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Background: Identifying host factors is key to understanding RNA virus pathogenicity. Besides proteins, RNAs can interact with virus genomes to impact replication., Results: Here, we use proximity ligation sequencing to identify virus-host RNA interactions for four strains of Zika virus (ZIKV) and one strain of dengue virus (DENV-1) in human cells. We find hundreds of coding and non-coding RNAs that bind to DENV and ZIKV viruses. Host RNAs tend to bind to single-stranded regions along the virus genomes according to hybridization energetics. Compared to SARS-CoV-2 interactors, ZIKV-interacting host RNAs tend to be downregulated upon virus infection. Knockdown of several short non-coding RNAs, including miR19a-3p, and 7SK RNA results in a decrease in viral replication, suggesting that they act as virus-permissive factors. In addition, the 3'UTR of DYNLT1 mRNA acts as a virus-restrictive factor by binding to the conserved dumbbell region on DENV and ZIKV 3'UTR to decrease virus replication. We also identify a conserved set of host RNAs that interacts with DENV, ZIKV, and SARS-CoV-2, suggesting that these RNAs are broadly important for RNA virus infection., Conclusions: This study demonstrates that host RNAs can impact virus replication in permissive and restrictive ways, expanding our understanding of host factors and RNA-based gene regulation during viral pathogenesis., (© 2023. The Author(s).)

AT-752 is a guanosine analogue prodrug active against dengue virus (DENV). In infected cells, it is metabolized into 2'-methyl-2'-fluoro guanosine 5'-triphosphate (AT-9010) which inhibits RNA synthesis in acting as a RNA chain terminator. Here we show that AT-9010 has several modes of action on DENV full-length NS5. AT-9010 does not inhibit the primer pppApG synthesis step significantly. However, AT-9010 targets two NS5-associated enzyme activities, the RNA 2'-O-MTase and the RNA-dependent RNA polymerase (RdRp) at its RNA elongation step. Crystal structure and RNA methyltransferase (MTase) activities of the DENV 2 MTase domain in complex with AT-9010 at 1.97 Å resolution shows the latter bound to the GTP/RNA-cap binding site, accounting for the observed inhibition of 2'-O but not N7-methylation activity. AT-9010 is discriminated ∼10 to 14-fold against GTP at the NS5 active site of all four DENV1-4 NS5 RdRps, arguing for significant inhibition through viral RNA synthesis termination. In Huh-7 cells, DENV1-4 are equally sensitive to AT-281, the free base of AT-752 (EC 50  ≈ 0.50 μM), suggesting broad spectrum antiviral properties of AT-752 against flaviviruses., Competing Interests: Declaration of competing interest S.G., K.L., A.M. and J.P.S. are employees of ATEA Pharmaceuticals, Inc. The other authors declare no competing interests., (Copyright © 2023. Published by Elsevier B.V.)

Zika virus (ZIKV) and dengue virus (DENV) are arthropod-borne pathogenic flaviviruses that co-circulate in many countries. To understand some of the pressures that influence ZIKV evolution, we mimic the natural transmission cycle by repeating serial passaging of ZIKV through cultured mosquito cells and either DENV-naive or DENV-immune mice. Compared with wild-type ZIKV, the strains passaged under both conditions exhibit increased pathogenesis in DENV-immune mice. Application of reverse genetics identifies an isoleucine-to-valine mutation (I39V) in the NS2B proteins of both passaged strains that confers enhanced fitness and escape from pre-existing DENV immunity. Introduction of I39V or I39T, a naturally occurring homologous mutation detected in recent ZIKV isolates, increases the replication of wild-type ZIKV in human neuronal precursor cells and laboratory-raised mosquitoes. Our data indicate that ZIKV strains with enhanced transmissibility and pathogenicity can emerge in DENV-naive or -immune settings, and that NS2B-I39 mutants may represent ZIKV variants of interest., Competing Interests: Declaration of interests The authors declare no conflicts of interest., (Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved.)

Dengue virus (DENV) is a mosquito-borne flavivirus that poses a threat to public health, yet no antiviral drug is available. We performed a high-throughput phenotypic screen using the Novartis compound library and identified candidate chemical inhibitors of DENV. This chemical series was optimized to improve properties such as anti-DENV potency and solubility. The lead compound, NITD-688, showed strong potency against all four serotypes of DENV and demonstrated excellent oral efficacy in infected AG129 mice. There was a 1.44-log reduction in viremia when mice were treated orally at 30 milligrams per kilogram twice daily for 3 days starting at the time of infection. NITD-688 treatment also resulted in a 1.16-log reduction in viremia when mice were treated 48 hours after infection. Selection of resistance mutations and binding studies with recombinant proteins indicated that the nonstructural protein 4B is the target of NITD-688. Pharmacokinetic studies in rats and dogs showed a long elimination half-life and good oral bioavailability. Extensive in vitro safety profiling along with exploratory rat and dog toxicology studies showed that NITD-688 was well tolerated after 7-day repeat dosing, demonstrating that NITD-688 may be a promising preclinical candidate for the treatment of dengue., (Copyright © 2021 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Monophosphate prodrug analogs of 2'-deoxy-2'-fluoro-2'- C -methylguanosine have been reported as potent inhibitors of hepatitis C virus (HCV) RNA-dependent RNA polymerase. These prodrugs also display potent anti-dengue virus activities in cellular assays although their prodrug moieties were designed to produce high levels of triphosphate in the liver. Since peripheral blood mononuclear cells (PBMCs) are among the major targets of dengue virus, different prodrug moieties were designed to effectively deliver 2'-deoxy-2'-fluoro-2'- C -methylguanosine monophosphate prodrugs and their corresponding triphosphates into PBMCs after oral administration. We identified a cyclic phosphoramidate, prodrug 17, demonstrating well-balanced anti-dengue virus cellular activity and in vitro stability profiles. We further determined the PBMC concentration of active triphosphate needed to inhibit virus replication by 50% (TP 50 ). Compound 17 was assessed in an AG129 mouse model and demonstrated 1.6- and 2.2-log viremia reductions at 100 and 300 mg/kg twice a day (BID), respectively. At 100 mg/kg BID, the terminal triphosphate concentration in PBMCs exceeded the TP 50 value, demonstrating TP 50 as the target exposure for efficacy. In dogs, oral administration of compound 17 resulted in high PBMC triphosphate levels, exceeding the TP 50 at 10 mg/kg. Unfortunately, 2-week dog toxicity studies at 30, 100, and 300 mg/kg/day showed that "no observed adverse effect level" (NOAEL) could not be achieved due to pulmonary inflammation and hemorrhage. The preclinical safety results suspended further development of compound 17. Nevertheless, present work has proven the concept that an efficacious monophosphate nucleoside prodrug could be developed for the potential treatment of dengue virus infection., (Copyright © 2020 American Society for Microbiology.)

