A Single Nonsynonymous Substitution in the RNA-Dependent RNA Polymerase of Potato virus Y Allows the Simultaneous Breakdown of Two Different Forms of Antiviral Resistance in Capsicum annuum.

The dominant Pvr4 gene in pepper (Capsicum annuum) confers resistance to members of six potyvirus species, all of which belong to the Potato virus Y (PVY) phylogenetic group. The corresponding avirulence factor in the PVY genome is the NIb cistron (i.e., RNA-dependent RNA polymerase). Here, we describe a new source of potyvirus resistance in the Guatemalan accession C. annuum cv. PM949. PM949 is resistant to members of at least three potyvirus species, a subset of those controlled by Pvr4. The F1 progeny between PM949 and the susceptible cultivar Yolo Wonder was susceptible to PVY, indicating that the resistance is recessive. The segregation ratio between resistant and susceptible plants observed in the F2 progeny matched preferably with resistance being determined by two unlinked recessive genes independently conferring resistance to PVY. Inoculations by grafting resulted in the selection of PVY mutants breaking PM949 resistance and, less efficiently, Pvr4-mediated resistance. The codon substitution E472K in the NIb cistron of PVY, which was shown previously to be sufficient to break Pvr4 resistance, was also sufficient to break PM949 resistance, a rare example of cross-pathogenicity effect. In contrast, the other selected NIb mutants showed specific infectivity in PM949 or Pvr4 plants. Comparison of Pvr4 and PM949 resistance, which share the same target in PVY, provides interesting insights into the determinants of resistance durability.

Anti-BVDV Activity of Traditional Chinese Medicine Monomers Targeting NS5B (RNA-Dependent RNA Polymerase) In Vitro and In Vivo.

Natural products have emerged as "rising stars" for treating viral diseases and useful chemical scaffolds for developing effective therapeutic agents. The nonstructural protein NS5B (RNA-dependent RNA polymerase) of NADL strain BVDV was used as the action target based on a molecular docking technique to screen herbal monomers for anti-BVDV viral activity. The in vivo and in vitro anti-BVDV virus activity studies screened the Chinese herbal monomers with significant anti-BVDV virus effects, and their antiviral mechanisms were initially explored. The molecular docking screening showed that daidzein, curcumin, artemisinine, and apigenin could interact with BVDV-NADL-NS5B with the best binding energy fraction. In vitro and in vivo tests demonstrated that none of the four herbal monomers significantly affected MDBK cell activity. Daidzein and apigenin affected BVDV virus replication mainly in the attachment and internalization phases, artemisinine mainly in the replication phase, and curcumin was active in the attachment, internalization, replication, and release phases. In vivo tests demonstrated that daidzein was the most effective in preventing and protecting BALB/C mice from BVDV infection, and artemisinine was the most effective in treating BVDV infection. This study lays the foundation for developing targeted Chinese pharmaceutical formulations against the BVDV virus.

Synthesis and structure-activity optimization of 7-azaindoles containing aza-ß-amino acids targeting the influenza PB2 subunit.

The PB2 subunit of influenza virus polymerase has been demonstrated as a promising drug target for anti-influenza therapy. In this work, 7-azaindoles containing aza-ß3- or ß2,3 -amino acids were synthesized possessing a good binding affinity of PB2. The aza-ß-amino acid moieties with diverse size, shape, steric hindrance and configuration were investigated. Then a lead HAA-09 was validated, and the attached aza-ß3-amino acid moiety with acyclic tertiary carbon side chain well occupied in the key hydrophobic cavity of PB2_cap binding domain. Importantly, HAA-09 displays potent polymerase inhibition capacity, low cytotoxicity (selectivity index up to 2915) as well as robust anti-viral activity against A/WSN/33 (H1N1) virus and oseltamivir-resistant H275Y variant. Moreover, HAA-09 exhibited druggability with high plasma stability (t1/2 ≥ 12 h) and no obvious hERG inhibition (IC50 > 10 µM). Also, HAA-09 demonstrated a favorable safety profile when orally administrated in healthy mice at a high dose of 40 mg/kg QD for consecutive 3 days. Besides, in vivo therapeutic efficacy (85.7% survival observed at the day 15 post infection) was demonstrated when HAA-09 was administrated orally at 12.5 mg/kg BID starting 48 h post infection for 9 days. These data support that exploring the interactions between side chains on aza-ß3- or ß2,3 -amino acid moieties and hydrophobic pocket of PB2_cap binding domain is a potential medicinal chemistry strategy for developing potent PB2 inhibitors.

