., Highlights • G4-T, a protein that mimics GLTSCR2, folds in an α-helical dimer structure. • G4-T suppresses type I IFN antiviral response. • G4-T promotes efficient proliferation of DNA and RNA viruses belonging to 7 families. • GLTSCR2-specific shRNA reduces the infection of viruses in mammalian and chicken cells. • Injection of GLTSCR2-specific shRNA into chicken embryo reduces the replication of NDV and AIV.

Due to diverse pathogenic potentials, there is a growing need for anti-HCMV agents. In this study, we show that treatment with DAPT, a γ-secretase inhibitor (GSI), impairs HCMV replication as assessed by a progeny assay based on immunostaining. This effect is not limited to DAPT because other GSIs with different structures and distinct mechanisms of action also exhibit a similar level of inhibitory effects on HCMV viral production, indicating that γ-secretase activity is required for efficient HCMV replication. Western blot and qPCR analyses reveal that DAPT does not interfere with the viral entry process, but reduces expression of the immediate early protein IE1 at the transcriptional level. Furthermore, we exclude the possible involvement of Notch signaling pathway during HCMV replication by showing that expression of the dominant-negative form of MAML1, which disrupts the transactivational ability of Notch intracellular domain (NICD), does not reduce viral particle formation, and that NICD cannot rescue the DAPT-treated outcomes. Taken together, these findings indicate that γ-secretase activity plays an important role in a key step of the HCMV life cycle and γ-secretase inhibition could potentially be used as a novel preventive and therapeutic strategy against HCMV infection and HCMV-related diseases.

Arboviral diseases caused by dengue (DENV), Zika (ZIKV) and chikungunya (CHIKV) viruses represent a major public health problem worldwide, especially in tropical areas where millions of infections occur every year. The aim of this research was to identify candidate molecules for the treatment of these diseases among the drugs currently available in the market, through in silico screening and subsequent in vitro evaluation with cell culture models of DENV and ZIKV infections. Numerous pharmaceutical compounds from antibiotics to chemotherapeutic agents presented high in silico binding affinity for the viral proteins, including ergotamine, antrafenine, natamycin, pranlukast, nilotinib, itraconazole, conivaptan and novobiocin. These five last compounds were tested in vitro, being pranlukast the one that exhibited the best antiviral activity. Further in vitro assays for this compound showed a significant inhibitory effect on DENV and ZIKV infection of human monocytic cells and human hepatocytes (Huh-7 cells) with potential abrogation of virus entry. Finally, intrinsic fluorescence analyses suggest that pranlukast may have some level of interaction with three viral proteins of DENV: envelope, capsid, and NS1. Due to its promising results, suitable accessibility in the market and reduced restrictions compared to other pharmaceuticals; the anti-asthmatic pranlukast is proposed as a drug candidate against DENV, ZIKV, and CHIKV, supporting further in vitro and in vivo assessment of the potential of this and other lead compounds that exhibited good affinity scores in silico as therapeutic agents or scaffolds for the development of new drugs against arboviral diseases. © 2019 Elsevier B.V. Universidad Tecnológica de Pereira, UTP: TRFCI-1P2016 National Institutes of Health, NIH National Institutes of Health, NIH: R01 AI24493 Department of Science, Information Technology and Innovation, Queensland Government, DSITI: 811-2018 Universidad Autónoma de Bucaramanga, UNAB The authors wish to thank the Administrative Department of Science, Technology and Innovation of Colombia [Grant: Colciencias No. 811-2018 ], Universidad Nacional Autónoma de México [Grant: Programa de Becas Posdoctorales en la UNAM 2016 ], Universidad Tecnológica de Bolívar [Grant: TRFCI-1P2016 ] and the National Institutes of Health [NIH grant R01 AI24493 ] for their financial support. Appendix A A continuación se relacionan los compuestos químicos y su número de registro CAS (Chemical Abstracts Service) antrafenine, 55300-29-3; conivaptan, 168626-94-6, 210101-16-9; ergotamine, 113-15-5, 52949-35-6; itraconazole, 84625-61-6; natamycin, 52882-37-8, 7681-93-8; nilotinib, 641571-10-0; novobiocin, 1476-53-5, 303-81-1, 39301-00-3, 4309-70-0; pranlukast, 103177-37-3