A series of substituted 4,6-dihydrospiro[[1,2,3]triazolo[4,5- b ]pyridine-7,3'-indoline]-2',5(3 H )-dione analogues were synthesized and evaluated as potent dengue virus inhibitors. Throughout a structure-activity relationship exploration on the amide of the indolone moiety, a wide range of substitutions were found to be well tolerated for chemical optimization at this position. Among these compounds, 15 ( JMX0254 ) displayed the most potent and broad inhibitory activities, effective against DENV-1 to -3 with EC 50 values of 0.78, 0.16, and 0.035 μM, respectively, while compounds 16 , 21 , 27 - 29 , 47 , and 70 exhibited relatively moderate to high activities with low micromolar to nanomolar potency against all four serotypes. The biotinylated compound 73 enriched NS4B protein from cell lysates in pull-down studies, and the findings together with the mutation investigations further validated dengue NS4B protein as the target of this class of compounds. More importantly, compound 15 exhibited good in vivo pharmacokinetic properties and efficacy in the A129 mouse model, indicating its therapeutic potential against the dengue virus infection as a drug candidate for further preclinical development.

Zika (ZIKV) and dengue (DENV) virus infections elicit a robust but cross-reactive antibody response against the viral envelope protein, while antibody responses against non-structural proteins (NS) are more virus specific. Building on this premise, we have previously developed a flavivirus multiplex microsphere immunoassay (MIA) for the serologic diagnosis of ZIKV and DENV infections. This assay significantly improved diagnostic accuracy; however, MIA could not differentiate more recent from past infections, which still represents a major diagnostic challenge. Therefore, an immunoglobulin G (IgG) based avidity assay was developed and its diagnostic performance evaluated. Specimens from New York State residents were submitted to the Wadsworth Center New York State Department of Health (NYSDOH) for routine clinical testing by Zika IgM ELISA and plaque reduction neutralization test (PRNT). Using our previously developed flavivirus MIA as a platform, we developed an IgG avidity assay to discriminate recent ZIKV from past DENV infections. Zika IgM positive specimens had an average Zika IgG avidity index of 14.8% (95% CI: 11.0-18.4%), while Zika IgM negative but flavivirus MIA and PRNT positive samples had an average Zika IgG avidity index of 34.9% (95% CI: 31.1-38.7%). Specimens positive for dengue antibodies by flavivirus MIA and PRNT had an average dengue IgG avidity index of 68.7% (95% CI: 62.7-75.0%). The IgG avidity assay accurately distinguished recent ZIKV from past DENV infections in patients who traveled to dengue endemic regions. This assay could be very useful in patients with high risk of Zika complications such as pregnant women and monitoring immune responses in vaccine trials., (© 2019 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.)

Dengue virus (DENV) is the most prevalent mosquito-transmitted viral pathogen in humans. The recently licensed dengue vaccine has major weaknesses. Therefore, there is an urgent need to develop improved dengue vaccines. Here, we report a virion assembly-defective DENV as a vaccine platform. DENV containing an amino acid deletion (K188) in nonstructural protein 2A (NS2A) is fully competent in viral RNA replication but is completely defective in virion assembly. When trans -complemented with wild-type NS2A protein, the virion assembly defect could be rescued, generating pseudoinfectious virus (PIV NS2A ) that could initiate single-round infection. The trans -complementation efficiency could be significantly improved through selection for adaptive mutations, leading to high-yield PIV NS2A production, with titers of >10 7 infectious-focus units (IFU)/ml. Mice immunized with a single dose of PIV NS2A elicited strong T cell immune responses and neutralization antibodies and were protected from wild-type-virus challenge. Collectively, the results proved the concept of using assembly-defective virus as a vaccine approach. The study also solved the technical bottleneck in producing high yields of PIV NS2A vaccine. The technology could be applicable to vaccine development for other viral pathogens. IMPORTANCE Many flaviviruses are significant human pathogens that pose global threats to public health. Although licensed vaccines are available for yellow fever, Japanese encephalitis, tick-borne encephalitis, and dengue viruses, new approaches are needed to develop improved vaccines. Using dengue virus as a model, we developed a vaccine platform using a virion assembly-defective virus. We show that such an assembly-defective virus could be rescued to higher titers and infect cells for a single round. Mice immunized with the assembly-defective virus were protected from wild-type-virus infection. This vaccine approach could be applicable to other viral pathogens., (Copyright © 2018 American Society for Microbiology.)

Flavivirus NS5 RNA-dependent RNA polymerase (RdRp) is an important drug target. Whilst a number of allosteric inhibitors have been described for Hepatitis C virus RdRp, few have been described for DENV RdRp. In addition, compound screening campaigns have not yielded suitable leads for this enzyme. Using fragment-based screening via X-ray crystallography, we identified a biphenyl acetic acid fragment that binds to a novel pocket of the dengue virus (DENV) RdRp, in the thumb/palm interface, close to its active site (termed "N pocket"). Structure-guided optimization yielded nanomolar inhibitors of the RdRp de novo initiation activity, with low micromolar EC 50 in DENV cell-based assays. Compound-resistant DENV replicons exhibited amino acid mutations that mapped to the N pocket. This is the first report of a class of pan-serotype and cell-active DENV RdRp inhibitors and provides a significant opportunity for rational design of novel therapeutics against this proven antiviral target.

Currently, the laboratory diagnosis of Zika virus (ZIKV) infection is primarily through the detection of ZIKV RNA or antibodies against ZIKV proteins. The detection of viral RNA is highly sensitive and specific, but periods of viremia and viruria are brief, limiting the utility of ZIKV RNA assays. Instead, most ZIKV infections are diagnosed serologically, using an IgM antibody capture enzyme-linked immunosorbent assay (MAC-ELISA) for screening, followed by a confirmatory plaque reduction neutralization test (PRNT). Typical turnaround times vary, due to assay incubation periods and a lack of clinical laboratories performing these tests. Recently, a novel luciferase-ZIKV- and -dengue virus (DENV)-based serological assay, which considerably improves the turnaround times and throughput for ZIKV diagnosis, was described. Using the traditional PRNT as a reference method, we evaluated the performance characteristics of the reporter virus neutralization test (RVNT) with 258 clinical serum specimens. The ZIKV RVNT produced primary ZIKV screening and secondary confirmation results in 4 days, with 100% reproducibility. As a screening assay, the ZIKV RVNT displayed excellent diagnostic accuracy, sensitivity, and specificity of 98.2%, 100%, and 98.1%, respectively. As a confirmatory assay, the ZIKV RVNT titers displayed 93.1% agreement with the traditional ZIKV PRNT titers. Overall, the RVNT accurately and reliably detects neutralizing antibodies in patient serum specimens, with improved turnaround times, and can be used for the serological detection of ZIKV infections. Due to the homogeneous 96-well format, the RVNT has also significantly improved the assay throughput to allow testing of a large number of specimens in a single run., (Copyright © 2017 Shan et al.)

