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Background: The largest dengue virus 2 (DENV2) outbreak occurred in Taiwan in 2015, resulting in many fatalities. We therefore aim to identify crucial genetic variations which determine the virulence of the 2015 Taiwan outbreak strains., Methods: We compared the 2015 Taiwan DENV2 sequences to the pre-2015 sequences. Reverse genetics (rg) viruses with substitutions were produced and the viral growth kinetics were investigated. We treated A549 cells with interferon (IFN) to determine the interferon-stimulated genes (ISGs) expression and STAT1 phosphorylation in the rg viral infection and plasmid transfection systems. IFN and pro-inflammatory cytokines levels were measured upon DENV infection using ELISA., Results: The rgNS1-K272R mutant showed faster replication in IFN-I producing cells compared to wildtype (WT) virus. Results revealed that NS1-K272R substitution contributed to higher soluble NS1 secretion and evade the antiviral response by suppressing the expression of ISGs and STAT1 phosphorylation compared to NS1-WT. Infection with rgNS1-K272R induced higher secretion of pro-inflammatory cytokines through the activation of canonical nuclear factor-kappa B (NF-κB) signaling pathway., Conclusions: Our results revealed that the DENV NS1 amino acid substitution affects the NS1 ability in immune evasion, which may contribute to the largest dengue outbreak in Taiwan since the 1990s., Competing Interests: Declarations. Ethics approval and consent to participate: Not applicable. Consent for publication: Not applicable. Competing interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2025. The Author(s).)

Dengue virus (DENV) causes approximately 390 million dengue infections worldwide every year. There were 22,777 reported DENV infections in Tainan, Taiwan in 2015. In this study, we sequenced the C-prM-E genes from 45 DENV 2015 strains, and phylogenetic analysis based on C-prM-E genes revealed that all strains were classified as DENV serotype 2 Cosmopolitan genotype. Sequence analysis comparing different DENV-2 genotypes and Cosmopolitan DENV-2 sequences prior to 2015 showed a clade replacement event in the DENV-2 Cosmopolitan genotype. Additionally, a major substitution C-A314G (K73R) was found in the capsid region which may have contributed to the clade replacement event. Reverse genetics virus rgC-A314G (K73R) showed slower replication in BHK-21 and C6/36 cells compared to wildtype virus, as well as a decrease in NS1 production in BHK-21-infected cells. After a series of passaging, the C-A314G (K73R) mutation reverted to wildtype and was thus considered to be unstable. Next generation sequencing (NGS) of three sera collected from a single DENV2-infected patient at 1-, 2-, and 5-days post-admission was employed to examine the genetic diversity over-time and mutations that may work in conjunction with C-A314G (K73R). Results showed that the number of haplotypes decreased with time in the DENV-infected patient. On the fifth day after admission, two new haplotypes emerged, and a single non-synonymous NS4A-L115I mutation was identified. Therefore, we have identified a persistent mutation C-A314G (K73R) in all of the DENV-2 isolates, and during the course of an infection, a single new non-synonymous mutation in the NS4A region appears in the virus population within a single host. The C-A314G (K73R) thus may have played a role in the DENV-2 2015 outbreak while the NS4A-L115I may be advantageous during DENV infection within the host., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Tsai et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

With approximately 3.8 billion people at risk of infection in tropical and sub-tropical regions, Dengue ranks among the top ten threats worldwide. Despite the potential for severe disease manifestation and the economic burden it places on endemic countries, there is a lack of approved antiviral agents to effectively treat the infection. Flavonoids, including baicalein, have garnered attention for their antimicrobial properties. In this study, we took a rational and iterative approach to develop a series of baicalein derivatives with improved antiviral activity against Dengue virus (DENV). Compound 11064 emerged as a promising lead candidate, exhibiting antiviral activity against the four DENV serotypes and representative strains of Zika virus (ZIKV) in vitro, with attractive selectivity indices. Mechanistic studies revealed that Compound 11064 did not prevent DENV attachment at the cell surface, nor viral RNA synthesis and viral protein translation. Instead, the drug was found to impair the post-receptor binding entry steps (endocytosis and/or uncoating), as well as the late stage of DENV infection cycle, including virus assembly/maturation and/or exocytosis. The inability to raise DENV resistant mutants, combined with significant antiviral activity against an unrelated RNA virus (Enterovirus-A71) suggested that Compound 11064 targets the host rather than a viral protein, further supporting its broad-spectrum antiviral potential. Overall, Compound 11064 represents a promising antiviral candidate for the treatment of Dengue and Zika., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Author(s). Published by Elsevier B.V. All rights reserved.)

