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Dengue viruses are human pathogens that are transmitted through mosquitoes. Apart from the typical symptoms associated with viral fevers, DENV infections are known to cause several neurological complications such as meningitis, encephalitis, intracranial haemorrhage, retinopathies along with the more severe, and sometimes fatal, vascular leakage and dengue shock syndrome. This study was designed to investigate, in detail, the predicted viral protein aggregation prone regions among all serotypes. Further, in order to understand the cross-talk between viral protein aggregation and aggregation of cellular proteins, cross-seeding experiments between the DENV NS1 (1-30), corresponding to the β-roll domain and the diabetes hallmark protein, amylin, were performed. Various techniques such as fluorescence spectroscopy, circular dichroism, atomic force microscopy and immunoblotting have been employed for this. We observe that the DENV proteomes have many predicted APRs and the NS1 (1-30) of DENV1-3, 2K and capsid anchor of DENV2 and DENV4 are capable of forming amyloids, in vitro. Further, the DENV NS1 (1-30), aggregates are also able to cross-seed and enhance amylin aggregation and vice-versa. This knowledge may lead to an opportunity for designing suitable inhibitors of protein aggregation that may be beneficial for viral infections and comorbidities. highlighting the questions asked and the observations made in the current manuscript. Amyloid formation by the DENV peptides, NS1 (1-30), 2K and capsid anchor was observed. Further, the aggregation of NS1 (1-30), could cross-seed and enhance the aggregation of human amylin and vice-versa. [Display omitted] • We've predicted the aggregation prone regions in the proteomes of the four DENV (1-4) serotypes using four online predictors. • NS1 (1-30) of DENV1-3 and the 2K and CA peptides of DENV2 and DENV4 are capable of aggregating and forming amyloid structures in vitro under physiological conditions, shown through various techniques such as fluorescence spectroscopy, circular dichroism, atomic force microscopy and immunoblotting • NS1 (1-30) aggregates also interact with the human amylin and cross-seed and enhance its aggregation and vice-versa. • We hypothesize that the pathogenicity of DENV infection could be a function of viral protein aggregation and that the adversity of symptoms observed in diabetic dengue patients could be due to the cross-talk between the aggregation pathways of both the DENV and diabetes systems. [ABSTRACT FROM AUTHOR]

Reverse genetics systems are critical tools in combating emerging viruses which enable a better understanding of the genetic mechanisms by which viruses cause disease. Traditional cloning approaches using bacteria are fraught with difficulties due to the bacterial toxicity of many viral sequences, resulting in unwanted mutations within the viral genome. Here, we describe a novel in vitro workflow that leverages gene synthesis and replication cycle reaction to produce a supercoiled infectious clone plasmid that is easy to distribute and manipulate. We developed two infectious clones as proof of concept: a low passage dengue virus serotype 2 isolate (PUO-218) and the USA-WA1/2020 strain of SARS-CoV-2, which replicated similarly to their respective parental viruses. Furthermore, we generated a medically relevant mutant of SARS-CoV-2, Spike D614G. Results indicate that our workflow is a viable method to generate and manipulate infectious clones for viruses that are notoriously difficult for traditional bacterial-based cloning methods. • The development of infectious clones using bacteria is difficult due to toxicity. • Current methods to mitigate toxicity lack plasmid recoverability or are leaky. • An in vitro replication cycle reaction (RCR) method can mitigate toxicity. • Using an RCR method, we produced clones of dengue and SARS-CoV-2. • Using an RCR method, we generated a SARS-CoV-2 mutant. [ABSTRACT FROM AUTHOR]

Type I interferon (IFN–I) evasion by Dengue virus (DENV) is key in DENV pathogenesis. The non-structural protein 5 (NS5) antagonizes IFN-I response through the degradation of the signal transducer and activator of transcription 2 (STAT2). We developed a K562 cell-based platform, for high throughput screening of compounds potentially counteracting the NS5-mediated antagonism of IFN-I signaling. Upon a screening with a library of 1220 approved drugs, 3 compounds previously linked to DENV inhibition (Apigenin, Chrysin, and Luteolin) were identified. Luteolin and Apigenin determined a significant inhibition of DENV2 replication in Huh7 cells and the restoration of STAT2 phosphorylation in both cell systems. Apigenin and Luteolin were able to stimulate STAT2 even in the absence of infection. Despite the "promiscuous" and "pan-assay-interfering" nature of Luteolin, Apigenin promotes STAT2 Tyr 689 phosphorylation and activation, highlighting the importance of screening for compounds able to interact with host factors, to counteract viral proteins capable of dampening innate immune responses. A) K562 cells were engineered by means of a double retroviral transduction followed by cell sorting to express the luciferase reporter gene under the control of a ISRE sequence-containing promoter, non targeting shRNA to mimic PRR stimulation elicited by a RNA virus infection and either GFP or GFP-NS5. After TPA-mediated megakaryocytes differentiation, compounds are administered before IFN-α stimulation. A day later, the luciferase assay is performed. Signals of different intensities are obtained in the luminescence assay, lower for cells containing the GFP-NS5 fusion protein compared to cells expressing GFP alone. The addition of a compound X to cells expressing GFP-NS5 fusion protein and potentially capable of inhibiting the anti-IFN function of the viral protein, should, at least partially, restore the signal obtained in control cells in response to IFN-α treatment. B) Schematic workflow of the 1220 compound library screening in a miniaturized format. At day 1, cells are seeded at a specific density, differetiated with TPA, and then seeded again in 384 cell plates. After 72 h of differentiation (day 4), cells were treated with the 1220 compound library and after 2 h (day 4 + 2 h) were stimulated with IFN-α for further 24 h. At day 5, cell were analyzed for light emission upon lysis, using a microplate luminometer, C) Possible mechanism of action of the identified anti-NS5 compound Apigenin as DENV NS5 inhibitor. Apigenin can determine the phosphorylation of STAT2 even in the absence of DENV infection or virus-induced IFN-I stimulation. Pre-activated STAT-2 may be more resistant to Dengue NS5 inhibition/degradation and, therefore, more prone to interact with activated STAT1 and interferon regulatory factor 9 (IRF-9) to promote interferon stimulated genes (ISGs) and therefore the antiviral state, All images of this graphical abstract were created with BioRender.com. [Display omitted] • We developed a luciferase-based K562 cell platform to identify inhibitors of anti–IFN–I activity of DENV NS5 protein. • Apigenin, Chrysin, and Luteolin were identified screening a library of approved drugs, with Luteolin behaving as PAINS. • Apigenin determines STAT2 Tyr 689 phosphorylation in Huh7 cells not infected and infected with DENV2. • The host factor STAT2 can be a valuable targets for therapeutic interventions aimed at inhibiting NS5 anti-IFN-I activity. • Our reporter K562 cellular platform, due to the successful miniaturization, can be exploited for future HTS campaigns. [ABSTRACT FROM AUTHOR]