Globally re-emerging dengue viruses are transmitted from human-to-human by Aedes mosquitoes. While viral determinants of human pathogenicity have been defined, there is a lack of knowledge of how dengue viruses influence mosquito transmission. Identification of viral determinants of transmission can help identify isolates with high epidemiological potential. Additionally, mechanistic understanding of transmission will lead to better understanding of how dengue viruses harness evolution to cycle between the two hosts. Here, we identified viral determinants of transmission and characterized mechanisms that enhance production of infectious saliva by inhibiting immunity specifically in salivary glands. Combining oral infection of Aedes aegypti mosquitoes and reverse genetics, we identified two 3' UTR substitutions in epidemic isolates that increased subgenomic flaviviral RNA (sfRNA) quantity, infectious particles in salivary glands and infection rate of saliva, which represents a measure of transmission. We also demonstrated that various 3'UTR modifications similarly affect sfRNA quantity in both whole mosquitoes and human cells, suggesting a shared determinism of sfRNA quantity. Furthermore, higher relative quantity of sfRNA in salivary glands compared to midgut and carcass pointed to sfRNA function in salivary glands. We showed that the Toll innate immune response was preferentially inhibited in salivary glands by viruses with the 3'UTR substitutions associated to high epidemiological fitness and high sfRNA quantity, pointing to a mechanism for higher saliva infection rate. By determining that sfRNA is an immune suppressor in a tissue relevant to mosquito transmission, we propose that 3'UTR/sfRNA sequence evolution shapes dengue epidemiology not only by influencing human pathogenicity but also by increasing mosquito transmission, thereby revealing a viral determinant of epidemiological fitness that is shared between the two hosts.

Dengue virus (DENV) is the most prevalent mosquito-borne virus pathogen in humans. There is currently no antiviral therapeutic or widely available vaccine against dengue infection. The DENV RNA genome is methylated on its 5' cap by its NS5 protein. DENV bearing a single E216A point mutation in NS5 loses 2'-O-methylation of its genome. While this mutant DENV is highly attenuated and immunogenic, the mechanism of this attenuation has not been elucidated. In this study, we find that replication of this mutant DENV is attenuated very early during infection. This early attenuation is not dependent on a functional type I interferon response and coincides with early activation of the innate immune response. Taken together, our data suggest that 2'-O-methylation of DENV genomic RNA is important for evasion of the host immune response during the very early stages of infection as the virus seeks to establish infection., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Dengue virus nonstructural protein 4B (NS4B) is a membrane protein consisting of 248 residues with a crucial role in virus replication and interference with the host innate immunity. The dengue virus serotype 3 NS4B was reconstituted into lyso-myristoyl phosphatidylglycerol (LMPG) micelles. Backbone resonance assignment of NS4B was obtained using conventional solution NMR experiments. Further studies suggested that NS4B contained eleven helices and six of them form five potential transmembrane regions. This study provides atomic level information for an important drug target to control flavivirus infections., (© 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Flaviviruses comprise major emerging pathogens such as dengue virus (DENV) or Zika virus (ZIKV). The flavivirus RNA genome is replicated by the RNA-dependent-RNA polymerase (RdRp) domain of non-structural protein 5 (NS5). This essential enzymatic activity renders the RdRp attractive for antiviral therapy. NS5 synthesizes viral RNA via a "de novo" initiation mechanism. Crystal structures of the flavivirus RdRp revealed a "closed" conformation reminiscent of a pre-initiation state, with a well ordered priming loop that extrudes from the thumb subdomain into the dsRNA exit tunnel, close to the "GDD" active site. To-date, no allosteric pockets have been identified for the RdRp, and compound screening campaigns did not yield suitable drug candidates. Using fragment-based screening via X-ray crystallography, we found a fragment that bound to a pocket of the apo-DENV RdRp close to its active site (termed "N pocket"). Structure-guided improvements yielded DENV pan-serotype inhibitors of the RdRp de novo initiation activity with nano-molar potency that also impeded elongation activity at micro-molar concentrations. Inhibitors exhibited mixed inhibition kinetics with respect to competition with the RNA or GTP substrate. The best compounds have EC50 values of 1-2 μM against all four DENV serotypes in cell culture assays. Genome-sequencing of compound-resistant DENV replicons, identified amino acid changes that mapped to the N pocket. Since inhibitors bind at the thumb/palm interface of the RdRp, this class of compounds is proposed to hinder RdRp conformational changes during its transition from initiation to elongation. This is the first report of a class of pan-serotype and cell-active DENV RdRp inhibitors. Given the evolutionary conservation of residues lining the N pocket, these molecules offer insights to treat other serious conditions caused by flaviviruses.

Nucleoside analogs represent the largest class of antiviral agents and have been actively pursued for potential therapy of dengue virus (DENV) infection. Early success in the treatment of human immunodeficiency virus (HIV) infection and the recent approval of sofosbuvir for chronic hepatitis C have provided proof of concept for this class of compounds in clinics. Here we review (i) nucleoside analogs with known anti-DENV activity; (ii) challenges of the nucleoside antiviral approach for dengue; and (iii) potential strategies to overcome these challenges. This article forms part of a symposium in Antiviral Research on flavivirus drug discovery., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Dengue fever is currently the most important mosquito-borne viral disease in Indonesia. In South Sulawesi province, most regions report dengue cases including the capital city, Makassar. Currently, no information is available on the serotypes and genotypes of the viruses circulating in the area. To understand the dynamic of dengue disease in Makassar, we carried out dengue fever surveillance study during 2007-2010. A total of 455 patients were recruited, in which antigen and serological detection revealed the confirmed dengue cases in 43.3% of patients. Molecular detection confirmed the dengue cases in 27.7% of patients, demonstrating that dengue places a significant disease burden on the community. Serotyping revealed that dengue virus serotype 1 (DENV-1) was the most predominant serotype, followed by DENV-2, -3, and -4. To determine the molecular evolution of the viruses, we conducted whole-genome sequencing of 80 isolates. Phylogenetic analysis grouped DENV-2, -3 and -4 to the Cosmopolitan genotype, Genotype I and Genotype II, respectively. Intriguingly, each serotype paints a different picture of evolution and transmission. DENV-1 appears to be undergoing a clade replacement with Genotype IV being supplanted by Genotype I. The Cosmopolitan DENV-2 isolates were found to be regionally endemic and is frequently being exchanged between countries in the region. By contrast, DENV-3 and DENV-4 isolates were related to strains with a long history in Indonesia although the DENV-3 strains appear to have been following a distinct evolutionary path since approximately 1998. To assess whether the various DENV serotypes/genotypes possess different growth characteristics, we performed growth kinetic assays on selected viruses. We observed the relatively higher rate of replication for DENV-1 and -2 compared to DENV-3 and -4. Within the DENV-1, viruses from Genotype I grow faster than that of Genotype IV. This higher replication rate may underlie their ability to replace the circulation of Genotype IV in the community., (Copyright © 2015 The Authors. Published by Elsevier B.V. All rights reserved.)