Marine microorganisms have been a resource for novel therapeutic drugs for decades. In addition to anticancer drugs, the drug acyclovir, derived from a marine sponge, is FDA-approved for the treatment of human herpes simplex virus-1 infections. Most alphaviruses that are infectious to terrestrial animals and humans, such as Venezuelan and eastern equine encephalitis viruses (VEEV and EEEV), lack efficient antiviral drugs and it is imperative to develop these remedies. To push the discovery and development of anti-alphavirus compounds forward, this study aimed to isolate and screen for potential antiviral compounds from cultured marine microbes originating from the marine environment. Compounds from marine microbes were of interest as they are prolific producers of bioactive compounds across the spectrum of human diseases and infections. Homoseongomycin, an actinobacteria isolated from a marine sponge displayed impressive activity against VEEV from a total of 76 marine bioactive products. The 50% effective concentration (EC50) for homoseongomycin was 8.6 μM for suppressing VEEV TC-83 luciferase reporter virus replication. Homoseongomycin was non-toxic up to 50 μM and partially rescued cells from VEEV induced cell death. Homoseongomycin exhibited highly efficient antiviral activity with a reduction of VEEV infectious titers by 8 log10 at 50 μM. It also inhibited EEEV replication with an EC50 of 1.2 μM. Mechanism of action studies suggest that homoseongomycin affects both early and late stages of the viral life cycle. Cells treated with 25 μM of homoseongomycin had a ~90% reduction in viral entry. In comparison, later stages showed a more robust reduction in infectious titers (6 log10) and VEEV extracellular viral RNA levels (4 log10), but a lesser impact on intracellular viral RNA levels (1.5 log10). In sum, this work demonstrates that homoseongomycin is a potential anti-VEEV and anti-EEEV compound due to its low cytotoxicity and potent antiviral activity.

Upon release of the ∼11 kb single-stranded positive polarity dengue virus genomic RNA (gRNA) into the cytoplasm of an infected cell, it serves as the template for translation of the viral polyprotein, which is cleaved into three structural and seven non-structural proteins. The structural organization of the viral replication complex and RNA is not known but it is increasingly becoming evident that the viral gRNA and replication intermediates adopt unique structural features and localize to discrete regions in the infected cell. Both structure and location play multiple roles ranging from evasion of innate immune response to recruitment of viral and host proteins for translation and replication of the message. This review visits the various transactions that the viral gRNA undergoes between entry and RNA synthesis with the view that some of these events may be targeted by antiviral compounds. This article forms part of a symposium on flavivirus drug discovery in Antiviral Research.

African swine fever virus (ASFV) is the etiological agent of a highly-contagious and fatal disease of domestic pigs, leading to serious socio-economic impact in affected countries. To date, neither a vaccine nor a selective anti-viral drug are available for prevention or treatment of African swine fever (ASF), emphasizing the need for more detailed studies at the role of ASFV proteins involved in viral DNA replication and transcription. Notably, ASFV encodes for a functional type II topoisomerase (ASFV-Topo II) and we recently showed that several fluoroquinolones (bacterial DNA topoisomerase inhibitors) fully abrogate ASFV replication in vitro . Here, we report that ASFV-Topo II gene is actively transcribed throughout infection, with transcripts being detected as early as 2 hpi and reaching a maximum peak concentration around 16 hpi, when viral DNA synthesis, transcription and translation are more active. siRNA knockdown experiments showed that ASFV-Topo II plays a critical role in viral DNA replication and gene expression, with transfected cells presenting lower viral transcripts (up to 89% decrease) and reduced cytopathic effect (−66%) when compared to the control group. Further, a significant decrease in the number of both infected cells (75.5%) and viral factories per cell and in virus yields (up to 99.7%, 2.5 log) was found only in cells transfected with siRNA targeting ASFV-Topo II. We also demonstrate that a short exposure to enrofloxacin during the late phase of infection (from 15 to 1 hpi) induces fragmentation of viral genomes, whereas no viral genomes were detected when enrofloxacin was added from the early phase of infection (from 2 to 16 hpi), suggesting that fluoroquinolones are ASFV-Topo II poisons. Altogether, our results demonstrate that ASFV-Topo II enzyme has an essential role during viral genome replication and transcription, emphasizing the idea that this enzyme can be a potential target for drug and vaccine development against ASF.