Wolbachia pipientis is known to block replication of positive sense RNA viruses. Previously, we created an Aedes aegypti Aag2 cell line (Aag2. w AlbB) transinfected with the w AlbB strain of Wolbachia and a matching tetracycline-cured Aag2.tet cell line. While dengue virus (DENV) was blocked in Aag2. w AlbB cells, we found significant inhibition of DENV in Aag2.tet cells. RNA-Seq analysis of the cells confirmed removal of Wolbachia and lack of expression of Wolbachia genes that could have been due to lateral gene transfer in Aag2.tet cells. However, we noticed a substantial increase in the abundance of phasi charoen-like virus (PCLV) in Aag2.tet cells. When RNAi was used to reduce the PCLV levels, DENV replication was significantly increased. Further, we found significant changes in the expression of antiviral and proviral genes in Aag2.tet cells. Overall, the results reveal an antagonistic interaction between DENV and PCLV and how PCLV-induced changes could contribute to DENV inhibition. • We explored the mechanism of dengue virus inhibition in Wolbachia -infected mosquito cells after the removal of Wolbachia. • Phasi charoen-like virus (PCLV) levels were significantly increased following the removal of Wolbachia. • Reducing PCLV using RNAi led to increased DENV replication. • PCLV changed the expression of antiviral and provial genes contributing to DENV replication. [ABSTRACT FROM AUTHOR]

Dengue infections pose a critical threat to public health worldwide. Since there are no clinically approved antiviral drugs to treat dengue infections caused by the four dengue virus (DENV) serotypes, there is an urgent need to develop effective antivirals. Peptides are promising antiviral candidates due to their specificity and non-toxic properties. The DENV envelope (E) protein was selected for the design of antiviral peptides due to its importance in receptor binding and viral fusion to the host cell membrane. Twelve novel peptides were designed to mimic regions containing critical amino acid residues of the DENV E protein required for interaction with the host. A total of four peptides were identified to exhibit potent inhibitory effects against at least three or all four DENV serotypes. Peptide 3 demonstrated all three modes of action: cell protection and inhibition of post-infection against all four DENV serotypes, whereas direct virus-inactivating effects were only observed against DENV-2, 3, and 4. Peptide 4 showed good direct virus-inactivating effects against DENV-2 (74.26%) as well as good inhibitions of DENV-1 (80.37%) and DENV-4 (72.22%) during the post-infection stage. Peptide 5 exhibited direct virus-inactivating effects against all four DENV serotypes, albeit at lower inhibition levels against DENV-1 and DENV-3. It also exhibited highly significant inhibition of DENV-4 (89.31%) during post-infection. Truncated peptide 5F which was derived from peptide 5 showed more significant inhibition of DENV-4 (91.58%) during post-infection and good direct virus-inactivating effects against DENV-2 (77.55%) at a lower concentration of 100 μM. Peptide 3 could be considered as the best antiviral candidate for pre- and post-infection treatments of DENV infections in regions with four circulating dengue serotypes. However, if the most predominant dengue serotype for a particular region could be identified, peptides with significantly high antiviral activities against that particular dengue serotype could serve as more suitable antiviral candidates. Thus, peptide 5F serves as a more suitable antiviral candidate for post-infection treatment against DENV-4. • Twelve novel peptides were designed to mimic regions of the DENV E protein containing critical amino acid residues. • These critical amino acid residues were identified to be important for viral attachment, entry, and membrane fusion. • Peptides 3, 4, 5, and 5F exhibited potent inhibitory effects against at least three or all four DENV serotypes. • Peptide 3 demonstrated three modes of action: cell protection, direct virus inactivation, and post-infection inhibition. • Peptides 4, 5, and 5F demonstrated two modes of action: direct virus inactivation and inhibition during post-infection. [ABSTRACT FROM AUTHOR]