Dengue virus (DENV) infection could lead to dengue fever (DF), dengue hemorrhagic fever (DHF) or dengue shock syndrome (DSS). The disease outcome is controlled by both viral and host factors. Inflammation mediators from DENV-infected cells could contribute to increased vascular permeability, leading to severe DHF/DSS. Therefore, suppression of inflammation could be a potential therapeutic approach for treatment of dengue patients. In this context, p38 MAPK (mitogen-activated protein kinase) is a key enzyme that modulates the initiation of stress and inflammatory responses. Here we show that SB203580, a p38 MAPK inhibitor, suppressed the over production of DENV-induced pro-inflammatory mediators such as TNF-α, IL-8, and RANTES from human PBMCs, monocytic THP-1, and granulocyte KU812 cell lines. Oral administration of SB203580 in DENV-infected AG129 mice prevented hematocrit rise and lymphopenia, limited the development of inflammation and pathology (including intestine leakage), and significantly improved survival. These results, for the first time, have provided experimental evidence to imply that a short term inhibition of p38 MAPK may be beneficial to reduce disease symptoms in dengue patients., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Dengue is the most common arboviral disease of humans. There is an unmet need for a therapeutic intervention that reduces the duration and severity of dengue symptoms and diminishes the likelihood of severe complications. To this end, there are active discovery efforts in industry and academia to develop interventions, with a focus on small molecule inhibitors of dengue virus replication that are suitable for therapy or chemoprophylaxis. Advancements in animal models of dengue virus infection together with the possibility of a dengue human infection model have further enhanced the platform for dengue drug discovery. Whilst drug discovery efforts gestate, there are ongoing clinical research designed to benefit today's patients, including trials of supportive care interventions, and descriptive studies that should improve the ability of clinicians to make an accurate diagnosis early in the illness course and to identify patients most at risk of progression to severe disease. This review provides a state of the art summary of dengue drug discovery, clinical trials, and supportive allied research and reflects discussions at the 2nd International Dengue Therapeutics Workshop held in Ho Chi Minh City, Vietnam, in December 2013.

Unlabelled: Dengue virus (DENV) infects an estimated 400 million people every year, causing prolonged morbidity and sometimes mortality. Development of an effective vaccine has been hampered by the lack of appropriate small animal models; mice are naturally not susceptible to DENV and only become infected if highly immunocompromised. Mouse models lacking both type I and type II interferon (IFN) receptors (AG129 mice) or the type I IFN receptor (IFNAR(-/-) mice) are susceptible to infection with mouse-adapted DENV strains but are severely impaired in mounting functional immune responses to the virus and thus are of limited use for study. Here we used conditional deletion of the type I IFN receptor (IFNAR) on individual immune cell subtypes to generate a minimally manipulated mouse model that is susceptible to DENV while retaining global immune competence. Mice lacking IFNAR expression on CD11c(+) dendritic cells and LysM(+) macrophages succumbed completely to DENV infection, while mice deficient in the receptor on either CD11c(+) or LysM(+) cells were susceptible to infection but often resolved viremia and recovered fully from infection. Conditional IFNAR mice responded with a swift and strong CD8(+) T-cell response to viral infection, compared to a weak response in IFNAR(-/-) mice. Furthermore, mice lacking IFNAR on either CD11c(+) or LysM(+) cells were also sufficiently immunocompetent to raise a protective immune response to a candidate subunit vaccine against DENV-2. These data demonstrate that mice with conditional deficiencies in expression of the IFNAR represent improved models for the study of DENV immunology and screening of vaccine candidates., Importance: Dengue virus infects 400 million people every year worldwide, causing 100 million clinically apparent infections, which can be fatal if untreated. Despite many years of research, there are no effective vaccine and no antiviral treatment available for dengue. Development of vaccines has been hampered in particular by the lack of a suitable small animal model. Mouse models used to test dengue vaccine are deficient in interferon (IFN) type I signaling and severely immunocompromised and therefore likely not ideal for the testing of vaccines. In this study, we explored alternative models lacking the IFN receptor only on certain cell types. We show that mice lacking the IFN receptor on either CD11c- or LysM-expressing cells (conditional IFNAR mice) are susceptible to dengue virus infection. Importantly, we demonstrate that conditional IFN receptor knockout mice generate a better immune response to live virus and a candidate dengue vaccine compared to IFNAR mice and are resistant to subsequent challenge., (Copyright © 2014, American Society for Microbiology. All Rights Reserved.)

Unlabelled: Flavivirus replication is mediated by a complex machinery that consists of viral enzymes, nonenzymatic viral proteins, and host factors. Many of the nonenzymatic viral proteins, such as NS4B, are associated with the endoplasmic reticulum membrane. How these membrane proteins function in viral replication is poorly understood. Here we report a robust method to express and purify dengue virus (DENV) and West Nile virus NS4B proteins. The NS4B proteins were expressed in Escherichia coli, reconstituted in dodecyl maltoside (DDM) detergent micelles, and purified to >95% homogeneity. The recombinant NS4B proteins dimerized in vitro, as evidenced by gel filtration, chemical cross-linking, and multiangle light scattering experiments. The dimeric form of NS4B was also detected when the protein was expressed alone in cells as well as in cells infected with DENV type 2 (DENV-2). Mutagenesis analysis showed that the cytosolic loop (amino acids 129 to 165) and the C-terminal region (amino acids 166 to 248) are responsible for NS4B dimerization. trans-Complementation experiments showed that (i) two genome-length RNAs containing distinct NS4B lethal mutations could not trans-complement each other, (ii) the replication defect of NS4B mutant RNA could be restored in cells containing DENV-2 replicons, and (iii) expression of wild-type NS4B protein alone was not sufficient to restore the replication of the NS4B mutant RNA. Collectively, the results indicate that trans-complementation of a lethal NS4B mutant RNA requires wild-type NS4B presented from a replication complex., Importance: The reported expression and purification system has made it possible to study the biochemistry and structure of flavivirus NS4B proteins. The finding of flavivirus NS4B dimerization and the mapping of regions important for NS4B dimerization provide the possibility to inhibit viral replication through blocking NS4B dimerization. The requirement of NS4B in the context of the replication complex for successful trans-complementation enhances our understanding of NS4B in flavivirus replication.