Chikungunya disease results from an infection with the arbovirus, chikungunya virus (CHIKV). Symptoms of CHIKV include fever and persistent, severe arthritis. In recent years, several antiviral drugs have been evaluated in clinical trials; however, no registered antivirals have been approved for clinical therapy. In this study, we established a high-throughput screening (HTS) system based on CHIKV 26S mediated insect cell fusion inhibition assay. Our screening system was able to search potential anti-CHIKV drugs in vitro. Using this system, four compounds (niclosamide, nitazoxanide, niflumic acid, tolfenamic acid) were identified. These compounds were then further analyzed using a microneutralization assay. We determined that niclosamide and nitazoxanide exhibit ability to against CHIKV-induced CPE. The anti-CHIKV abilities of these compounds were further confirmed by RT-qPCR and IFA. Moreover, niclosamide and nitazoxanide were found to (1) limit virus entry, (2) inhibit both viral release and cell-to-cell transmission, and (3) possess broad anti-alphavius activities, including against two clinical CHIKV isolates and two alphaviruses: Sindbis virus (SINV) and Semliki forest virus (SFV). In conclusion, our findings suggested that niclosamide and nitazoxanide were able to inhibit CHIKV entry and transmission, which might provide a basis for the development of novel human drug therapies against CHIKV and other alphavirus infections., Highlights • Fusion inhibition assay was successfully established an anti-CHIKV drugs HTS system. • Niclosamide and nitazoxanide were found and verified their ability to against CHIKV entry and transmission. • Both of niclosamide and nitazoxanide also possessed broad anti-alphavirus abilities.

Human cytomegalovirus (HCMV) infects up to 90–100% of the world population. Although HCMV infection is not a concern for immunocompetent patients, it can be life threatening for immunocompromised individuals. Additionally, congenital HCMV infections can cause serious neurological deficits in neonates. Since viral resistance mutations arise for all current treatments, new treatments targeting novel processes are needed. A well-defined target for HCMV is heparan sulfate, a highly sulfated glycosaminoglycan (GAG) necessary for virion/host cell attachment. In this study, we investigated as possible antiviral agents substitution-inert cationic polynuclear platinum complexes (PPCs) that demonstrate charge-dependent high affinity for GAGs (Katner et al., 2018; Peterson et al., 2017). Certain PPCs had anti-HCMV activities in low micromolar concentrations and antiviral activity correlated with their GAG-binding affinity. Time of addition, removal, and mechanistic studies were consistent with PPCs binding to cells and blocking HCMV virion attachment; however, evidence also suggested that PPC/virion interactions could inhibit fibroblast but not epithelial cell infection. We hypothesize that the PPC-heparan sulfate interaction described here is a general approach to inhibition of virion/host cell attachment and viral entry mediated by other anionic GAGs and sialic acids on the cell surface. Through metalloshielding of the critical sulfate receptors, PPCs offer an attractive alternative to current antiviral compounds, with the potential to target a broad spectrum of viruses that utilize GAGs for attachment and entry.

Severe fever with thrombocytopenia syndrome (SFTS), an emerging viral infectious disease with a high case fatality rate, is caused by the SFTS virus (SFTSV). Although several cellular molecules involved in viral entry have been identified, the entry mechanisms of SFTSV remain unclear. In this study, we screened the protein kinase inhibitors in inhibitory effects on the infection of Vero cells with SFTSV using InhibitorSelect™ Protein Kinase Library Series (Merck & Co., Inc., Kenilworth, NJ, USA). Several types of inhibitors targeted to platelet-derived growth factor receptor β (PDGFRβ) inhibited the infection of Vero, Huh7, and NIH3T3 cells with SFTSV in a dose-dependent manner within the noncytotoxic range. In addition, these protein kinase inhibitors also inhibited the infection of the target cells with SFTSV glycoprotein (SFTSV-GP) pseudotyped virus (SFTSVpv). Activation of PDGFRβ phosphorylation was detected in SFTSV-treated cells. The infectivities of SFTSVpv were specifically decreased not only in NIH3T3 cells treated with siRNA for PDGFRβ but also in NIH3T3 cells treated with anti-PDGFRβ neutralizing antibody in a dose-dependent manner. SFTSV growth and entry of SFTSVpv were also inhibited by Akt inhibitors. Activation of Akt phosphorylation was also detected in SFTSV-treated cells. These data indicate that PDGFRβ is one of the important host factors in the entry steps of SFTSV.