Platelet count is widely used for the diagnosis and follow-up of patients with dengue. Despite its close viral structural and symptomatic homology, ZIKV infection does not typically induce significant thrombocytopenia. To determine the effect of DENV-2 and ZIKV infection on human platelet precursors we utilized MEG-01 cell line to evaluate the viral infection, viability, innate gene expression and release of platelet-like particles (PLPs). DENV-2 induced a higher proportion of cell death at 48–72 h post-infection than ZIKV. The median range of intracellular NS1+/E+ cells was 11.2% (3.3%–25%) and 5% (3%–8.1%) for DENV-2 and ZIKV, respectively (p = 0.03). MEG-01 cells infected with DENV-2 quickly expressed higher levels of IFN-β, indolamine 2,3-dioxygenase and CXCL10 mRNA compared to ZIKV infected cells and DENV-2 but not ZIKV infection reduced the number PLPs from stimulated MEG-01 cells. The results shed light into mechanisms including thrombocytopenia present in patients with DENV but absent in ZIKV infections. • DENV-2 but not ZIKV affects the viability of platelet precursors. • DENV-2 infects more efficiently MEG-01 cells than ZIKV. • Platelet precursors could be a partial source of circulating innate biomarkers during flavivirus infection. • DENV-2 but not ZIKV infection decreases the number of PLPs generated during induced-thrombopoiesis. [ABSTRACT FROM AUTHOR]

Dengue virus (DENV) is a prevalent mosquito-transmitted human pathogen, causing about 100 million cases of acute dengue fever and 21,000 deaths annually worldwide. Therapeutic neutralizing antibodies against dengue virus might be effective to treat severe dengue fever. Here, we showed that human monoclonal antibody (HMAb) 9C7 bound to all four intact serotypes of DENV but not to the recombinant envelope protein, suggesting HMAb 9C7 recognized a conformational epitope of the envelope protein. Taken together our results suggested that HMAb 9C7 neutralized all four serotypes of DENV in vitro and, for DENV-1, indicated activity at the pre- and post-attachment steps in the viral life cycle. HMAb 9C7 potently protected suckling mice from lethal challenge with all four serotypes of DENV. FcγRII-mediated uptake of immune complexes and antibody-dependent enhancement at low doses of the antibody were abolished by two Leu-to-Ala (9C7-LALA) mutations or deletion of nine amino acids (9C7-9del) in HMAb 9C7 Fc. Therefore, HMAb 9C7 represented a promising prophylactic and therapeutic agent against all four serotypes of DENV. • We generated and characterized a novel DENV-specific HMAb 9C7 recognized a conformational epitope of the envelope protein. • HMAb 9C7 potently neutralized all four serotypes DENV in vitro at the pre- and post-attachment steps in the viral life cycle. • HMAb 9C7 potently protected model mice from lethal challenge with all four serotypes of DENV. • FcγRII-mediated ADE were abolished by two Leu-to-Ala mutations or deletion of nine amino acids in HMAb 9C7 Fc. [ABSTRACT FROM AUTHOR]

Dengue fever is caused by four related dengue virus serotypes, DENV-1 to DENV-4, where each serotype comprises distinct genotypes and lineages. The last major outbreak in Mexico occurred during 2012 and 2013, when 112,698 confirmed cases were reported (DENV-1 and DENV-2 were predominant). Following partial E, NS2A and NS5 gene sequencing, based on the virus genome variability, we analyzed 396 DENV-1 and 248 DENV-2 gene sequences from serum samples from dengue acute clinical cases from 13 Mexican states, Mutations were identified, and their genetic variability estimated, along with their evolutionary relationship with DENV sequences sampled globally. DENV-1 genotype V and DENV-2 Asian-American genotype V were the only genotypes circulating during the outbreak. Mutations in NS2A and NS5 proteins were widely disseminated and suggested local emergence of new lineages. Phylogeographic analysis suggested viral spread occurred from coastal regions, and tourist destinations, such as Yucatan and Quintana Roo, which played important roles in disseminating these lineages. • We document that hypervariable regions of DENV can recapitulate the full-CDS phylogeny. • DENV-1 genotype V and DENV-2 Asian-American were the only genotypes circulating. • Mutations in NS2A and NS5 were disseminated and suggest the emergence of new lineages. • Phylogeographic analysis showed viral spread from coastal and tourist destinations. [ABSTRACT FROM AUTHOR]

The enveloped positive-sense RNA viruses including Zika virus (ZIKV) need host lipids to successfully replicate. The nature of the lipids and the replication step(s) where lipids are utilized often vary amongst viruses. In this study, we demonstrate that ZIKV particle envelope is significantly enriched in distinct sphingolipid species. To determine the role of sphingolipids in ZIKV replication, we leveraged a panel of sphingolipid-deficient cell lines. Notably, knockout of glucosylceramide and lactosylceramide synthase encoding genes (GCSKO; B4G5KO) resulted in a marked decrease in ZIKV titers. GCSKO or pharmacological inhibition of GCS also led to a significant decrease in ZIKV genome replication. Further analysis indicated that GCSKO reduced intracellular virus titers but had minimal impact on ZIKV binding. Restoration of B4G5 expression in B4G5KO cells or supplementing PDMP-treated cells with glucosylceramide led to a significant rescue of ZIKV replication. Altogether, our findings suggest that ZIKV needs glycosphingolipids to facilitate virus replication. • Quantitative analysis of partially purified ZIKV particles showed an enrichment of sphingolipid and glycosphingolipid species. • Transcription activator-like effector nucleases (TALENs)-based knockout of glucosylceramide synthase (GCS) gene and pharmacological inhibition of GCS activity resulted in a significant reduction in ZIKV titers and genome replication. • Supplementing cells with glucosylceramide glycosphingolipid led to a rescue of ZIKV replication in cells treated with a pharmacological inhibitor of GCS activity. • SPharmacological inhibition of GCS activity at both early and late stages of ZIKV infection cycle led to a significant reduction in ZIKV titers, implying a role for glycosphingolipids in ZIKV genome replication and post replication steps. • Our findings reveal the importance of GCS, and its related glycosphingolipids, in ZIKV genome replication and post replication steps. [ABSTRACT FROM AUTHOR]