Dengue fever is the most important arthropod-transmitted viral disease affecting humans. It is a blood-borne disease characterized by persistent fever and joint pain. In the blood, primary peripheral blood mononuclear cells (PBMCs), in particular monocytes, are the main target of the dengue virus (DENV). These cells are poorly permissive for in vitro dengue virus infection and their infectivity varies from donor to donor. To overcome this barrier, an anti-dengue antibody was used to improve the infectivity of DENV-2 clinical isolates to PBMCs, the monocytic leukemia cell line, THP-1 and the granulocyte cell line, KU812. A higher throughput 96-well-format assay based on a fluorescent-activated cell sorter could potentially be developed to evaluate the antiviral potency of compounds in DENV-infected PBMCs in vitro. The results correlate well with data obtained by a standard plaque assay. Altogether, an assay has been developed that enables evaluation of the antiviral activity of test compounds in a physiologically-relevant cell system (PBMCs). These screening processes are urgently needed for dengue drug discovery., (Copyright © 2013 Elsevier B.V. All rights reserved.)

In a recent clinical trial, balapiravir, a prodrug of a cytidine analog (R1479), failed to achieve efficacy (reducing viremia after treatment) in dengue patients, although the plasma trough concentration of R1479 remained above the 50% effective concentration (EC(50)). Here, we report experimental evidence to explain the discrepancy between the in vitro and in vivo results and its implication for drug development. R1479 lost its potency by 125-fold when balapiravir was used to treat primary human peripheral blood mononuclear cells (PBMCs; one of the major cells targeted for viral replication) that were preinfected with dengue virus. The elevated EC(50) was greater than the plasma trough concentration of R1479 observed in dengue patients treated with balapiravir and could possibly explain the efficacy failure. Mechanistically, dengue virus infection triggered PBMCs to generate cytokines, which decreased their efficiency of conversion of R1479 to its triphosphate form (the active antiviral ingredient), resulting in decreased antiviral potency. In contrast to the cytidine-based compound R1479, the potency of an adenosine-based inhibitor of dengue virus (NITD008) was much less affected. Taken together, our results demonstrate that viral infection in patients before treatment could significantly affect the conversion of the prodrug to its active form; such an effect should be calculated when estimating the dose efficacious for humans.

Dengue virus (DENV) non-structural protein 4B (NS4B) has been demonstrated to be an attractive antiviral target. Due to its nature as an integral membrane protein, NS4B remains poorly characterized. In this study, we generated and characterized two monoclonal antibodies (mAb) that selectively bind to DENV NS4B protein. One mAb, 10-3-7, is specific for DENV-2 NS4B, and its epitope was mapped to residues 5-15 of NS4B. The other mAb, 44-4-7, cross-reacts with all the four serotypes of DENV NS4B, and its epitope was mapped to residues 141-147 of NS4B. Using the mAbs, we probed the intracellular orientation of the epitopes of NS4B by an epitope accessibility assay. The results showed that the N-terminus of NS4B is located in the ER lumen, whereas amino acids 130-148 of NS4B are located in the cytosol. The study demonstrates that the two anti-NS4B mAbs will be useful for future structural and functional analyses of DENV NS4B., (Copyright © 2014 Elsevier Inc. All rights reserved.)

The development of a safe and efficient dengue vaccine represents a global challenge in public health. Chimeric dengue viruses (DENV) based on an attenuated flavivirus have been well developed as vaccine candidates by using reverse genetics. In this study, based on the full-length infectious cDNA clone of the well-known Japanese encephalitis virus live vaccine strain SA14-14-2 as a backbone, a novel chimeric dengue virus (named ChinDENV) was rationally designed and constructed by replacement with the premembrane and envelope genes of dengue 2 virus. The recovered chimeric virus showed growth and plaque properties similar to those of the parental DENV in mammalian and mosquito cells. ChinDENV was highly attenuated in mice, and no viremia was induced in rhesus monkeys upon subcutaneous inoculation. ChinDENV retained its genetic stability and attenuation phenotype after serial 15 passages in cultured cells. A single immunization with various doses of ChinDENV elicited strong neutralizing antibodies in a dose-dependent manner. When vaccinated monkeys were challenged with wild-type DENV, all animals except one that received the lower dose were protected against the development of viremia. Furthermore, immunization with ChinDENV conferred efficient cross protection against lethal JEV challenge in mice in association with robust cellular immunity induced by the replicating nonstructural proteins. Taken together, the results of this preclinical study well demonstrate the great potential of ChinDENV for further development as a dengue vaccine candidate, and this kind of chimeric flavivirus based on JE vaccine virus represents a powerful tool to deliver foreign antigens.

To combat neglected diseases, the Novartis Institute of Tropical Diseases (NITD) was founded in 2002 through private-public funding from Novartis and the Singapore Economic Development Board. One of NITD's missions is to develop antivirals for dengue virus (DENV), the most prevalent mosquito-borne viral pathogen. Neither vaccine nor antiviral is currently available for DENV. Here we review the progress in dengue drug discovery made at NITD as well as the major discoveries made by academia and other companies. Four strategies have been pursued to identify inhibitors of DENV through targeting both viral and host proteins: (i) HTS (high-throughput screening) using virus replication assays; (ii) HTS using viral enzyme assays; (iii) structure-based in silico docking and rational design; (iv) repurposing hepatitis C virus inhibitors for DENV. Along the developmental process from hit finding to clinical candidate, many inhibitors did not advance beyond the stage of hit-to-lead optimization, due to their poor selectivity, physiochemical or pharmacokinetic properties. Only a few compounds showed efficacy in the AG129 DENV mouse model. Two nucleoside analogs, NITD-008 and Balapiravir, entered preclinical animal safety study and clinic trial, but both were terminated due to toxicity and lack of potency, respectively. Celgosivir, a host alpha-glucosidase inhibitor, is currently under clinical trial; its clinical efficacy remains to be determined. The knowledge accumulated during the past decade has provided a better rationale for ongoing dengue drug discovery. Though challenging, we are optimistic that this continuous, concerted effort will lead to an effective dengue therapy., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Dengue virus (DENV) infection is one of the most important public health threats globally; however, no vaccines or effective antivirals are currently available. The bioluminescence imaging technique has emerged as a powerful tool for studies on viral pathogenesis in vitro and in vivo. In this study, using a recombinant DENV that stably expressed Renilla luciferase (Rluc-DENV), we used bioluminescence for imaging of DENV infection in the brain of A129 mice that lacked type I interferon receptors. Upon intracranial inoculation with Rluc-DENV, A129 mice developed typical neurological symptoms and rapidly succumbed to viral infection. Real-time bioluminescence intensity analysis revealed the replication kinetics of Rluc-DENV in the brain of A129 mice. Linear regression analyses showed a good correlation between photon flux and viral titers (R(2) = 0.9923). Finally, the bioluminescence model was validated using a known mouse monoclonal antibody, 2A10G6, and the therapeutic effects of this neutralizing antibody were readily monitored by live imaging in the same animal. The noninvasive bioluminescence imaging of DENV infection as described here shows distinct advantages over traditional animal models and provides a powerful tool for potential antiviral or vaccine assays against DENV infection in vivo.