A computational study of cooperative binding to multiple SARS-CoV-2 proteins.

Structure-based drug design targeting the SARS-CoV-2 virus has been greatly facilitated by available virus-related protein structures. However, there is an urgent need for effective, safe small-molecule drugs to control the spread of the virus and variants. While many efforts are devoted to searching for compounds that selectively target individual proteins, we investigated the potential interactions between eight proteins related to SARS-CoV-2 and more than 600 compounds from a traditional Chinese medicine which has proven effective at treating the viral infection. Our original ensemble docking and cooperative docking approaches, followed by a total of over 16-micorsecond molecular simulations, have identified at least 9 compounds that may generally bind to key SARS-CoV-2 proteins. Further, we found evidence that some of these compounds can simultaneously bind to the same target, potentially leading to cooperative inhibition to SARS-CoV-2 proteins like the Spike protein and the RNA-dependent RNA polymerase. These results not only present a useful computational methodology to systematically assess the anti-viral potential of small molecules, but also point out a new avenue to seek cooperative compounds toward cocktail therapeutics to target more SARS-CoV-2-related proteins.

Cordycepin Inhibits Virus Replication in Dengue Virus-Infected Vero Cells.

Dengue virus (DENV) infection causes mild to severe illness in humans that can lead to fatality in severe cases. Currently, no specific drug is available for the treatment of DENV infection. Thus, the development of an anti-DENV drug is urgently required. Cordycepin (3'-deoxyadenosine), which is a major bioactive compound in Cordyceps (ascomycete) fungus that has been used for centuries in Chinese traditional medicine, was reported to exhibit antiviral activity. However, the anti-DENV activity of cordycepin is unknown. We hypothesized that cordycepin exerts anti-DENV activity and that, as an adenosine derivative, it inhibits DENV replication. To test this hypothesis, we investigated the anti-DENV activity of cordycepin in DENV-infected Vero cells. Cordycepin treatment significantly decreased DENV protein at a half-maximal effective concentration (EC50) of 26.94 µM. Moreover, DENV RNA was dramatically decreased in cordycepin-treated Vero cells, indicating its effectiveness in inhibiting viral RNA replication. Via in silico molecular docking, the binding of cordycepin to DENV non-structural protein 5 (NS5), which is an important enzyme for RNA synthesis, at both the methyltransferase (MTase) and RNA-dependent RNA polymerase (RdRp) domains, was predicted. The results of this study demonstrate that cordycepin is able to inhibit DENV replication, which portends its potential as an anti-dengue therapy.

Natural plant products as potential inhibitors of RNA dependent RNA polymerase of Severe Acute Respiratory Syndrome Coronavirus-2.

Drug repurposing studies targeting inhibition of RNA dependent RNA polymerase (RdRP) of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) have exhibited the potential effect of small molecules. In the present work a detailed interaction study between the phytochemicals from Indian medicinal plants and the RdRP of SARS-CoV-2 has been performed. The top four phytochemicals obtained through molecular docking were, swertiapuniside, cordifolide A, sitoindoside IX, and amarogentin belonging to Swertia chirayita, Tinospora cordifolia and Withania somnifera. These ligands bound to the RdRP were further studied using molecular dynamics simulations. The principal component analysis of these systems showed significant conformational changes in the finger and thumb subdomain of the RdRP. Hydrogen bonding, salt-bridge and water mediated interactions supported by MM-GBSA free energy of binding revealed strong binding of cordifolide A and sitoindoside IX to RdRP. The ligand-interacting residues belonged to either of the seven conserved motifs of the RdRP. These residues were polar and charged amino acids, namely, ARG 553, ARG 555, ASP 618, ASP 760, ASP 761, GLU 811, and SER 814. The glycosidic moieties of the phytochemicals were observed to form favourable interactions with these residues. Hence, these phytochemicals may hold the potential to act as RdRP inhibitors owing to their stability in binding to the druggable site.

Potential effect of Maxing Shigan decoction against coronavirus disease 2019 (COVID-19) revealed by network pharmacology and experimental verification.