Dengue virus (DENV) is a major global health concern, causing millions of infections annually. Understanding the cellular response to DENV infection is crucial for developing effective therapies. This study provides an in-depth analysis of the cellular response to Dengue virus (DENV) infection, with a specific focus on the interplay between microRNAs (miRNAs), apoptosis, and viral load across different DENV serotypes. Utilizing a variety of cell lines infected with four DENV serotypes, the research methodically quantifies viral load, and the expression levels of miRNA-15, miRNA-16, and BCL2 protein, alongside measuring apoptosis markers. Methodologically, the study employs quantitative PCR for viral load and miRNA expression analysis, and Western blot for apoptosis and BCL2 detection, with a statistical framework that includes ANOVA and correlation analysis to discern significant differences and relationships. The findings reveal that despite similar viral loads across DENV serotypes, DENV-2 exhibits a marginally higher load. A notable upregulation of miRNA-15 and miRNA-16 correlates positively with increased viral load, suggesting their potential role in modulating viral replication. Concurrently, a marked activation of caspases 3 and 7, along with changes in BCL2 protein levels, underscores the role of apoptosis in the cellular response to DENV infection. Conclusively, the study enhances the understanding of miRNA involvement in DENV pathogenesis, highlighting miRNA-15 and miRNA-16 as potential regulatory agents in viral replication and apoptosis. These findings pave the way for further exploration into miRNA-based therapeutic strategies against DENV infection. • Similar viral loads were observed among different dengue virus serotypes. • Significant alterations in miRNAs, apoptosis, and BCL2 occurred in Dengue infection. • Relationships exist between miRNAs and apoptotic pathways during Dengue infection. • BCL2 expression correlates with increased viral load across all dengue serotypes. [ABSTRACT FROM AUTHOR]

Septins are a family of GTP-binding proteins identified in insects and mammals. Septins are components of the cytoskeleton and participate in cytokinesis, chromosomal segregation, intracellular vesicular traffic, and response to pathogens. Human septin 6 was identified as necessary for hepatitis C virus replication. Information about host factors necessary for flavivirus replication in mosquitoes is scarce. Thus, the role of septins in the replicative cycle of dengue virus in Aedes spp. derived cells was investigated. Through bioinformatic analysis, sequences of septin-like proteins were identified. Infected mosquito cells showed increased expression of Sep2. Colocalization analysis, proximity ligation and immunoprecipitation assays indicated that Sep2 interacts with proteins E, NS3 and NS5, but not NS1. Immunoelectron microscopy evidenced the presence of AalSep2 in replicative complexes. Finally, silencing of Sep2 expression resulted in a significant decrease in virus progeny, indicating that Sep2 is a host factor participating in dengue virus replication in mosquito cells. • Aedes albopictus and A. aegypti cell lines have four genes coding for canonical Septin proteins. • Mosquito DNV infected cells increase Septin2 protein production. • In dengue virus infected Aedes spp. cell lines, Septin 2 protein binds to E, NS3 and NS5, but not to NS1. • Sep2 silencing decreases viral progeny but not genome molecules production. [ABSTRACT FROM AUTHOR]

To date, there are no broad-spectrum antivirals available to treat infections with flaviviruses such as dengue (DENV) and Zika virus (ZIKV). In this study, we determine the broad antiviral activity of the lantibiotic Labyrinthopeptin A1. We show that Laby A1 inhibits all DENV serotypes and various ZIKV strains with IC 50 around 1 μM. The structurally related Laby A2 also displayed a consistent, but about tenfold lower, antiviral activity. Furthermore, Laby A1 inhibits many viruses from divergent families such as HIV, YFV, RSV and Punta Torovirus. Of interest, Laby A1 does not show activity against non-enveloped viruses. Its antiviral activity is independent of the cell line or the used evaluation method, and can also be observed in MDDC, a physiologically relevant primary cell type. Furthermore, Laby A1 demonstrates low cellular toxicity and has a more favorable SI compared to duramycin, a well-described lantibiotic with broad-spectrum antiviral activity. Time-of-drug addition experiments demonstrate that Laby A1 inhibits infection and entry processes of ZIKV and DENV. We reveal that Laby A1 performs its broad antiviral activity by interacting with a viral factor rather than a cellular factor, and that it has virucidal properties. Finally, using SPR interaction studies we demonstrate that Laby A1 interacts with several phospholipids (i.e. PE and PS) present in the viral envelope. Together with other recent Labyrinthopeptin antiviral publications, this work validates the activity of Laby A1 as broad antiviral entry inhibitor with a unique mechanism of action and demonstrates its potential value as antiviral agent against emerging flaviviruses. • The lantibiotic Labyrinthopeptin A1 exerts broad antiviral activity against various enveloped viruses. • Using SPR, we demonstrate that Labyrinthopeptin A1 interacts with phospholipids present in the viral envelope. • Labyrinthopeptin A1 has virucidal properties, which inhibits viral entry into target cells. [ABSTRACT FROM AUTHOR]

Envelope phosphatidylserine (PtdSer) and phosphatidylethanolamine (PtdEtr) have been shown to mediate binding of enveloped viruses. However, commonly used PtdSer binding molecules such as Annexin V cannot block PtdSer-mediated viral infection. Lack of reagents that can conceal envelope PtdSer and PtdEtr and subsequently inhibit infection hinders elucidation of the roles of the envelope phospholipids in viral infection. Here, we developed sTIM1dMLDR801, a reagent capable of blocking PtdSer- and PtdEtr-dependent infection of enveloped viruses. Using sTIM1dMLDR801, we found that envelope PtdSer and/or PtdEtr can support ZIKV infection of not only human but also mosquito cells. In a mouse model for ZIKV infection, sTIM1dMLDR801 reduced ZIKV load in serum and the spleen, indicating envelope PtdSer and/or PtdEtr support in viral infection in vivo. sTIM1dMLDR801 will enable elucidation of the roles of envelope PtdSer and PtdEtr in infection of various virus species, thereby facilitating identification of their receptors and transmission mechanisms. • We have created a biologics, sTIM1dMLDR801, that can specifically inhibit PtdSer- and PtdEtr-dependent viral entry mechanisms. • Using this reagent, we showed that envelope PtdSer and/or PtdEtr support ZIKV viral replication in human and mosquito cells in vitro as well as in vivo replication in mice. • This reagent can facilitate and provide new insights in the identification of viral receptors, host ranges, and mechanisms of transmission by identifying roles of envelope PtdSer and PtdEtr. [ABSTRACT FROM AUTHOR]