Dengue virus is transmitted by Aedes mosquitoes and infects at least 100 million people every year. Progressive urbanization in Asia and South-Central America and the geographic expansion of Aedes mosquito habitats have accelerated the global spread of dengue, resulting in a continuously increasing number of cases. A cost-effective, safe vaccine conferring protection with ideally a single injection could stop dengue transmission. Current vaccine candidates require several booster injections or do not provide protection against all four serotypes. Here we demonstrate that dengue virus mutants lacking 2'-O-methyltransferase activity are highly sensitive to type I IFN inhibition. The mutant viruses are attenuated in mice and rhesus monkeys and elicit a strong adaptive immune response. Monkeys immunized with a single dose of 2'-O-methyltransferase mutant virus showed 100% sero-conversion even when a dose as low as 1,000 plaque forming units was administrated. Animals were fully protected against a homologous challenge. Furthermore, mosquitoes feeding on blood containing the mutant virus were not infected, whereas those feeding on blood containing wild-type virus were infected and thus able to transmit it. These results show the potential of 2'-O-methyltransferase mutant virus as a safe, rationally designed dengue vaccine that restrains itself due to the increased susceptibility to the host's innate immune response.

Dengue fever is an emerging mosquito-borne flaviviral disease that threatens 2.5 billion people worldwide. No clinically approved vaccine and antiviral therapy are currently available to prevent or treat dengue virus (DENV) infection. Vertebrate animals other than primates are not normally infectable with DENV; however, a small animal dengue infection model would greatly facilitate the development of a vaccine or an antiviral therapy. To this end, a rodent model for DENV infection has been established in IFN-α/β and IFN-γ receptor-deficient (AG129) mice. This chapter describes the protocol for the DENV infection model in AG129 mice and testing of antiviral compounds by oral gavage or parenteral injection.

Background: Dengue virus surface proteins, envelope (E) and pre-membrane (prM), undergo rearrangement during the maturation process at acidic condition., Results: prM-stem region binds tighter to both E protein and lipid membrane when environment becomes acidic., Conclusion: At acidic condition, E proteins are attracted to the membrane-associated prM-stem., Significance: prM-stem region induces virus structural changes during maturation. Newly assembled dengue viruses (DENV) undergo maturation to become infectious particles. The maturation process involves major rearrangement of virus surface premembrane (prM) and envelope (E) proteins. The prM-E complexes on immature viruses are first assembled as trimeric spikes in the neutral pH environment of the endoplasmic reticulum. When the virus is transported to the low pH environment of the exosomes, these spikes rearrange into dimeric structures, which lie parallel to the virus lipid envelope. The proteins involved in driving this process are unknown. Previous cryoelectron microscopy studies of the mature DENV showed that the prM-stem region (residues 111-131) is membrane-associated and may interact with the E proteins. Here we investigated the prM-stem region in modulating the virus maturation process. The binding of the prM-stem region to the E protein was shown to increase significantly at low pH compared with neutral pH in ELISAs and surface plasmon resonance studies. In addition, the affinity of the prM-stem region for the liposome, as measured by fluorescence correlation spectroscopy, was also increased when pH is lowered. These results suggest that the prM-stem region forms a tight association with the virus membrane and attracts the associated E protein in the low pH environment of exosomes. This will lead to the surface protein rearrangement observed during maturation.

Viral replication relies on the host to supply nucleosides. Host enzymes involved in nucleoside biosynthesis are potential targets for antiviral development. Ribavirin (a known antiviral drug) is such an inhibitor that suppresses guanine biosynthesis; depletion of the intracellular GTP pool was shown to be the major mechanism to inhibit flavivirus. Along similar lines, inhibitors of the pyrimidine biosynthesis pathway could be targeted for potential antiviral development. Here we report on a novel antiviral compound (NITD-982) that inhibits host dihydroorotate dehydrogenase (DHODH), an enzyme required for pyrimidine biosynthesis. The inhibitor was identified through screening 1.8 million compounds using a dengue virus (DENV) infection assay. The compound contains an isoxazole-pyrazole core structure, and it inhibited DENV with a 50% effective concentration (EC(50)) of 2.4 nM and a 50% cytotoxic concentration (CC(50)) of >5 μM. NITD-982 has a broad antiviral spectrum, inhibiting both flaviviruses and nonflaviviruses with nanomolar EC(90)s. We also show that (i) the compound inhibited the enzymatic activity of recombinant DHODH, (ii) an NITD-982 analogue directly bound to the DHODH protein, (iii) supplementing the culture medium with uridine reversed the compound-mediated antiviral activity, and (iv) DENV type 2 (DENV-2) variants resistant to brequinar (a known DHODH inhibitor) were cross resistant to NITD-982. Collectively, the results demonstrate that the compound inhibits DENV through depleting the intracellular pyrimidine pool. In contrast to the in vitro potency, the compound did not show any efficacy in the DENV-AG129 mouse model. The lack of in vivo efficacy is likely due to the exogenous uptake of pyrimidine from the diet or to a high plasma protein-binding activity of the current compound.

Cellular α-glucosidases I and II are enzymes that sequentially trim the three terminal glucoses in the N-linked oligosaccharides of viral envelope glycoproteins. This process is essential for the proper folding of viral glycoproteins and subsequent assembly of many enveloped viruses, including dengue virus (DENV). Imino sugars are substrate mimics of α-glucosidases I and II. In this report, we show that two oxygenated alkyl imino sugar derivatives, CM-9-78 and CM-10-18, are potent inhibitors of both α-glucosidases I and II in vitro and in treated animals, and efficiently inhibit DENV infection of cultured human cells. Pharmacokinetic studies reveal that both compounds are well tolerated at doses up to 100mg/kg in rats and have favorable pharmacokinetic properties and bioavailability in mice. Moreover, we showed that oral administration of either CM-9-78 or CM-10-18 reduces the peak viremia of DENV in mice. Interestingly, while treatment of DENV infected mice with ribavirin alone did not reduce the viremia, combination therapy of ribavirin with sub-effective dose of CM-10-18 demonstrated a significantly enhanced antiviral activity, as indicated by a profound reduction of the viremia. Our findings thus suggest that combination therapy of two broad-spectrum antiviral agents may provide a practically useful approach for the treatment of DENV infection., (Copyright © 2010 Elsevier B.V. All rights reserved.)

