Blocking NS3-NS4B interaction inhibits dengue virus in non-human primates.

Dengue is a major health threat and the number of symptomatic infections caused by the four dengue serotypes is estimated to be 96 million1 with annually around 10,000 deaths2. However, no antiviral drugs are available for the treatment or prophylaxis of dengue. We recently described the interaction between non-structural proteins NS3 and NS4B as a promising target for the development of pan-serotype dengue virus (DENV) inhibitors3. Here we present JNJ-1802-a highly potent DENV inhibitor that blocks the NS3-NS4B interaction within the viral replication complex. JNJ-1802 exerts picomolar to low nanomolar in vitro antiviral activity, a high barrier to resistance and potent in vivo efficacy in mice against infection with any of the four DENV serotypes. Finally, we demonstrate that the small-molecule inhibitor JNJ-1802 is highly effective against viral infection with DENV-1 or DENV-2 in non-human primates. JNJ-1802 has successfully completed a phase I first-in-human clinical study in healthy volunteers and was found to be safe and well tolerated4. These findings support the further clinical development of JNJ-1802, a first-in-class antiviral agent against dengue, which is now progressing in clinical studies for the prevention and treatment of dengue.

A pan-serotype dengue virus inhibitor targeting the NS3-NS4B interaction.

Dengue virus causes approximately 96 million symptomatic infections annually, manifesting as dengue fever or occasionally as severe dengue1,2. There are no antiviral agents available to prevent or treat dengue. Here, we describe a highly potent dengue virus inhibitor (JNJ-A07) that exerts nanomolar to picomolar activity against a panel of 21 clinical isolates that represent the natural genetic diversity of known genotypes and serotypes. The molecule has a high barrier to resistance and prevents the formation of the viral replication complex by blocking the interaction between two viral proteins (NS3 and NS4B), thus revealing a previously undescribed mechanism of antiviral action. JNJ-A07 has a favourable pharmacokinetic profile that results in outstanding efficacy against dengue virus infection in mouse infection models. Delaying start of treatment until peak viraemia results in a rapid and significant reduction in viral load. An analogue is currently in further development.

Safety, Tolerability, and Pharmacokinetics of JNJ-1802, a Pan-serotype Dengue Direct Antiviral Small Molecule, in a Phase 1, Double-Blind, Randomized, Dose-Escalation Study in Healthy Volunteers.

BACKGROUND: Dengue is a growing global health threat with no specific antiviral drugs available for treatment or prophylaxis. This first-in-human, double-blind, randomized, placebo-controlled study aimed to examine the safety, tolerability, and pharmacokinetics of increasing single and multiple oral doses of JNJ-1802, a pan-serotype dengue antiviral small molecule. METHODS: Eligible healthy participants (18-55 years of age) were randomized to receive oral JNJ-1802 in fasted conditions as (1) single doses (50-1200 mg; n = 29) or placebo (n = 10); or (2) once-daily doses (50-560 mg for 10 consecutive days or 400 mg for 31 days; n = 38) or placebo (n = 9). Safety and tolerability were evaluated throughout the study. Plasma and urine samples were collected at predetermined time points to characterize pharmacokinetics. RESULTS: JNJ-1802 was generally safe and well-tolerated. One grade 3 adverse event (depression) was reported but not considered drug-related by the investigator. Two grade 2 events of rash occurred (multiple-dose part) that were considered very likely related to JNJ-1802 by the investigator and resolved. No clinically relevant changes were observed in laboratory tests, electrocardiograms, or vital signs.JNJ-1802 exposure after single or multiple doses increased dose-proportionally from 50 to 150 mg and less than dose-proportionally for higher doses. The terminal elimination half-life was 6.3-9.2 days and the accumulation factor was 4.3-7.3 after 10 days and 14.6 after 31 days with low amounts of unchanged drug in urine (<0.001% of the 400 mg dose). CONCLUSIONS: Pharmacokinetics and safety results of JNJ-1802 support further clinical development for the treatment and prevention of dengue infection.

Development and optimization of a high-throughput screening assay for in vitro anti-SARS-CoV-2 activity: Evaluation of 5676 Phase 1 Passed Structures.