Antibodies (Abs) recognizing the Dengue virus (DENV) E dimer epitope (EDE) that potently neutralize all DENV serotypes are promising templates for vaccine design. As an important feature for some Abs is their bivalency, we sought to define the role avidity plays in neutralization by EDE Abs. We compared neutralization activity between bivalent IgGs and monovalent Ab fragments (Fabs) for two EDE Abs, A11 and C10. IgG forms of both Abs exhibited more potent neutralization activity than their counterpart Fabs, yet only for C10 was this enhanced activity associated with bivalent binding. A11 and C10 also exhibited differential binding profiles to DENV virus-like particles under acidic conditions mimicking the environment that triggers viral membrane fusion, suggesting that EDE Abs employ diverse neutralization mechanisms despite sharing an epitope. Delineating the full range of Ab binding modes and neutralization mechanisms against a single epitope may inform therapeutic approaches and refine vaccine design. • Broadly-neutralizing antibodies target the Dengue virus E dimer epitope. • Monovalent antibody fragments neutralize Dengue less potently than whole antibodies. • Not all antibodies are capable of binding the Dengue E dimer epitope bivalently. • Antibodies targeting the same epitope may exhibit diverse neutralization mechanisms. [ABSTRACT FROM AUTHOR]

In tropical and subtropical countries, dengue virus (DENV) infections have been increasing; however, we still lack effective therapy. In the present study, we aimed to engineer a bispecific antibody (subsequently named LUZ-8F2–6B1), based on monoclonal antibody 6B1, which has anti DENV-1, 2, and 3 activity, and 8F2, which has anti DENV-4 activity. LUZ-8F2–6B1 displayed potent neutralization activity against four serotypes of DENV by binding to the envelop protein. In vivo , we demonstrated that LUZ-8F2–6B1 could provide protection against infection by four serotypes of DENV in a mouse model. In addition, the deletion of nine amino acids in the Fc region (LUZ-8F2–6B1-9del) completely abolished the antibody-dependent enhancement observed at lower doses of the antibody. Thus, LUZ-8F2–6B1 is a promising, safe, and effective agent for the prophylaxis and treatment of DENV infection. • An engineered bispecific antibody LUZ-8F2-6B1 showed potent neutralization activity against all four serotypes of DENV. • In vivo, a mouse model showed that LUZ-8F2-6B1 provided protection against infection by all four serotypes of DENVs. • LUZ-8F2-6B1-9del completely abrogated antibody dependent enhancement, which occurs when using lower doses of the antibody. • LUZ-8F2-6B1 could be an effective and safe agent for the prophylaxis and therapy of DENV infection. [ABSTRACT FROM AUTHOR]

Dengue virus (DENV) infection elevates intracellular Ca2+ concentration ([Ca2+] i), but it is unknown whether Ca2+ and calmodulin (CaM) are involved in DENV infection. We conducted immunofluorescence and western blot experiments and measured [Ca2+] i examining the effects of DENV infection and drugs that alter Ca2+/CaM functions on CaM translocation, DENV2 infection, protein expression, virus-inducible STAT2 protein abundance, and CREB phosphorylation in H9c2 cells. DENV infection increased CaM expression, its nuclear translocation and NS3 and E viral proteins expression and colocalization in a manner that could be blocked by the ryanodine receptor antagonist dantrolene. DENV infection also increased CREB phosphorylation, an effect inhibited by either dantrolene or the CaM inhibitor W7. Dantrolene substantially hindered infection as assessed by focus assays in Vero cells. These results suggest that Ca2+ and CaM play an important role in DENV infection of cardiac cells and that dantrolene may protect against severe DENV cardiac morbidity. Image 1 • Dengue virus infection increases calmodulin expression and nuclear translocation. • Dengue virus infection increases phosphorylation of the transcription factor CREB. • Dantrolene inhibits upregulation and translocation of calmodulin by dengue. • Dantrolene inhibits pCREB expression and dengue viral protein expression. • Dantrolene may be protective against severe dengue virus infection in the heart. [ABSTRACT FROM AUTHOR]

Drugs against flaviviruses such as dengue (DENV) and Zika (ZIKV) virus are urgently needed. We previously demonstrated that three fluoroquinolones, ciprofloxacin, enoxacin, and difloxacin, suppress replication of six flaviviruses. To investigate the barrier to resistance and mechanism(s) of action of these drugs, DENV-4 was passaged in triplicate in HEK-293 cells in the presence or absence of each drug. Resistance to ciprofloxacin was detected by the seventh passage and to difloxacin by the tenth, whereas resistance to enoxacin did not occur within ten passages. Two putative resistance-conferring mutations were detected in the envelope gene of ciprofloxacin and difloxacin-resistant DENV-4. In the absence of ciprofloxacin, ciprofloxacin-resistant viruses sustained a significantly higher viral titer than control viruses in HEK-293 and HuH-7 cells and resistant viruses were more stable than control viruses at 37 °C. These results suggest that the mechanism of action of ciprofloxacin and difloxacin involves interference with virus binding or entry. Image 1 • Dengue virus evolved resistance to ciprofloxacin after 7 passages with the drug. • Ciprofloxacin-resistant dengue virus was resistant to difloxacin but not enoxacin. • Two mutations in the viral envelope were associated with ciprofloxacin resistance. • Ciprofloxacin-resistant viruses gained fitness in cultured human cells. • Ciprofloxacin-resistant virions showed higher stability than controls. [ABSTRACT FROM AUTHOR]