Dengue virus (DENV) is the cause of dengue fever, infecting 390 million people worldwide per year. It is transmitted to humans through the bites of mosquitoes and could potentially develop severe symptoms. In spite of the rising social and economic impact inflicted by the disease on the global population, a conspicuous lack of efficacious therapeutics against DENV still persists. In this study, catechin, a natural polyphenol compound, was evaluated as a DENV infection inhibitor in vitro. Through time-course studies, catechin was shown to inhibit a post-entry stage of the DENV replication cycle. Further investigation revealed its role in affecting viral protein translation. Catechin inhibited the replication of all four DENV serotypes and chikungunya virus (CHIKV). Together, these results demonstrate the ability of catechin to inhibit DENV replication, hinting at its potential to be used as a starting scaffold for further development of antivirals against DENV infection.

Dengue and Zika viruses are mosquito-borne flaviviruses burdening millions every year with hemorrhagic fever and neurological symptoms. Baicalein was previously reported as a potential anti-flaviviral candidate and halogenation of flavones and flavanones potentiated their antiviral efficacies. Here, we reported that a chemically modified 8-bromobaicalein effectively inhibited all dengue serotypes and Zika viruses at 0.66-0.88 micromolar in cell-based system. The compound bound to dengue serotype 2 conserved pocket and inhibited the dengue RdRp activity with 6.93 fold more than the original baicalein. Moreover, the compound was mildly toxic against infant and adult C57BL/6 mice despite administering continuously for 7 days. Therefore, the 8-bromobaicalein should be investigated further in pharmacokinetics and efficacy in an animal model., (© 2023. The Author(s).)

Early prognosis of abnormal vasculopathy is essential for effective clinical management of patients with severe dengue. An exaggerated interferon (IFN) response and release of vasoactive factors from endothelial cells cause vasculopathy. This study shows that dengue virus 2 (DENV2) infection of human umbilical vein endothelial cells (HUVEC) results in differentially regulated microRNAs (miRNAs) important for endothelial function. miR-573 was significantly downregulated in DENV2-infected HUVEC due to decreased peroxisome proliferator activator receptor gamma (PPARγ) activity. Restoring miR-573 expression decreased endothelial permeability by suppressing the expression of vasoactive angiopoietin 2 (ANGPT2). We also found that miR-573 suppressed the proinflammatory IFN response through direct downregulation of Toll-like receptor 2 (TLR2) expression. Our study provides a novel insight into miR-573-mediated regulation of endothelial function during DENV2 infection, which can be further translated into a potential therapeutic and prognostic agent for severe dengue patients. IMPORTANCE We need to identify molecular factors that can predict the onset of endothelial dysfunction in dengue patients. Increase in endothelial permeability during severe dengue infections is poorly understood. In this study, we focus on factors that regulate endothelial function and are dysregulated during DENV2 infection. We show that miR-573 rescues endothelial permeability and is downregulated during DENV2 infection in endothelial cells. This finding can have both diagnostic and therapeutic applications.