Although vaccines are currently used to control the coronavirus disease 2019 (COVID-19) pandemic, treatment options are urgently needed for those who cannot be vaccinated and for future outbreaks involving new severe acute respiratory syndrome coronavirus virus 2 (SARS-CoV-2) strains or coronaviruses not covered by current vaccines. Thus far, few existing antivirals are known to be effective against SARS-CoV-2 and clinically successful against COVID-19. As part of an immediate response to the COVID-19 pandemic, a high-throughput, high content imaging-based SARS-CoV-2 infection assay was developed in VeroE6 African green monkey kidney epithelial cells expressing a stable enhanced green fluorescent protein (VeroE6-eGFP cells) and was used to screen a library of 5676 compounds that passed Phase 1 clinical trials. Eight drugs (nelfinavir, RG-12915, itraconazole, chloroquine, hydroxychloroquine, sematilide, remdesivir, and doxorubicin) were identified as inhibitors of in vitro anti-SARS-CoV-2 activity in VeroE6-eGFP and/or Caco-2 cell lines. However, apart from remdesivir, toxicity and pharmacokinetic data did not support further clinical development of these compounds for COVID-19 treatment.

In vitro activity of itraconazole against SARS-CoV-2.

Although vaccination campaigns are currently being rolled out to prevent coronavirus disease (COVID-19), antivirals will remain an important adjunct to vaccination. Antivirals against coronaviruses do not exist, hence global drug repurposing efforts have been carried out to identify agents that may provide clinical benefit to patients with COVID-19. Itraconazole, an antifungal agent, has been reported to have activity against animal coronaviruses. Using cell-based phenotypic assays, the in vitro antiviral activity of itraconazole and 17-OH itraconazole was assessed against clinical isolates from a German and Belgian patient infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Itraconazole demonstrated antiviral activity in human Caco-2 cells (EC50 = 2.3 µM; 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay). Similarly, its primary metabolite, 17-OH itraconazole, showed inhibition of SARS-CoV-2 activity (EC50 = 3.6 µM). Remdesivir inhibited viral replication with an EC50 = 0.4 µM. Itraconazole and 17-OH itraconazole resulted in a viral yield reduction in vitro of approximately 2-log10 and approximately 1-log10 , as measured in both Caco-2 cells and VeroE6-eGFP cells, respectively. The viral yield reduction brought about by remdesivir or GS-441524 (parent nucleoside of the antiviral prodrug remdesivir; positive control) was more pronounced, with an approximately 3-log10 drop and >4-log10 drop in Caco-2 cells and VeroE6-eGFP cells, respectively. Itraconazole and 17-OH itraconazole exert in vitro low micromolar activity against SARS-CoV-2. Despite the in vitro antiviral activity, itraconazole did not result in a beneficial effect in hospitalized COVID-19 patients in a clinical study (EudraCT Number: 2020-001243-15).

An Industry Perspective on Dengue Drug Discovery and Development.

Dengue is the most important mosquito-borne viral disease in the world, representing a major unmet medical need and a growing public health concern. The disease imposes a heavy burden to the affected individuals, to the health care systems, and to the economies of endemic countries. Vector control is the most widespread tool to curb dengue epidemics, but has been insufficient. Therefore, additional means such as vaccines and antivirals are required to aid in a coordinated response. The discovery and development of small molecule dengue virus inhibitors as a tool to prevent and/or treat dengue disease faces major hurdles in combining pan-serotypic efficacy, safety, and optimal drug-like properties. Moreover, the financial return of dengue drug projects may not compensate for the initial investment in research and development. This review article addresses the efforts undertaken to face the dengue epidemics, focusing on antiviral drug development. The dengue drug research and development process is described in detail and a dengue antiviral target product profile is proposed. The article discusses collaborations between the different players in the research field: government and government-sponsored organizations, pharmaceutical and biotechnology companies, academia, and non-profit and philanthropic organizations. Public-private partnerships are proposed as a model to boost dengue research and development towards an approved antiviral drug in the near future.

Human cytomegalovirus miR-UL112-1 promotes the down-regulation of viral immediate early-gene expression during latency to prevent T-cell recognition of latently infected cells.