Dengue virus (DENV) infects roughly 400 million people annually, causing febrile and hemorrhagic disease. While preexisting inter-serotype immunity (PISI) provides transient protection, it may drive severe disease over time. PISI's impact on virus evolution, however, is less understood. Retrospective epidemiological analyses suggest that PISI may drive DENV evolution. Using in vitro directed evolution, we explored how DENV2 evolves in the presence of DENV3/4 convalescent serum. Two post-passaging mutations (E-I6M and E-N203D) were then studied for fitness effects in mammalian and insect hosts and immune escape. E-I6M resisted neutralization, altered fitness in mammalian cell culture models, and had no effect in Aedes albopictus mosquitoes. E-N203D showed no change in neutralization sensitivity, reduced fitness in a DENV-naïve epithelial model, and no effects in the other models. These results align with surveillance data, where E-I6M emerged and disappeared, while E−203D and E−203 N cocirculate, thus suggesting that PISI can drive DENV evolution. • Pre-existing inter-serotype immunity is critical in DENV pathology but is under-studied in evolution. • We replicated the emergence of natural DENV mutations by passaging in the presence of cross-reactive neutralizing antibodies. • Neutralization escape did not correlate with increased fitness in immune environments. • DENV, without cross-reactive pressures, gained fitness in naïve environments but became more sensitive to neutralization. • These findings highlight the importance of PISI on DENV evolution. [ABSTRACT FROM AUTHOR]

The four serotypes of dengue virus (DENV) are the leading etiologic agent of disease caused by arthropod-borne viruses (arboviruses) in the world, with billions at risk of DENV infection spread by infected mosquitoes. DENV causes illness ranging from dengue fever (DF) to life-threatening dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS). DENV proliferates well in two different host systems, an invertebrate mosquito vector and vertebrate primate host, which have a distinct difference in their preference of codon pairs (CP) for translation (different " codon pair bias "). Consequently, arboviruses must delicately balance the use of codon pairs between mammals and arthropods, which presents an Achilles' heel that we have exploited by specifically shifting the codon pair preference in the E and NS3 ORFs away from mammals while keeping the CPB favorable for mosquito ORFs. Here we report that recoding of the ORFs has led to variants that were over-attenuated in rhesus macaques although induction of protective antibodies in animals vaccinated with the smallest recoded ORF (E) was observed. The flexibility of our synthetic vaccine design (by decreasing the number of unfavorable CPs in the E ORF), allowed us to construct two new vaccine candidates (EhminA and EhminB) with intermediate attenuation in cell culture and neonatal mice, a result demonstrating proof of concept. New DENV vaccine candidates are being developed based on selective attenuation by dramatic recoding, with flexibility in balancing the attenuation and immunogenicity by marrying rational design and empirical modification. [ABSTRACT FROM AUTHOR]

Abstract Subgenomic flavivirus RNA (sfRNA) is essential for flavivirus-induced cytopathicity. sfRNA-deficient mutant dengue virus (DENV) M8 harboring a deleted domain II in the 3′ untranslated region (UTR) has severely decreased cytopathicity and replication efficiency. Here, recovered viruses acquired from M8 by serial passages replicated similarly to the wild type (WT) but produced two types of sfRNA, whereas only one was detected in WT infected cells, resulting in mildly decreased cytopathicity. Sequencing and structure prediction showed that recovered viruses acquired a novel additional partial domain I sequence duplication and formed analogous RNA secondary structures. This was the first experimental evidence verifying that a longer precursor may exist in the 3′ UTR of DENV. Additionally, the duplicated structures could be compensated by the homologous structures from other flaviviruses, rather than heterologous viruses, implying that homologous secondary structures or higher tertiary structures played more important roles in sfRNA production and viral replication. [ABSTRACT FROM AUTHOR]

Flaviviruses depend on multiple host pathways during their life cycles and have evolved strategies to avoid the innate immune response. Previously, we showed that the West Nile virus capsid protein plays a role in this process by blocking apoptosis. In this study, we examined how expression of capsid proteins from several flaviviruses affects apoptosis and other host processes that impact virus replication. All of the tested capsid proteins protected cells from Fas-dependent apoptosis through a mechanism that requires activated Akt. Capsid expression upregulated other Akt-dependent cellular processes including expression of glucose transporter 1 and mitochondrial metabolism. Protein phosphatase 1, which is known to inactivate Akt, was identified as a DENV capsid interacting protein. This suggests that DENV capsid expression activates Akt by sequestering phosphatases that downregulate phospho-Akt. Capsid-dependent upregulation of Akt would enhance downstream signalling pathways that affect cell survival and metabolism, thus providing a favourable environment for virus replication. [ABSTRACT FROM AUTHOR]

Dengue virus (DENV) is an arbovirus that belongs to the Flaviviridae family. Studies reveal that peptides secreted by amphibians have many functions, such as antiviral and antimicrobial activities. As there is no antiviral drug effective against the DENV, the antiviral activity of a synthetic peptide called HS-1, derived from the secretion of the anuran Hypsiboas semilineatus, has been evaluated. The assays of neutralization in the Vero cells show a complete inhibition of infection of the serotypes 2 and 3. Furthermore, the direct action of peptides on the viral particle can be observed through atomic force microscopy. In vivo tests display 80% protection against the dengue-2 virus due to the presence of HS-1, which reveals its potential as an antiviral against the DENV. [ABSTRACT FROM AUTHOR]