Arboviruses, especially Dengue virus (DENV) and Zika virus (ZIKV), have been a severe threat to human health in the last few years due to uncontrollable transmission. There are no approved vaccines or clinical drugs available for use to prevent and treat their infections. Transient receptor potential mucolipin 2 and 3 (TRPML2 and TRPML3) were reported to modulate viral entry, but the antiviral function of these modulators was unknown. Here, we reported that ML-SA1, a TRPML agonist, inhibited DENV2 and ZIKV in vitro in a dose-dependent manner. Time-of-drug-addition experiments showed that ML-SA1 mainly restricted viral entry. Moreover, the selective TRPML3 activator SN-2 was found to share a similar antiviral effect against DENV2 and ZIKV, but the specific TRPML1 agonist MK6-83 was not effective. Although ML-SA1 was further revealed to induce autophagy, its antiviral role was independent of autophagy induction. Finally, ML-SA1 was found to inhibit DENV2 and ZIKV by promoting lysosome acidification and protease activity to cause viral degradation. Together, our study identifies two TRPML agonists, ML-SA1 and SN-2, as potent inhibitors of DENV2 and ZIKV, which may lead to the discovery of new candidates against viruses.

We have recently identified three molecules (tilorone, quinacrine and pyronaridine tetraphosphate) which all demonstrated efficacy in the mouse model of infection with mouse-adapted Ebola virus (EBOV) model of disease and had similar in vitro inhibition of an Ebola pseudovirus (VSV-EBOV-GP), suggesting they interfere with viral entry. Using a machine learning model to predict lysosomotropism these compounds were evaluated for their ability to possess a lysosomotropic mechanism in vitro. We now demonstrate in vitro that pyronaridine tetraphosphate is an inhibitor of Lysotracker accumulation in lysosomes (IC50 = 0.56 μM). Further, we evaluated antiviral synergy between pyronaridine and artesunate (Pyramax®), which are used in combination to treat malaria. Artesunate was not found to have lysosomotropic activity in vitro and the combination effect on EBOV inhibition was shown to be additive. Pyramax® may represent a unique example of the repurposing of a combination product for another disease., Highlights • We describe a machine learning model to predict lysosomotropic activity. • We demonstrate that pyronaridine, quinacrine and tilorone all inhibit lysotracker accumulation as predicted. • Artesunate does not appear to inhibit lysostracker accumulation as predicted. • We demonstrate additivity between pyronaridine and artesunate for their in vitro EBOV activity. • These findings suggest pyronaridine and artesunate may be combined and repurposed against EBOV.

Enterovirus D68 (EV-D68) is a member of the Picornavirus family and a causative agent of respiratory diseases in children. The incidence of EV-D68 infection has increased worldwide in recent years. Thus far, there are no approved antiviral agents or vaccines for EV-D68. Here, we show that methyl-β-cyclodextrin (MβCD), a common drug that disrupts lipid rafts, specifically inhibits EV-D68 infection without producing significant cytotoxicity at virucidal concentrations. The addition of exogenous cholesterol attenuated the anti-EV-D68 activity of MβCD. MβCD treatment had a weak influence on the attachment of viral particles to the cell membrane but significantly inhibited EV-D68 entry into host cells. We demonstrated that EV-D68 facilitated the translocation of the viral receptor ICAM-5 to membrane rafts in infected cells. The colocalization of viral particles with ICAM-5 in lipid rafts was thoroughly abolished in cells after treatment with MβCD. Finally, we showed that MβCD inhibited the replication of isolated circulating EV-D68 strains. In summary, our results demonstrate that MβCD suppresses EV-D68 replication by perturbing the accumulation of virus particles and ICAM-5 in lipid rafts. This mechanism represents a promising strategy for drug development.