Human cytomegalovirus, a member of the herpesvirus family, can cause significant morbidity and mortality in immune compromised patients resulting from either primary lytic infection or reactivation from latency. Latent infection is associated with a restricted viral transcription programme compared to lytic infection which consists of defined protein coding RNAs but also includes a number of virally encoded microRNAs (miRNAs). One of these, miR-UL112-1, is known to target the major lytic IE72 transcript but, to date, a functional role for miR-UL112-1 during latent infection has not been shown. To address this, we have analysed latent infection in myeloid cells using a virus in which the target site for miR-UL112-1 in the 3' UTR of IE72 was removed such that any IE72 RNA present during latent infection would no longer be subject to regulation by miR-UL112-1 through the RNAi pathway. Our data show that removal of the miR-UL112-1 target site in IE72 results in increased levels of IE72 RNA in experimentally latent primary monocytes. Furthermore, this resulted in induction of immediate early (IE) gene expression that is detectable by IE-specific cytotoxic T-cells (CTLs); no such CTL recognition of monocytes latently infected with wild-type virus was observed. We also recapitulated these findings in the more tractable THP-1 cell line model of latency. These observations argue that an important role for miR-UL112-1 during latency is to ensure tight control of lytic viral immediate early (IE) gene expression thereby preventing recognition of latently infected cells by the host's potent pre-existing anti-viral CTL response.

High-throughput analysis of human cytomegalovirus genome diversity highlights the widespread occurrence of gene-disrupting mutations and pervasive recombination.

Human cytomegalovirus is a widespread pathogen of major medical importance. It causes significant morbidity and mortality in the immunocompromised and congenital infections can result in severe disabilities or stillbirth. Development of a vaccine is prioritized, but no candidate is close to release. Although correlations of viral genetic variability with pathogenicity are suspected, knowledge about strain diversity of the 235kb genome is still limited. In this study, 96 full-length human cytomegalovirus genomes from clinical isolates were characterized, quadrupling the available information for full-genome analysis. These data provide the first high-resolution map of human cytomegalovirus interhost diversity and evolution. We show that cytomegalovirus is significantly more divergent than all other human herpesviruses and highlight hotspots of diversity in the genome. Importantly, 75% of strains are not genetically intact, but contain disruptive mutations in a diverse set of 26 genes, including immunomodulative genes UL40 and UL111A. These mutants are independent from culture passaging artifacts and circulate in natural populations. Pervasive recombination, which is linked to the widespread occurrence of multiple infections, was found throughout the genome. Recombination density was significantly higher than in other human herpesviruses and correlated with strain diversity. While the overall effects of strong purifying selection on virus evolution are apparent, evidence of diversifying selection was found in several genes encoding proteins that interact with the host immune system, including UL18, UL40, UL142 and UL147. These residues may present phylogenetic signatures of past and ongoing virus-host interactions. IMPORTANCE: Human cytomegalovirus has the largest genome of all viruses that infect humans. Currently, there is a great interest in establishing associations between genetic variants and strain pathogenicity of this herpesvirus. Since the number of publicly available full-genome sequences is limited, knowledge about strain diversity is highly fragmented and biased towards a small set of loci. Combined with our previous work, we have now contributed 101 complete genome sequences. We have used these data to conduct the first high-resolution analysis of interhost genome diversity, providing an unbiased and comprehensive overview of cytomegalovirus variability. These data are of major value to the development of novel antivirals and a vaccine and to identify potential targets for genotype-phenotype experiments. Furthermore, they have enabled a thorough study of the evolutionary processes that have shaped cytomegalovirus diversity.

Glucocorticosteroids trigger reactivation of human cytomegalovirus from latently infected myeloid cells and increase the risk for HCMV infection in D+R+ liver transplant patients.

Graft rejection in transplant patients is managed clinically by suppressing T-cell function with immunosuppressive drugs such as prednisolone and methylprednisolone. In such immunocompromised hosts, human cytomegalovirus (HCMV) is an important opportunistic pathogen and can cause severe morbidity and mortality. Currently, the effect of glucocorticosteroids (GCSs) on the HCMV life cycle remains unclear. Previous reports showed enhanced lytic replication of HCMV in vitro in the presence of GCSs. In the present study, we explored the implications of steroid exposure on latency and reactivation. We observed a direct effect of several GCSs used in the clinic on the activation of a quiescent viral major immediate-early promoter in stably transfected THP-1 monocytic cells. This activation was prevented by the glucocorticoid receptor (GR) antagonist Ru486 and by shRNA-mediated knockdown of the GR. Consistent with this observation, prednisolone treatment of latently infected primary monocytes resulted in HCMV reactivation. Analysis of the phenotype of these cells showed that treatment with GCSs was correlated with differentiation to an anti-inflammatory macrophage-like cell type. On the basis that these observations may be pertinent to HCMV reactivation in post-transplant settings, we retrospectively evaluated the incidence, viral kinetics and viral load of HCMV in liver transplant patients in the presence or absence of GCS treatment. We observed that combination therapy of baseline prednisolone and augmented methylprednisolone, upon organ rejection, significantly increased the incidence of HCMV infection in the intermediate risk group where donor and recipient are both HCMV seropositive (D+R+) to levels comparable with the high risk D+R- group.