Dengue virus (DENV) is an arthropod-borne virus that has developed into a prominent global health threat in recent decades. The main causative agent of dengue fever, the virus infects an estimated 390 million individuals across the globe each year. Despite the sharply increasing social and economic burden on global society caused by the disease, there is still a glaring lack of effective therapeutics against DENV. In this study, betulinic acid, a naturally occurring pentacyclic triterpenoid was established as an inhibitor of DENV infection in vitro. Time-course studies revealed that betulinic acid inhibits a post-entry stage of the DENV replication cycle and subsequent analyses also showed that the compound is able to inhibit viral RNA synthesis and protein production. Betulinic acid also demonstrated antiviral efficacy against other serotypes of DENV, as well as against other mosquito-borne RNA viruses such as Zika virus and Chikungunya virus, which are commonly found co-circulating together with DENV. As such, betulinic acid may serve as a valuable starting point for the development of antivirals to combat potential DENV outbreaks, particularly in tropical and subtropical regions which make up a large majority of documented infections., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Dengue virus, the causative agent for the dengue fever, infects approximately 50-100 million people worldwide per year. The high incidence of dengue fever, along with its potential to develop into a severe, life-threatening form, resulted in great interest in the discovery of an antiviral against it. In this study, we constructed a DENV2-EGFP infectious clone, established a fluorescence-based, high-throughput screening platform, and conducted a screen for anti-DENV compounds on a flavonoid-derivative library, Amongst the hits identified, ST081006 was found to be a strong inhibitor of the DENV replication. Time-course studies suggest that the presence of ST081006 is necessary to inhibit successive rounds of virus replication. Further investigations demonstrated that ST081006 affects the synthesis of both viral protein and viral RNA, and one anti-DENV mechanism is the direct inhibition of viral protein synthesis. The replication of all dengue serotypes, along with that of the enterovirus EV-A71, was shown to be affected by ST081006. Attempts to generate ST081006-resistant DENV were unsuccessful, and thus hints at host factors as potential drug target. Together, these results suggest that ST081006 affect DENV replication, likely by acting on a target involved in the viral protein and/or RNA synthesis pathway., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Dengue virus (DENV) disease outbreaks continue to develop across the globe with significant associated mortality and economic burden, yet no treatment has been approved to combat this virus. In an attempt to identify novel drug candidates as therapeutics for DENV infection, we evaluated four US Food and Drug Administration (FDA) approved drugs including aminolevullic acid, azelaic acid, mitoxantrone hydrochloride, and quinine sulfate, and tested their ability to inhibit DENV replication using focus-forming unit assay to quantify virus production. Of the four investigated compounds, quinine was found to have the most pronounced anti-DENV activity. Quinine inhibited DENV production of DENV by about 80% compared to untreated controls, while the other three drugs decreased virus production by only about 50%. Moreover, quinine inhibited DENV production of all four serotypes of DENV. Reduction in virus production was documented in three different cell lines of human origin. Quinine significantly inhibited DENV replication by reducing DENV RNA and viral protein synthesis in a dose-dependent manner. In addition, quinine ameliorated expression of genes related to innate immune response. These findings suggest the efficacy of quinine for stimulating antiviral genes to reduce DENV replication. The antiviral activity of quinine observed in this study may have applicability in the development of new drug therapies against DENV., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Hepatic dysfunction is a feature of dengue virus (DENV) infection. Hepatic biopsy specimens obtained from fatal cases of DENV infection show apoptosis, which relates to the pathogenesis of DENV infection. However, how DENV induced liver injury is not fully understood. In this study, we aim to identify the factors that influence cell death by employing an apoptosis-related siRNA library screening. Our results show the effect of 558 gene silencing on caspase 3-mediated apoptosis in DENV-infected Huh7 cells. The majority of genes that contributed to apoptosis were the apoptosis-related kinase enzymes. Tumor necrosis factor superfamily member 12 (TNFSF12), and sphingosine kinase 2 (SPHK2), were selected as the candidate genes to further validate their influences on DENV-induced apoptosis. Transfection of siRNA targeting SPHK2 but not TNFSF12 genes reduced apoptosis determined by Annexin V/PI staining. Knockdown of SPHK2 did not reduce caspase 8 activity; however, did significantly reduce caspase 9 activity, suggesting its involvement of SPHK2 in the intrinsic pathway of apoptosis. Treatment of ABC294649, an inhibitor of SPHK2, reduced the caspase 3 activity, suggesting the involvement of its kinase activity in apoptosis. Knockdown of SPHK2 significantly reduced caspase 3 activity not only in DENV-infected Huh7 cells but also in DENV-infected HepG2 cells. Our results were consistent across all of the four serotypes of DENV infection, which supports the pro-apoptotic role of SPHK2 in DENV-infected liver cells.

The polypyrimidine tract-binding protein (PTB) functions primarily as an IRES trans-acting factor in the propagation of hepatitis C virus and picornaviruses. PTB interacts with secondary structures within the 3'- and 5'-untranslated regions of these viral genomes to mediate efficient IRES-mediated viral translation. PTB has also been reported to bind to the untranslated region of the single-stranded RNA dengue virus (DENV), suggesting a similar function for PTB in flaviviruses. Indeed small interfering RNA-mediated PTB knockdown inhibited the production of infectious DENV, and this inhibition was specific to PTB knockdown and not due to a nonspecific anti-viral state. In fact, PTB depletion did not inhibit the production infectious yellow fever virus, another flavivirus. Nevertheless, whereas PTB knockdown led to a significant decrease in the accumulation of DENV viral RNAs, it did not impair translation. Moreover, PTB was shown to interact with the DENV nonstructural 4A protein, a known component of the viral replication complex, and with the DENV genome during infection. These data suggest that PTB interacts with the replication complex of DENV and is acting at the level of viral RNA replication.