Dengue virus (DENV) is an RNA virus transmitted among humans by mosquito vectors, mainly Aedes aegypti . DENV transmission requires viral dissemination from the mosquito midgut to the salivary glands. During this process the virus undergoes several population bottlenecks, which are stochastic reductions in population size that restrict intra-host viral genetic diversity and limit the efficiency of natural selection. Despite the implications for virus transmission and evolution, DENV replication in salivary glands has not been directly demonstrated. Here, we used a strand-specific quantitative RT-PCR assay to demonstrate that negative-strand DENV RNA is produced in Ae. aegypti salivary glands, providing conclusive evidence that viral replication occurs in this tissue. Furthermore, we showed that the concentration of DENV genomic RNA in salivary glands increases significantly over time, indicating that active replication likely replenishes DENV genetic diversity prior to transmission. These findings improve our understanding of the biological determinants of DENV fitness and evolution. [ABSTRACT FROM AUTHOR]

Mitochondria are highly dynamic organelles that undergo continuous cycles of fission and fusion to maintain essential cellular functions. An imbalance between these two processes can result in many pathophysiological outcomes. Dengue virus (DENV) interacts with cellular organelles, including mitochondria, to successfully replicate in cells. This study used live-cell imaging and found an increase in mitochondrial length and respiration during DENV infection. The level of mitochondrial fission protein, Dynamin-related protein 1 (Drp1), was decreased on mitochondria during DENV infection, as well as Drp1 phosphorylated on serine 616, which is important for mitochondrial fission. DENV proteins NS4b and NS3 were also associated with subcellular fractions of mitochondria. Induction of fission through uncoupling of mitochondria or overexpression of Drp1 wild-type and Drp1 with a phosphomimetic mutation (S616D) significantly reduced viral replication. These results demonstrate that DENV infection causes an imbalance in mitochondrial dynamics by inhibiting Drp1-triggered mitochondrial fission, which promotes viral replication. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Although infections of vertebrate hosts by arthropod-borne viruses may lead to pathogenic outcomes, infections of vector mosquitoes result in persistent infections, where the virus replicates in the host without causing apparent pathological effects. It is unclear how persistent infections are established and maintained in mosquitoes. Several reports revealed the presence of flavivirus-like DNA sequences in the mosquito genome, and recent studies have shown that DNA forms of RNA viruses restrict virus replication in Drosophila , suggesting that DNA forms may have a role in developing persistent infections. Here, we sought to investigate whether arboviruses generate DNA forms following infection in mosquitoes. Our results with West Nile, Dengue, and La Crosse viruses demonstrate that DNA forms of the viral RNA genome are generated in mosquito cells; however, not the entire viral genome, but patches of viral RNA in DNA forms can be detected 24 h post infection. [ABSTRACT FROM AUTHOR]

Arboviruses are a large group of viruses that are transmitted by arthropods including ticks and mosquitoes. The global diversity of arboviruses is unknown; however, theoretical studies have estimated that over 2,000 mosquito-borne flaviviruses may exist. An increasing number of flaviviruses can only infect insect cells. We hypothesize that insect-specific flaviviruses (ISFVs) represent model genetic precursors to pathogenic flaviviruses, although the genetic mechanisms required for adaptation to vertebrate hosts are unclear. In this study, we determined that Kamiti River virus (KRV) infection was inhibited by innate immunity pathways in vertebrate cells. KRV infection of IRF3,5,7 -/- mouse embryonic fibroblasts led to low levels of viral protein production and shedding of infectious progeny. These data suggest that ISFVs cannot evade vertebrate innate immune pathways. Identifying cellular pathways and genetic changes that are required for adaptation of arthropod-specific arboviruses to vertebrate hosts is critical to understanding emerging infectious disease. [ABSTRACT FROM AUTHOR]

Flaviviruses are positive-stranded RNA viruses that incorporate envelope (E) and premembrane (prM) proteins into the virion. Furin-mediated cleavage of prM defines a required maturation step in the flavivirus lifecycle. Inefficient prM cleavage results in structurally heterogeneous virions with unique antigenic and functional characteristics. Recent studies with dengue virus suggest that viruses produced in tissue culture cells are less mature than those produced in primary cells. In this study, we describe a Vero cell line that ectopically expresses high levels of human furin (Vero-furin) for use in the production of more homogenous mature flavivirus populations. Laboratory-adapted and clinical dengue virus isolates grow efficiently in Vero-furin cells. Biochemical and structural techniques demonstrate efficient prM cleavage in Vero-furin derived virus preparations. These virions also were less sensitive to neutralization by antibodies that bind efficiently to immature virions. This furin-expressing cell line will be of considerable utility for flavivirus neutralization and structural studies. [ABSTRACT FROM AUTHOR]

Megakaryocyte–erythrocyte progenitor (MEP) cells are potential in vivo targets of dengue virus (DENV); the virus has been found associated with megakaryocytes ex vivo and platelets during DENV-induced thrombocytopenia. We report here that DENV serotype 2 (DENV2) propagates well in human nondifferentiated MEP cell lines (Meg01 and K562). In comparison to virus propagated in Vero cells, viruses from MEP cell lines had similar structure and buoyant density. However, differences in MEP-DENV2 stability and composition were suggested by distinct protein patterns in western blot analysis. Also, antibody neutralization of envelope domain I/II on MEP-DENV2 was reduced relative to that on Vero-DENV2. Infectious DENV2 was produced at comparable kinetics and magnitude in MEP and Vero cells. However, fewer virion structures appeared in electron micrographs of MEP cells. We propose that DENV2 infects and produces virus efficiently in megakaryocytes and that megakaryocyte impairment might contribute to dengue disease pathogenesis. [ABSTRACT FROM AUTHOR]