A flexible, image-based, high-throughput platform encompassing in-depth cell profiling to identify broad-spectrum coronavirus antivirals with limited off-target effects.

The recent pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) posed a major threat to global health. Although the World Health Organization ended the public health emergency status, antiviral drugs are needed to address new variants of SARS-CoV-2 and future pandemics. To identify novel broad-spectrum coronavirus drugs, we developed a high-content imaging platform compatible with high-throughput screening. The platform is broadly applicable as it can be adapted to include various cell types, viruses, antibodies, and dyes. We demonstrated that the antiviral activity of compounds against SARS-CoV-2 variants (Omicron BA.5 and Omicron XBB.1.5), SARS-CoV, and human coronavirus 229E could easily be assessed. The inclusion of cellular dyes and immunostaining in combination with in-depth image analysis enabled us to identify compounds that induced undesirable phenotypes in host cells, such as changes in cell morphology or in lysosomal activity. With the platform, we screened ∼900K compounds and triaged hits, thereby identifying potential candidate compounds carrying broad-spectrum activity with limited off-target effects. The flexibility and early-stage identification of compounds with limited host cell effects provided by this high-content imaging platform can facilitate coronavirus drug discovery. We anticipate that its rapid deployability and fast turnaround can also be applied to combat future pandemics.

A pan-serotype antiviral to prevent and treat dengue: A journey from discovery to clinical development driven by public-private partnerships.

While progress has been made in fighting diseases disproportionally affecting underserved populations, unmet medical needs persist for many neglected tropical diseases. The World Health Organization has encouraged strong public-private partnerships to address this issue and several public and private organizations have set an example in the past showing a strong commitment to combat these diseases. Pharmaceutical companies are contributing in different ways to address the imbalance in research efforts. With this review, we exemplify the role of a public-private partnership in research and development by the journey of our dengue antiviral molecule that is now in early clinical development. We detail the different steps of drug development and outline the contribution of each partner to this process. Years of intensive collaboration resulted in the identification of two antiviral compounds, JNJ-A07 and JNJ-1802, the latter of which has advanced to clinical development.

Discovery of Acyl-Indole Derivatives as Pan-Serotype Dengue Virus NS4B Inhibitors.

In the absence of any approved dengue-specific treatment, the discovery and development of a novel small-molecule antiviral for the prevention or treatment of dengue are critical. We previously reported the identification of a novel series of 3-acyl-indole derivatives as potent and pan-serotype dengue virus inhibitors. We herein describe our optimization efforts toward preclinical candidates 24a and 28a with improved pan-serotype coverage (EC50's against the four DENV serotypes ranging from 0.0011 to 0.24 µM for 24a and from 0.00060 to 0.084 µM for 28a), chiral stability, and oral bioavailability in preclinical species, as well as showing a dose-proportional increase in efficacy against DENV-2 infection in vivo in mice.

Identification of Z-Tyr-Ala-CHN2, a Cathepsin L Inhibitor with Broad-Spectrum Cell-Specific Activity against Coronaviruses, including SARS-CoV-2.

The ongoing COVID-19 pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is partly under control by vaccination. However, highly potent and safe antiviral drugs for SARS-CoV-2 are still needed to avoid development of severe COVID-19. We report the discovery of a small molecule, Z-Tyr-Ala-CHN2, which was identified in a cell-based antiviral screen. The molecule exerts sub-micromolar antiviral activity against SARS-CoV-2, SARS-CoV-1, and human coronavirus 229E. Time-of-addition studies reveal that Z-Tyr-Ala-CHN2 acts at the early phase of the infection cycle, which is in line with the observation that the molecule inhibits cathepsin L. This results in antiviral activity against SARS-CoV-2 in VeroE6, A549-hACE2, and HeLa-hACE2 cells, but not in Caco-2 cells or primary human nasal epithelial cells since the latter two cell types also permit entry via transmembrane protease serine subtype 2 (TMPRSS2). Given their cell-specific activity, cathepsin L inhibitors still need to prove their value in the clinic; nevertheless, the activity profile of Z-Tyr-Ala-CHN2 makes it an interesting tool compound for studying the biology of coronavirus entry and replication.

A phylogenetic study of dengue virus in urban Vietnam shows long-term persistence of endemic strains.