ETHNOPHARMACOLOGICAL RELEVANCE: Since the occurrence of coronavirus disease 2019 (COVID-19) in Wuhan, China in December 2019, COVID-19 has been quickly spreading out to other provinces and countries. Considering that traditional Chinese medicine (TCM) played an important role during outbreak of SARS and H1N1, finding potential alternative approaches for COVID-19 treatment is necessary before vaccines are developed. According to previous studies, Maxing Shigan decoction (MXSGD) present a prominent antivirus effect and is often used to treat pulmonary diseases. Furthermore, we collected 115 open prescriptions for COVID-19 therapy from the National Health Commission, State Administration of TCM and other organizations, MXSGD was identified as the key formula. However, the underlying molecular mechanism of MXSGD against COVID-19 is still unknown. AIM OF THE STUDY: The present study aimed to evaluate the therapeutic mechanism of MXSGD against COVID-19 by network pharmacology and in vitro experiment verification, and screen the potential components which could bind to key targets of COVID-19 via molecular docking method. MATERIALS AND METHODS: Multiple open-source databases related to TCM or compounds were employed to screen active ingredients and potential targets of MXSGD. Network pharmacology analysis methods were used to initially predict the antivirus and anti-inflammatory effects of MXSGD against COVID-19. IL-6 induced rat lung epithelial type Ⅱ cells (RLE-6TN) damage was established to explore the anti-inflammatory damage activity of MXSGD. After MXSGD intervention, the expression level of related proteins and their phosphorylation in the IL-6 mediated JAK-STAT signaling pathway were detected by Western blot. Molecular docking technique was used to further identify the potential substances which could bind to three key targets (ACE2, Mpro and RdRp) of COVID-19. RESULTS: In this study, 105 active ingredients and 1025 candidate targets were selected for MXSGD, 83 overlapping targets related to MXSGD and COVID-19 were identified, and the protein-protein interaction (PPI) network of MXSGD against COVID-19 was constructed. According to the results of biological enrichment analysis, 63 significant KEGG pathways were enriched, and most of them were related to signal transduction, immune system and virus infection. Furthermore, according the relationship between signal pathways, we confirmed MXSGD could effectively inhibit IL-6 mediated JAK-STAT signal pathway related protein expression level, decreased the protein expression levels of p-JAK2, p-STAT3, Bax and Caspase 3, and increased the protein expression level of Bcl-2, thereby inhibiting RLE-6TN cells damage. In addition, according to the LibDock scores screening results, the components with strong potential affinity (Top 10) with ACE2, Mpro and RdRp are mainly from glycyrrhiza uralensis (Chinese name: Gancao) and semen armeniacae amarum (Chinese name: Kuxingren). Among them, amygdalin was selected as the optimal candidate component bind to all three key targets, and euchrenone, glycyrrhizin, and glycyrol also exhibited superior affinity interactions with ACE2, Mpro and RdRp, respectively. CONCLUSION: This work explained the positive characteristics of multi-component, multi-target, and multi-approach intervention with MXSGD in combating COVID-19, and preliminary revealed the antiviral and anti-inflammatory pharmacodynamic substances and mechanism of MXSGD, which might provide insights into the vital role of TCM in the prevention and treatment of COVID-19.

A fluorescence-based high throughput-screening assay for the SARS-CoV RNA synthesis complex.

The Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV) emergence in 2003 introduced the first serious human coronavirus pathogen to an unprepared world. To control emerging viruses, existing successful anti(retro)viral therapies can inspire antiviral strategies, as conserved viral enzymes (eg., viral proteases and RNA-dependent RNA polymerases) represent targets of choice. Since 2003, much effort has been expended in the characterization of the SARS-CoV replication/transcription machinery. Until recently, a pure and highly active preparation of SARS-CoV recombinant RNA synthesis machinery was not available, impeding target-based high throughput screening of drug candidates against this viral family. The current Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) pandemic revealed a new pathogen whose RNA synthesis machinery is highly (>96 % aa identity) homologous to SARS-CoV. This phylogenetic relatedness highlights the potential use of conserved replication enzymes to discover inhibitors against this significant pathogen, which in turn, contributes to scientific preparedness against emerging viruses. Here, we report the use of a purified and highly active SARS-CoV replication/transcription complex (RTC) to set-up a high-throughput screening of Coronavirus RNA synthesis inhibitors. The screening of a small (1520 compounds) chemical library of FDA-approved drugs demonstrates the robustness of our assay and will allow to speed-up drug discovery against the SARS-CoV-2.