Despite the development of direct-acting antivirals (DAAs), hepatitis C virus (HCV) infection remains a major cause for liver disease and cancer worldwide. Entry inhibitors block virus host cell entry and, therefore, prevent establishment of chronic infection and liver disease. Due to their unique mechanism of action, entry inhibitors provide an attractive antiviral strategy in organ transplantation. In this study, we developed an innovative approach in preventing HCV infection using a synergistic combination of a broadly neutralizing human monoclonal antibody (HMAb) targeting the HCV E2 protein and a host-targeting anti-claudin 1 (CLDN1) humanized monoclonal antibody. An in vivo proof-of-concept study in human liver-chimeric FRG-NOD mice proved the efficacy of the combination therapy at preventing infection by an HCV genotype 1b infectious serum. While administration of individual antibodies at lower doses only showed a delay in HCV infection, the combination therapy was highly protective. Furthermore, the combination proved to be effective in preventing infection of primary human hepatocytes by neutralization-resistant HCV escape variants selected during liver transplantation, suggesting that a combination therapy is suited for the neutralization of difficult-to-treat variants. In conclusion, our findings suggest that the combination of two HMAbs targeting different steps of virus entry improves treatment efficacy while simultaneously reducing treatment duration and costs. Our approach not only provides a clinical perspective to employ HMAb combination therapies to prevent graft re-infection and its associated liver disease but may also help to alleviate the urgent demand for organ transplants by allowing the transplantation of organs from HCV-positive donors.

Herpes simplex virus type-1 (HSV-1) is a significant pathogen that affects vision by targeting multiple regions in the human eye including iris. Using a focused library of synthetic non-saccharide glycosaminoglycan mimetics (NSGMs), we identified sulfated pentagalloylglucoside (SPGG) as a potent inhibitor of HSV-1 entry and cell-to-cell spread in the primary cultures of human iris stromal (HIS) cells isolated from eye donors. Using in vitro β-galactosidase reporter assay and plaque reduction assay, SPGG was found to inhibit HSV-1 entry in a dosage-dependent manner (IC50 ∼6.0 μM). Interestingly, a pronounced inhibition in HSV-1 entry and spread was observed in HIS cells, or a cell line expressing specific gD-receptor, when virions were pre-treated with mimetics suggesting a possible interaction between SPGG and the HSV-1 glycoprotein. To examine the significance of gD-SPGG interaction, HIS cells were pretreated with SPGG, which showed a significant reduction in gD binding. Taken together, our results provide strong evidence of SPGG being a novel viral entry inhibitor against ocular HSV infection.

Amiodarone and other cationic amphiphilic drugs (CADs) inhibit cell entry by diverse human pathogenic viruses including Filoviruses, Dengue virus and Japanese encephalitis virus. They are thus considered potential broad spectrum antiviral agents. Here we report the unexpected finding that amiodarone and other CADs markedly enhance rabies virus (RABV) glycoprotein- (GP-) mediated cell entry of pseudotyped lentiviruses into non-neuronal cells but not in neuronal cells. Increased cell entry can also be elicited when CADs are added several hours after pseudoviral attachment. Perturbing endosomal processing with phosphoinosite-3-kinase inhibitors wortmannin and LY294002 mimics the effects of CADs on RABV GP-mediated cell entry. Thus, CADs may enhance RABV GP-mediated cell entry of pseudotyped lentiviruses by promoting a late step of the pseudoviral cell entry process, possibly release from an endosomal compartment into the cytosol. In contrast to the pseudotyped lentiviruses, infection by fully infectious RABV was not enhanced by CADs, indicating, that the observed stimulation of RABV GP mediated lentivirus entry also depended on the used lentivirus vector backbone. In conclusion, we show that while CADs inhibit cell entry of diverse viruses they can also have a paradoxical enhancing effect on the ability of a viral glycoprotein to mediate cell entry depending on the cellular and viral context. Although, we show CAD-mediated enhancement of entry only for pseudoviruses, but not fully infectious RABV, the potential to unexpectedly enhance viral entry should be taken into account when considering use of CADs as antiviral agents.