Dengue virus (DENV) causes repeated outbreaks of disease in endemic areas, with patterns of local transmission strongly influenced by seasonality, importation via human movement, immunity, and vector control efforts. An understanding of how each of these interacts to enable endemic transmission (continual circulation of local virus strains) is largely unknown. There are times of the year when no cases are reported, often for extended periods of time, perhaps wrongly implying the successful eradication of a local strain from that area. Individuals who presented at a clinic or hospital in four communes in Nha Trang, Vietnam, were initially tested for DENV antigen presence. Enrolled positive individuals then had their corresponding household members invited to participate, and those who enrolled were tested for DENV. The presence of viral nucleic acid in all samples was confirmed using quantitative polymerase chain reaction, and positive samples were then whole-genome sequenced using an amplicon and target enrichment library preparation techniques and Illumina MiSeq sequencing technology. Generated consensus genome sequences were then analysed using phylogenetic tree reconstruction to categorise sequences into clades with a common ancestor, enabling investigations of both viral clade persistence and introductions. Hypothetical introduction dates were additionally assessed using a molecular clock model that calculated the time to the most recent common ancestor (TMRCA). We obtained 511 DENV whole-genome sequences covering four serotypes and more than ten distinct viral clades. For five of these clades, we had sufficient data to show that the same viral lineage persisted for at least several months. We noted that some clades persisted longer than others during the sampling time, and by comparison with other published sequences from elsewhere in Vietnam and around the world, we saw that at least two different viral lineages were introduced into the population during the study period (April 2017-2019). Next, by inferring the TMRCA from the construction of molecular clock phylogenies, we predicted that two of the viral lineages had been present in the study population for over a decade. We observed five viral lineages co-circulating in Nha Trang from three DENV serotypes, with two likely to have remained as uninterrupted transmission chains for a decade. This suggests clade cryptic persistence in the area, even during periods of low reported incidence.

From Oxetane to Thietane: Extending the Antiviral Spectrum of 2'-Spirocyclic Uridines by Substituting Oxygen with Sulfur.

In continuation of our efforts of finding novel nucleoside inhibitors for the treatment of viral diseases, we initiated a discovery research program aimed at identifying novel nucleos(t)ide inhibitors for emerging diseases like Dengue and Chikungunya. Based on the previously reported 2'-spiro-oxetane uridine derivatives active against Hepatitis C Virus (HCV), we envisaged its sulfur analogue as an interesting congener both from a synthetic as well as biological point of view. Surprisingly, we found the 2'-spirothietane uridine derivatives not only to be active against HCV and Dengue virus (DENV), viruses belonging to the flavivirus family, but also to demonstrate activity against alphaviruses like Chikungunya virus (CHIKV) and Sindbis virus (SINV).

Evolution of inflammation and immunity in a dengue virus 1 human infection model.

Dengue virus (DENV) infections are major causes of morbidity and mortality throughout the tropics and subtropics. More than 400 million infections are estimated to occur every year, resulting in nearly 100 million symptomatic infections and more than 20,000 deaths. Early immune response kinetics to infection remain unclear, in large part due to the variable incubation period exhibited by the DENVs after introduction into a susceptible host. To fill this knowledge gap, we performed a comprehensive virologic and immunologic analysis of individuals experimentally infected with the underattenuated DENV-1 strain 45AZ5. This analysis captured both the kinetics and composition of the innate, humoral, and cellular immune responses elicited by experimental DENV-1 infection, as well as virologic and clinical features. We observed a robust DENV-specific immunoglobulin A (IgA) antibody response that manifested between the appearance of DENV-specific IgM and IgG in all challenged individuals, as well as the presence of a non-neutralizing/NS1-specific antibody response that was delayed relative to the appearance of DENV virion-specific humoral immunity. RNA sequencing analysis revealed discrete and temporally restricted gene modules that correlated with acute viremia and the induction of adaptive immunity. Our analysis provides a detailed description, in time and space, of the evolving matrix of DENV-elicited human inflammation and immunity and reveals several previously unappreciated immunological aspects of primary DENV-1 infection that can inform countermeasure development and evaluation.

Combining rapid diagnostic tests to estimate primary and post-primary dengue immune status at the point of care.