COSMO-RS-Based Descriptors for the Machine Learning-Enabled Screening of Nucleotide Analogue Drugs against SARS-CoV-2.

Chemical similarity-based approaches employed to repurpose or develop new treatments for emerging diseases, such as COVID-19, correlates molecular structure-based descriptors of drugs with those of a physiological counterpart or clinical phenotype. We propose novel descriptors based on a COSMO-RS (short for conductor-like screening model for real solvents) σ-profiles for enhanced drug screening enabled by machine learning (ML). The descriptors' performance is hereby illustrated for nucleotide analogue drugs that inhibit the ribonucleic acid-dependent ribonucleic acid polymerase, key to viral transcription and genome replication. The COSMO-RS-based descriptors account for both chemical reactivity and structure, and are more effective for ML-based screening than fingerprints based on molecular structure and simple physical/chemical properties. The descriptors are evaluated using principal component analysis, an unsupervised ML technique. Our results correlate with the active monophosphate forms of the leading drug remdesivir and the prospective drug EIDD-2801 with nucleotides, followed by other promising drugs, and are superior to those from molecular structure-based descriptors and molecular docking. The COSMO-RS-based descriptors could help accelerate drug discovery for the treatment of emerging diseases.

Unraveling the anti-influenza effect of flavonoids: Experimental validation of luteolin and its congeners as potent influenza endonuclease inhibitors.

The biological effects of flavonoids on mammal cells are diverse, ranging from scavenging free radicals and anti-cancer activity to anti-influenza activity. Despite appreciable effort to understand the anti-influenza activity of flavonoids, there is no clear consensus about their precise mode-of-action at a cellular level. Here, we report the development and validation of a screening assay based on AlphaScreen technology and illustrate its application for determination of the inhibitory potency of a large set of polyols against PA N-terminal domain (PA-Nter) of influenza RNA-dependent RNA polymerase featuring endonuclease activity. The most potent inhibitors we identified were luteolin with an IC50 of 72 ± 2 nM and its 8-C-glucoside orientin with an IC50 of 43 ± 2 nM. Submicromolar inhibitors were also evaluated by an in vitro endonuclease activity assay using single-stranded DNA, and the results were in full agreement with data from the competitive AlphaScreen assay. Using X-ray crystallography, we analyzed structures of the PA-Nter in complex with luteolin at 2.0 Å resolution and quambalarine B at 2.5 Å resolution, which clearly revealed the binding pose of these polyols coordinated to two manganese ions in the endonuclease active site. Using two distinct assays along with the structural work, we have presumably identified and characterized the molecular mode-of-action of flavonoids in influenza-infected cells.

Analysis of SARS-CoV-2 RNA-dependent RNA polymerase as a potential therapeutic drug target using a computational approach.

BACKGROUND: The Severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2) outbreak originating in Wuhan, China, has raised global health concerns and the pandemic has now been reported on all inhabited continents. Hitherto, no antiviral drug is available to combat this viral outbreak. METHODS: Keeping in mind the urgency of the situation, the current study was designed to devise new strategies for drug discovery and/or repositioning against SARS-CoV-2. In the current study, RNA-dependent RNA polymerase (RdRp), which regulates viral replication, is proposed as a potential therapeutic target to inhibit viral infection. RESULTS: Evolutionary studies of whole-genome sequences of SARS-CoV-2 represent high similarity (> 90%) with other SARS viruses. Targeting the RdRp active sites, ASP760 and ASP761, by antiviral drugs could be a potential therapeutic option for inhibition of coronavirus RdRp, and thus viral replication. Target-based virtual screening and molecular docking results show that the antiviral Galidesivir and its structurally similar compounds have shown promise against SARS-CoV-2. CONCLUSIONS: The anti-polymerase drugs predicted here-CID123624208 and CID11687749-may be considered for in vitro and in vivo clinical trials.

Discovery of novel Hepatitis C virus inhibitor targeting multiple allosteric sites of NS5B polymerase.