Graphical abstract, Highlights • Large clusters of repeated G-quadruplex folding sequences were identified in the HSV-1 genome. • BRACO-19, a G-quadruplex ligand, was able to bind and stabilize the HSV-1 G-quadruplexes. • Stabilization of HSV-1 G-quadruplexes by BRACO-19 inhibited DNA polymerase processing and viral DNA replication. • BRACO-19 diminished virus production in infected cells. • HSV-1 G-quadruplex may be regarded as alternative and innovative targets for antiviral therapy., Guanine-rich nucleic acids can fold into G-quadruplexes, secondary structures implicated in important regulatory functions at the genomic level in humans, prokaryotes and viruses. The remarkably high guanine content of the Herpes Simplex Virus-1 (HSV-1) genome prompted us to investigate both the presence of G-quadruplex forming sequences in the viral genome and the possibility to target them with G-quadruplex ligands to obtain anti-HSV-1 effects with a novel mechanism of action. Using biophysical, molecular biology and antiviral assays, we showed that the HSV-1 genome displays multiple clusters of repeated sequences that form very stable G-quadruplexes. These sequences are mainly located in the inverted repeats of the HSV-1 genome. Treatment of HSV-1 infected cells with the G-quadruplex ligand BRACO-19 induced inhibition of virus production. BRACO-19 was able to inhibit Taq polymerase processing at G-quadruplex forming sequences in the HSV-1 genome, and decreased intracellular viral DNA in infected cells. The last step targeted by BRACO-19 was viral DNA replication, while no effect on virus entry in the cells was observed. This work, presents the first evidence of extended G-quadruplex sites in key regions of the HSV-1 genome, indicates the possibility to block viral DNA replication by G-quadruplex-ligand and therefore provides a proof of concept for the use of G-quadruplex ligands as new anti-herpetic therapeutic options.

Highlights • 2H6-antigen binding fragment forms a multimeric complex with non-structural 1 protein. • 2H6 antibody binds tightly to non-structural 1 protein. • The binding affinity reduces significantly when threonine 49 is substituted with Alanine. • Intracellular expression of 2H6-scFv in mammalian cells reduces viral replication and release of progeny virus., The emergence of resistant influenza A viruses highlights the continuous requirement of new antiviral drugs that can treat the viral infection. Non-structural 1 (NS1) protein, an indispensable component for efficient virus replication, can be used as a potential target for generating new antiviral agents. Here, we study the interaction of 2H6 monoclonal antibody with NS1 protein and also determine whether influenza virus replication can be inhibited by blocking NS1. The 2H6-antigen binding fragment (Fab) forms a multimeric complex with the NS1 RNA-binding domain (RBD). T49, a residue which forms a direct hydrogen bond with double stranded RNA, in NS1 protein was found to be critical for its interaction with 2H6 antibody. NS1(RBD) has high affinity to 2H6 with KD of 43.5 ± 4.24 nM whereas NS1(RBD)-T49A has more than 250 times lower affinity towards 2H6. Interestingly, the intracellular expression of 2H6-single-chain variable fragment (scFv) in mammalian cells caused a reduction in viral growth and the M1 viral protein level was significantly reduced in 2H6-scFv transfected cells in comparison to vector transfected cells at 12 h post infection. These results indicate that the tight binding of 2H6 to NS1 could lead to reduction in viral replication and release of progeny virus. In future, 2H6 antibody in combination with other neutralizing antibodies can be used to increase the potency of viral inhibition.

Enterovirus 71 (EV-A71) is a major cause of hand, foot, and mouth disease (HFMD). Infection with EV-A71 is more often associated with neurological complications in children and is responsible for the majority of fatalities, but currently there is no approved antiviral therapy for treatment. Here, we identified auraptene, formononetin, and yangonin as effective inhibitors of EV-A71 infection in the low-micromolar range from screening of a natural product library. Among them, formononetin and yangonin selectively inhibited EV-A71 while auraptene could inhibit viruses within the enterovirus species A. Time of addition studies showed that all the three inhibitors inhibit both attachment and postattachment step of entry. We found mutations conferring the resistance to these inhibitors in the VP1 and VP4 capsid proteins and confirmed the target residues using a reverse genetic approach. Interestingly, auraptene- and formononetin-resistant viruses exhibit cross-resistance to other inhibitors while yangonin-resistant virus still remains susceptible to auraptene and formononetin. Moreover, auraptene and formononetin, but not yangonin protected EV-A71 against thermal inactivation, indicating a direct stabilizing effect of both compounds on virion capsid conformation. Finally, neither biochanin A (an analog of formononetin) nor DL-Kavain (an analog of yangonin) exhibited anti-EV-A71 activity, suggesting the structural elements required for anti-EV-A71 activity. Taken together, these compounds could become potential lead compounds for anti-EV-A71 drug development and also serve as tool compounds for studying virus entry.