BACKGROUND: Characterising dengue virus (DENV) infection history at the point of care is challenging as it relies on intensive laboratory techniques. We investigated how combining different rapid diagnostic tests (RDTs) can be used to accurately determine the primary and post-primary DENV immune status of reporting patients during diagnosis. METHODS AND FINDINGS: Serum from cross-sectional surveys of acute suspected dengue patients in Indonesia (N:200) and Vietnam (N: 1,217) were assayed using dengue laboratory assays and RDTs. Using logistic regression modelling, we determined the probability of being DENV NS1, IgM and IgG RDT positive according to corresponding laboratory viremia, IgM and IgG ELISA metrics. Laboratory test thresholds for RDT positivity/negativity were calculated using Youden's J index and were utilized to estimate the RDT outcomes in patients from the Philippines, where only data for viremia, IgM and IgG were available (N:28,326). Lastly, the probabilities of being primary or post-primary according to every outcome using all RDTs, by day of fever, were calculated. Combining NS1, IgM and IgG RDTs captured 94.6% (52/55) and 95.4% (104/109) of laboratory-confirmed primary and post-primary DENV cases, respectively, during the first 5 days of fever. Laboratory test predicted, and actual, RDT outcomes had high agreement (79.5% (159/200)). Among patients from the Philippines, different combinations of estimated RDT outcomes were indicative of post-primary and primary immune status. Overall, IgG RDT positive results were confirmatory of post-primary infections. In contrast, IgG RDT negative results were suggestive of both primary and post-primary infections on days 1-2 of fever, yet were confirmatory of primary infections on days 3-5 of fever. CONCLUSION: We demonstrate how the primary and post-primary DENV immune status of reporting patients can be estimated at the point of care by combining NS1, IgM and IgG RDTs and considering the days since symptoms onset. This framework has the potential to strengthen surveillance operations and dengue prognosis, particularly in low resource settings.

Lack of antiviral activity of darunavir against SARS-CoV-2.

OBJECTIVES: Given the high need and the absence of specific antivirals for treatment of COVID-19 (the disease caused by severe acute respiratory syndrome-associated coronavirus-2 [SARS-CoV-2]), human immunodeficiency virus (HIV) protease inhibitors are being considered as therapeutic alternatives. METHODS: Prezcobix/Rezolsta is a fixed-dose combination of 800 mg of the HIV protease inhibitor darunavir (DRV) and 150 mg cobicistat, a CYP3A4 inhibitor, which is indicated in combination with other antiretroviral agents for the treatment of HIV infection. There are currently no definitive data on the safety and efficacy of DRV/cobicistat for the treatment of COVID-19. The in vitro antiviral activity of darunavir against a clinical isolate from a patient infected with SARS-CoV-2 was assessed. RESULTS: DRV showed no antiviral activity against SARS-CoV-2 at clinically relevant concentrations (EC50 > 100 µM). Remdesivir, used as a positive control, demonstrated potent antiviral activity (EC50 = 0.38 µM). CONCLUSIONS: Overall, the data do not support the use of DRV for the treatment of COVID-19.

Phylogenetically based establishment of a dengue virus panel, representing all available genotypes, as a tool in dengue drug discovery.

Dengue fever is the most widespread of the human arbovirus diseases, with approximately one third of the world's population at risk of infection. Dengue viruses are members of the genus Flavivirus (family Flaviviridae) and, antigenically, they separate as four closely related serotypes (1-4) that share 60-75% amino acid homology. This genetic diversity complicates the process of antiviral drug discovery. Thus, currently no approved dengue-specific therapeutic treatments are available. With the aim of providing an efficient tool for dengue virus drug discovery, a collection of nineteen dengue viruses, representing the genotypic diversity within the four serotypes, was developed. After phylogenetic analysis of the full-length genomes, we selected relevant strains from the EVAg collection at Aix-Marseille University and completed the virus collection, using a reverse genetic system based on the infectious sub-genomic amplicons technique. Finally, we evaluated this dengue virus collection against three published dengue inhibitory compounds. NITD008, which targets the highly conserved active site of the viral NS5 polymerase enzyme, exhibited similar antiviral potencies against each of the different dengue genotypes in the panel. Compounds targeting less conserved protein subdomains, such as the capsid inhibitor ST-148, or SDM25N, a ∂ opioid receptor antagonist which indirectly targets NS4B, exhibited larger differences in potency against the various genotypes of dengue viruses. These results illustrate the importance of a phylogenetically based dengue virus reference panel for dengue antiviral research. The collection developed in this study, which includes such representative dengue viruses, has been made available to the scientific community through the European Virus Archive to evaluate novel DENV antiviral candidates.