HCV is a viral infection posing a severe global threat when left untreated progress to end-stage liver disease, including cirrhosis and HCC. The NS5B polymerase of HCV is the most potent target that harbors four allosteric binding sites that could interfere with the HCV infection. We present the discovery of a novel synthetic compound that harbors the potential of NS5B polymerase inhibition. All eight compounds belonging to the benzothiazine family of heterocycles displayed no cellular cytotoxicity in HepG2 cells at nontoxic dose concentration (200 µM). Subsequently, among eight compounds of the series, merely compound 5b exhibited significant inhibition of the expression of the HCV NS5B gene as compared to DMSO control in semi-quantitative PCR. Based on our western blot result, 5b at the range of 50, 100 and 200 µM induced 20, 40, and 70% inhibition of NS5B protein respectively. To estimate the binding potential, 5b was docked at respective allosteric sites followed by molecular dynamics (MD) simulations for a period of 20 ns. In addition, binding free energy calculation by MM-GB/PBSA method revealed a conserved interaction profile of residues lining the allosteric sites in agreement with the reported NS5B co-crystallized inhibitors. The presented results provide important information about a novel compound 5b which may facilitate the the discovery of novel inhibitors that tends to target multiple sites on NS5B polymerase.

Potential therapeutic targets and promising drugs for combating SARS-CoV-2.

As of April 9, 2020, a novel coronavirus (SARS-CoV-2) had caused 89,931 deaths and 1,503,900 confirmed cases worldwide, which indicates an increasingly severe and uncontrollable situation. Initially, little was known about the virus. As research continues, we now know the genome structure, epidemiological and clinical characteristics, and pathogenic mechanisms of SARS-CoV-2. Based on this knowledge, potential targets involved in the processes of virus pathogenesis need to be identified, and the discovery or development of drugs based on these potential targets is the most pressing need. Here, we have summarized the potential therapeutic targets involved in virus pathogenesis and discuss the advances, possibilities, and significance of drugs based on these targets for treating SARS-CoV-2. This review will facilitate the identification of potential targets and provide clues for drug development that can be translated into clinical applications for combating SARS-CoV-2.

The potential chemical structure of anti-SARS-CoV-2 RNA-dependent RNA polymerase.

An outbreak of coronavirus disease 2019 (COVID-19) occurred in Wuhan and it has rapidly spread to almost all parts of the world. For coronaviruses, RNA-dependent RNA polymerase (RdRp) is an important polymerase that catalyzes the replication of RNA from RNA template and is an attractive therapeutic target. In this study, we screened these chemical structures from traditional Chinese medicinal compounds proven to show antiviral activity in severe acute respiratory syndrome coronavirus (SARS-CoV) and the similar chemical structures through a molecular docking study to target RdRp of SARS-CoV-2, SARS-CoV, and Middle East respiratory syndrome coronavirus (MERS-CoV). We found that theaflavin has a lower idock score in the catalytic pocket of RdRp in SARS-CoV-2 (-9.11 kcal/mol), SARS-CoV (-8.03 kcal/mol), and MERS-CoV (-8.26 kcal/mol) from idock. To confirm the result, we discovered that theaflavin has lower binding energy of -8.8 kcal/mol when it docks in the catalytic pocket of SARS-CoV-2 RdRp by using the Blind Docking server. Regarding contact modes, hydrophobic interactions contribute significantly in binding and additional hydrogen bonds were found between theaflavin and RdRp. Moreover, one π-cation interaction was formed between theaflavin and Arg553 from the Blind Docking server. Our results suggest that theaflavin could be a potential SARS-CoV-2 RdRp inhibitor for further study.

Antiviral activity of iridoid glycosides extracted from Fructus Gardeniae against influenza A virus by PACT-dependent suppression of viral RNA replication.

Epidemic and pandemic influenza A virus (IAV) poses a significant threat to human populations worldwide. Iridoid glycosides are principal bioactive components from the Gardenia jasminoides J. Ellis fruit that exhibit antiviral activity against several strains of IAV. In the present study, we evaluated the protective effect of Fructus Gardeniae iridoid glycoside extracts (IGEs) against IAV by cytopathogenic effect(CPE), MTT and a plaque formation assay in vitro and examined the reduction in the pulmonary index (PI), restoration of body weight, reduction in mortality and increases in survival time in vivo. As a host factor, PACT provides protection against the pathogenic influenza A virus by interacting with IAV polymerase and activating the IFN-I response. To verify the whether IGEs suppress IAV replication in a PACT-dependent manner, IAV RNA replication, expression of PACT and the phosphorylation of eIF2α in A549 cells were detected; the levels of IFNß, PACT and PKR in mouse lung tissues were determined; and the activity of IAV polymerase was evaluated in PACT-compromised cells. The results indicated that IGEs sufficiently alleviated cell damage and suppressed IAV replication in vitro, protecting mice from IAV-induced injury and lethal IAV infection. These anti-IAV effects might be related to disrupted interplay between IVA polymerase and PACT and/or prevention of a PACT-dependent overactivated IFN-I antiviral response. Taken together, our findings reveal a new facet of the mechanisms by which IGEs fight the influenza A virus in a PACT-dependent manner.