Dengue virus (DENV) is the most prevalent mosquito-borne viral pathogen that infects humans. Neither a vaccine nor an antiviral therapy is currently available for DENV. Here, we report an adenosine nucleoside prodrug that potently inhibits DENV replication both in cell culture and in a DENV mouse model. NITD449 (2'-C-acetylene-7-deaza-7-carbamoyladenosine) was initially identified as a parental compound that inhibits all four serotypes of DENV with low cytotoxicity. However, in vivo pharmacokinetic studies indicated that NITD449 had a low level of exposure in plasma when dosed orally. To increase the oral bioavailability, we covalently linked isobutyric acids to the 3'- and 5'-hydroxyl groups of ribose via ester linkage to NITD449, leading to the prodrug NITD203 (3',5'-O-diisobutyryl-2'-C-acetylene-7-deaza-7-carbamoyl-adenosin). Pharmacokinetic analysis showed that upon oral dosing of the prodrug, NITD203 was readily converted to NITD449, resulting in improved exposure of the parental compound in plasma in both mouse and rat. In DENV-infected AG129 mice, oral dosing of the prodrug at 25 mg/kg of body weight reduced peak viremia by 30-fold. Antiviral spectrum analysis showed that NITD203 inhibited various flaviviruses (DENV, yellow fever virus, and West Nile virus) and hepatitis C virus but not Chikungunya virus (an alphavirus). Mode-of-action analysis, using a luciferase-reporting replicon, indicated that NITD203 inhibited DENV RNA synthesis. Although NITD203 exhibited potent in vitro and in vivo efficacies, the compound could not reach a satisfactory no-observable-adverse-effect level (NOAEL) in a 2-week in vivo toxicity study. Nevertheless, our results demonstrate that a prodrug approach using a nucleoside analog could potentially be developed for flavivirus antiviral therapy.

Dengue virus (DENV) is the most prevalent mosquito-borne viral pathogen in humans. Neither vaccine nor antiviral therapy is currently available for DENV. We report here that N-sulfonylanthranilic acid derivatives are allosteric inhibitors of DENV RNA-dependent RNA polymerase (RdRp). The inhibitor was identified through high-throughput screening of one million compounds using a primer extension-based RdRp assay [substrate poly(C)/oligo(G)(20)]. Chemical modification of the initial "hit" improved the compound potency to an IC(50) (that is, a concentration that inhibits 50% RdRp activity) of 0.7 microM. In addition to suppressing the primer extension-based RNA elongation, the compound also inhibited de novo RNA synthesis using a DENV subgenomic RNA, but at a lower potency (IC(50) of 5 microM). Remarkably, the observed anti-polymerase activity is specific to DENV RdRp; the compound did not inhibit WNV RdRp and exhibited IC(50)s of >100 microM against hepatitis C virus RdRp and human DNA polymerase alpha and beta. UV cross-linking and mass spectrometric analysis showed that a photoreactive inhibitor could be cross-linked to Met343 within the RdRp domain of DENV NS5. On the crystal structure of DENV RdRp, Met343 is located at the entrance of RNA template tunnel. Biochemical experiments showed that the order of addition of RNA template and inhibitor during the assembly of RdRp reaction affected compound potency. Collectively, the results indicate that the compound inhibits RdRp through blocking the RNA tunnel. This study has provided direct evidence to support the hypothesis that allosteric pockets from flavivirus RdRp could be targeted for antiviral development.

Dengue virus (DENV), a mosquito-borne flavivirus, is a major public health threat. The virus poses risk to 2.5 billion people worldwide and causes 50 to 100 million human infections each year. Neither a vaccine nor an antiviral therapy is currently available for prevention and treatment of DENV infection. Here, we report a previously undescribed adenosine analog, NITD008, that potently inhibits DENV both in vitro and in vivo. In addition to the 4 serotypes of DENV, NITD008 inhibits other flaviviruses, including West Nile virus, yellow fever virus, and Powassan virus. The compound also suppresses hepatitis C virus, but it does not inhibit nonflaviviruses, such as Western equine encephalitis virus and vesicular stomatitis virus. A triphosphate form of NITD008 directly inhibits the RNA-dependent RNA polymerase activity of DENV, indicating that the compound functions as a chain terminator during viral RNA synthesis. NITD008 has good in vivo pharmacokinetic properties and is biologically available through oral administration. Treatment of DENV-infected mice with NITD008 suppressed peak viremia, reduced cytokine elevation, and completely prevented the infected mice from death. No observed adverse effect level (NOAEL) was achieved when rats were orally dosed with NITD008 at 50 mg/kg daily for 1 week. However, NOAEL could not be accomplished when rats and dogs were dosed daily for 2 weeks. Nevertheless, our results have proved the concept that a nucleoside inhibitor could be developed for potential treatment of flavivirus infections.

West Nile virus (WNV) is an emerging flavivirus that has caused frequent epidemics since 1996. Besides natural transmission by mosquitoes, WNV can also be transmitted through blood transfusion and organ transplantation, thus heightening the urgency of development of a specific and rapid serologic assay of WNV infection. The current immunoassays lack specificity because they are based on detection of antibodies against WNV structural proteins and immune responses to structural proteins among flaviviruses cross-react to each other. Here, we describe microsphere immunoassays that detect antibodies to nonstructural proteins 3 and 5 (NS3 and NS5). In contrast to immunoassays based on viral envelope and NS3 proteins, the NS5-based assay (i) reliably discriminates between WNV infections and dengue virus or St. Louis encephalitis virus infections, (ii) differentiates between flavivirus vaccination and natural WNV infection, and (iii) indicates recent infections. These unique features of the NS5-based immunoassay will be very useful for both clinical and veterinary diagnosis of WNV infection.

Dengue virus is the most prevalent mosquito-borne virus, causing approximately 390 million infections and 25,000 deaths per year. Aedes aegypti , the primary mosquito vector of dengue virus, is well-established throughout the state of Florida, United States. Autochthonous transmission of dengue virus to humans in Florida has been increasing since 2009, alongside consistent importation of dengue cases. However, most cases of first infection with dengue are asymptomatic and the virus can be maintained in mosquito populations, complicating surveillance and leading to an underestimation of disease risk. Metagenomic sequencing of A. aegypti mosquitoes in Manatee County, Florida revealed the presence of dengue virus serotype 4 (DENV-4) genomes in mosquitoes from multiple trapping sites over 2years, in the absence of a human DENV-4 index case, and even though a locally acquired case of DENV-4 has never been reported in Florida. This finding suggested that: (i) DENV-4 may circulate among humans undetected; (ii) the virus was being maintained in the mosquito population, or (iii) the detected complete genome sequence may not represent a viable virus. This study demonstrates that an infectious clone generated from the Manatee County DENV-4 (DENV-4M) sequence is capable of infecting mammalian and insect tissue culture systems, as well as adult female A. aegypti mosquitoes when fed in a blood meal. However, the virus is subject to a dose dependent infection barrier in mosquitoes, and has a kinetic delay compared to a phylogenetically related wild-type (WT) control virus from a symptomatic child, DENV-4H (strain Homo sapiens /Haiti-0075/2015, GenBank accession MK514144.1). DENV-4M disseminates from the midgut to the ovary and saliva at 14days post-infection. Viral RNA was also detectable in the adult female offspring of DENV-4M infected mosquitoes. These results demonstrate that the virus is capable of infecting vector mosquitoes, is transmissible by bite, and is vertically transmitted, indicating a mechanism for maintenance in the environment without human-mosquito transmission. These findings suggest undetected human-mosquito transmission and/or long-term maintenance of the virus in the mosquito population is occurring in Florida, and underscore the importance of proactive surveillance for viruses in mosquitoes. GRAPHICAL ABSTRACT In order to better assess the public health risk posed by a detection of DENV-4 RNA in Manatee County, FL Aedes aegypti , we produced an infectious clone using the sequence from the wild-caught mosquitoes and characterized it via laboratory infections of mosquitoes and mosquito tissues. [ABSTRACT FROM AUTHOR]

Dengue virus (DENV) is one of the most widespread arboviruses. The four DENV serotypes infect about 400 million people every year, causing 96 million clinical dengue cases, of which approximately 500’000 are severe and potentially life-threatening. The only licensed vaccine has a limited efficacy and is only recommended in regions with high endemicity. We previously reported that 2’-O-methyltransferase mutations in DENV-1 and DENV-2 block their capacity to inhibit type I IFNs and render the viruses attenuated in vivo, making them amenable as vaccine strains; here we apply this strategy to all four DENV serotypes to generate a tetravalent, non-chimeric live-attenuated dengue vaccine. 2’-O-methyltransferase mutants of all four serotypes are highly sensitive to type I IFN inhibition in human cells. The tetravalent formulation is attenuated and immunogenic in mice and cynomolgus macaques and elicits a response that protects from virus challenge. These results show the potential of 2’-O-methyltransferase mutant viruses as a safe, tetravalent, non-chimeric dengue vaccine. [ABSTRACT FROM AUTHOR]

Reporter virus neutralization test (RVNT) has been used as an alternative to the more laborious and time-demanding conventional PRNT assay for both DENV and ZIKV. However, few studies have investigated how these techniques would perform in epidemic areas with the circulation of multiple flavivirus. Here, we evaluate the performance of ZIKV and DENV Rluc RVNT and ZIKV mCh RVNT assays in comparison to the conventional PRNT assay against patient sera collected before and during ZIKV outbreak in Brazil. These samples were categorized into groups based on (1) acute and convalescent samples according to the time of disease, and (2) laboratorial diagnostic results (DENV and ZIKV RT-PCR and IgM-capture ELISA). Our results showed that DENV Rluc assay presented 100% and 78.3% sensitivity and specificity, respectively, with 93.3% accuracy, a similar performance to the traditional PRNT. ZIKV RVNT90, on the other hand, showed much better ZIKV antibody detection performance (around nine-fold higher) when compared to PRNT, with 88% clinical sensitivity. Specificity values were on average 76.8%. Even with these results, however, ZIKV RVNT90 alone was not able to reach a final diagnostic conclusion for secondary infection in human samples due to flavivirus cross reaction. As such, in regions where the flavivirus differential diagnosis represents a challenge, we suggest the establishment of a RVNT panel including other flaviviruses circulating in the region, associated with the other serological techniques such as IgM ELISA and the investigation of seroconversion, in order to help define an accurate diagnostic conclusion using serology. [ABSTRACT FROM AUTHOR]

The flavivirus nonstructural 4B protein (NS4B) has recently emerged as a valid antiviral target for drug discovery. Here we review (i) the current understanding of the structure and function of DENV NS4B, (ii) the approaches that have been taken to identify NS4B inhibitors, and (iii) the known inhibitors of flavivirus NS4B protein. This article forms part of a symposium in Antiviral Research on flavivirus drug discovery. [ABSTRACT FROM AUTHOR]

Abstract: A series of new pyrimido[4,5-b]indole ribonucleosides bearing phenyl or hetaryl group at position 4 has been prepared by selective Pd-catalyzed cross-coupling reactions of the corresponding protected 4,6-dichloropyrimido[4,5-b]indole ribonucleoside with (het)arylboronic acids or stannanes followed by deprotection. Further cross-couplings under harsher conditions and employing X-Phos ligand proceeded at the position 6 leading to 4,6-disubstituted pyrimido[4,5-b]indole ribonucleosides. Some of these compounds displayed antiviral activity against Dengue virus. [Copyright &y& Elsevier]

Abstract: Methyltransferases (MTases) from the genus Flavivirus encode both N-7 and 2′-O activities needed for type 1 (m7GpppNm) cap structure formation. We performed kinetic studies to understand the mechanisms of its progressive N-7 and 2′-O methylations. Sequential N-7 to 2′-O methylation occurred via a random bi bi and processive mechanism that does not involve enzyme–RNA dissociation. Analyses of steady state kinetic parameters showed that N-7 precedes 2′-O methylation as it turnovers RNA faster (k cat) resulting in 2.4-fold higher catalytic efficiency. Michaelis constants for S-adenosyl-methionine (AdoMet) in both reactions were about 10-fold lower than for their respective RNA substrates, suggesting that the rate-limiting steps in methylase reactions were associated with RNA templates. In the context of long viral RNA sequences, and compared to S-adenosyl-homocysteine, sinefungin was about 60- and 12-folds more potent against dengue N-7 and 2′-O MTase activity, exhibiting IC50 values of 30 and 41nM, respectively. [Copyright &y& Elsevier]

Abstract: Dengue virus (DENV) is a mosquito-borne flavivirus responsible for 50–100 million human infections each year. The development of DENV chemotherapy requires high-throughput screening (HTS) assays. A dengue virus-like particle (VLP) has been constructed using viral structural proteins to package a Renilla luciferase reporter replicon. VLP could be produced by either the sequential electroporation of the replicon RNAs and the structural gene RNAs or by electroporating replicon RNA into a stable cell line expressing the structural proteins. In both approaches, the key to produce high titer VLP (3×106 foci-forming unit/ml) is to use low temperature (30°C) in the packaging step. In addition, exogenous expression of host protease furin increased VLP infectivity. The infection could be blocked by antibodies against viral envelope protein and by an inhibitor of viral NS5 polymerase, but not by an inhibitor of host alpha-glucosidase (castanospermine). The VLP infection assay was optimized for HTS in a 384-well format with consistent and robust signal, providing a simple and rapid cell-based assay for screening inhibitors against DENV entry, translation, and replication in an HTS format. [Copyright &y& Elsevier]

Based on genome-scale loss-of-function screens we discovered that Topoisomerase III-β (TOP3B), a human topoisomerase that acts on DNA and RNA, is required for yellow fever virus and dengue virus-2 replication. Remarkably, we found that TOP3B is required for efficient replication of all positive-sense-single stranded RNA viruses tested, including SARS-CoV-2. While there are no drugs that specifically inhibit this topoisomerase, we posit that TOP3B is an attractive anti-viral target. • Topoisomerase III-ß (TOP3B) is a host factor for all single stranded positive strand RNA viruses. • TOP3B is not a host factor for Ebola and influenza viruses, which are negative strand RNA viruses. • TOP3B acts directly on the viral genome of DENV2. • TOP3B could be a target for antiviral development. [ABSTRACT FROM AUTHOR]