Discovery of a small molecule inhibitor targeting dengue virus NS5 RNA-dependent RNA polymerase.

Dengue is a mosquito-borne viral infection that has spread globally in recent years. Around half of the world's population, especially in the tropics and subtropics, is at risk of infection. Every year, 50-100 million clinical cases are reported, and more than 500,000 patients develop the symptoms of severe dengue infection: dengue haemorrhagic fever and dengue shock syndrome, which threaten life in Asia and Latin America. No antiviral drug for dengue is available. The dengue virus (DENV) non-structural protein 5 (NS5), which possesses the RNA-dependent RNA polymerase (RdRp) activity and is responsible for viral replication and transcription, is an attractive target for anti-dengue drug development. In the present study, 16,240 small-molecule compounds in a fragment library were screened for their capabilities to inhibit the DENV type 2 (DENV2) RdRp activities in vitro. Based on in cellulo antiviral and cytotoxity assays, we selected the compound RK-0404678 with the EC50 value of 6.0 µM for DENV2. Crystallographic analyses revealed two unique binding sites for RK-0404678 within the RdRp, which are conserved in flavivirus NS5 proteins. No resistant viruses emerged after nine rounds of serial passage of DENV2 in the presence of RK-0404678, suggesting the high genetic barrier of this compound to the emergence of a resistant virus. Collectively, RK-0404678 and its binding sites provide a new framework for antiviral drug development.

A Parallel Phenotypic Versus Target-Based Screening Strategy for RNA-Dependent RNA Polymerase Inhibitors of the Influenza A Virus.

Influenza A virus infections cause significant morbidity and mortality, and novel antivirals are urgently needed. Influenza RNA-dependent RNA polymerase (RdRp) activity has been acknowledged as a promising target for novel antivirals. In this study, a phenotypic versus target-based screening strategy was established to identify the influenza A virus inhibitors targeting the virus RNA transcription/replication steps by sequentially using an RdRp-targeted screen and a replication-competent reporter virus-based approach using the same compounds. To demonstrate the utility of this approach, a pilot screen of a library of 891 compounds derived from natural products was carried out. Quality control analysis indicates that the primary screen was robust for identification of influenza A virus inhibitors targeting RdRp activity. Finally, two hit candidates were identified, and one was validated as a putative RdRp inhibitor. This strategy can greatly reduce the number of false positives and improve the accuracy and efficacy of primary screening, thereby providing a powerful tool for antiviral discovery.

Nucleoside Analogs with Selective Antiviral Activity against Dengue Fever and Japanese Encephalitis Viruses.

Dengue virus (DENV) and Japanese encephalitis virus (JEV) are important arthropod-borne viruses from the Flaviviridae family. DENV is a global public health problem with significant social and economic impacts, especially in tropical and subtropical areas. JEV is a neurotropic arbovirus endemic to east and southeast Asia. There are no U.S. FDA-approved antiviral drugs available to treat or to prevent DENV and JEV infections, leaving nearly one-third of the world's population at risk for infection. Therefore, it is crucial to discover potent antiviral agents against these viruses. Nucleoside analogs, as a class, are widely used for the treatment of viral infections. In this study, we discovered nucleoside analogs that possess potent and selective anti-JEV and anti-DENV activities across all serotypes in cell-based assay systems. Both viruses were susceptible to sugar-substituted 2'-C-methyl analogs with either cytosine or 7-deaza-7-fluoro-adenine nucleobases. Mouse studies confirmed the anti-DENV activity of these nucleoside analogs. Molecular models were assembled for DENV serotype 2 (DENV-2) and JEV RNA-dependent RNA polymerase replication complexes bound to nucleotide inhibitors. These models show similarities between JEV and DENV-2, which recognize the same nucleotide inhibitors. Collectively, our findings provide promising compounds and a structural rationale for the development of direct-acting antiviral agents with dual activity against JEV and DENV infections.