Dengue virus NS1 is a glycoprotein of 46–50 kDa which associates as a dimer to internal and cytoplasmic membranes and is also secreted, as a hexamer, to the extracellular milieu. However, the notion exist that NS1 is secreted only from infected vertebrate and not mosquito cells. In this work, evidence is presented showing that NS1 is secreted efficiently by infected mosquito cells. NS1 was detected in cell supernatants starting at 6 hpi with a continuous concentration increase up to 24 hpi. Nevertheless, cell viability showed an average cell survival of 97%. At variance with observations with vertebrate cells, NS1 does not seems to associate with the cytoplasmic membrane of insect cells. Finally, evidence is presented indicating that NS1 is secreted from insect cells as a barrel-shaped hexamer. These findings provide new insights into the biology of NS1 and open questions about the role of secreted NS1 in the vector mosquito. [ABSTRACT FROM AUTHOR]

Here we investigated the binding of Dengue virus envelope glycoprotein domain III (DIII) by two broadly neutralizing antibodies (bNAbs), 4E11 and 4E5A. There are four serotypes of Dengue virus (DENV-1 to -4), whose DIII sequences vary by up to 49%. We used combinatorial alanine scanning mutagenesis, a phage display approach, to map functional epitopes (those residues that contribute most significantly to the energetics of antibody–antigen interaction) on these four serotypes. Our results showed that 4E11, which binds strongly to DENV-1, -2, and -3, and moderately to DENV-4, recognized a common conserved core functional epitope involving DIII residues K310, L/I387, L389, and W391. There were also unique recognition features for each serotype, suggesting that 4E11 has flexible recognition requirements. Similar scanning studies for the related bNAb 4E5A, which binds more tightly to DENV-4, identified broader functional epitopes on DENV-1. These results provide useful information for immunogen and therapeutic antibody design. [ABSTRACT FROM AUTHOR]

Productive arbovirus infections require mechanisms to suppress or circumvent the cellular RNA interference (RNAi) pathway, a major antiviral response in mosquitoes. In this study, we demonstrate that two flaviviruses, Dengue virus and Kunjin virus, significantly repress siRNA-mediated RNAi in infected human cells as well as during infection of the mosquito vector Culex quinquefasciatus . Arthropod-borne flaviviruses generate a small structured non-coding RNA from the viral 3′ UTR referred to as sfRNA. Analysis of infections with a mutant Kunjin virus that is unable to generate appreciable amounts of the major sfRNA species indicated that RNAi suppression was associated with the generation of the non-coding sfRNA. Co-immunoprecipitation of sfRNA with RNAi mediators Dicer and Ago2 suggest a model for RNAi suppression. Collectively, these data help to establish a clear role for sfRNA in RNAi suppression and adds to the emerging impact of viral long non-coding RNAs in modulating aspects of anti-viral immune processes. [ABSTRACT FROM AUTHOR]

It is well established that +ssRNA viruses manipulate cellular lipid homoeostasis and distribution to facilitate efficient replication. Here, we show that the cellular lipid ceramide is redistributed to the West Nile virus strain Kunjin virus (WNV KUN ) replication complex (RC) but not to the dengue virus serotype 2 strain New Guinea C (DENV NGC ) RC. We show that prolonged chemical inhibition of serine palmitoyltransferase with myriocin had a significant deleterious effect on WNV KUN replication but enhanced DENV NGC replication. Additionally, inhibition of ceramide synthase with Fumonisin B 1 had a detrimental effect on WNV KUN replication and release of infectious virus particles but contrastingly an enhancing effect on DENV NGC replication and virus production. These observations suggest that ceramide production via the de novo and salvage pathway is a requirement for WNV KUN replication but inhibitory for DENV NGC replication. Thus, although these two viruses are from the same genus, they have a differential ceramide requirement for replication. [ABSTRACT FROM AUTHOR]

Remarkable progress in structural biology has equipped virologists with insight into structures of viral proteins and virions at increasingly high resolution. Structural information has been used extensively to address fundamental questions about virtually all aspects of how viruses replicate in cells, interact with the host, and in the design of antiviral compounds. However, many critical aspects of virology exist outside the snapshots captured by traditional methods used to generate high-resolution structures. Like all proteins, viral proteins are not static structures. The conformational flexibility and dynamics of proteins play a significant role in protein–protein interactions, and in the structure and biology of virus particles. This review will discuss the implications of the dynamics of viral proteins on the biology, antigenicity, and immunogenicity of flaviviruses. [ABSTRACT FROM AUTHOR]

The predicted secondary structure of sub-genomic RNA in dengue virus defective interfering (D.I.) particles from patients, or generated in vitro , resembled that of the 3′ and 5′ regions of wild type dengue virus (DENV) genomes. While these structures in the sub-genomic RNA were found to be essential for its replication, their nucleotide sequences were not, so long as any new sequences maintained wild type RNA secondary structure. These observations suggested that these sub-genomic fragments of RNA from dengue viruses were replicated in the same manner as the full length genomes of their wild type, “helper”, viruses and that they probably represent the smallest fragments of DENV RNA that can be replicated during a natural infection. While D.I. particles containing sub-genomic RNA are completely parasitic, the relationship between wild type and D.I. DENV may be symbiotic, with the D.I. particles enhancing the transmission of infectious DENV. [ABSTRACT FROM AUTHOR]

Abstract: Antibody-dependent enhancement (ADE) of infection may cause severe illness in patients suffering a secondary infection by a heterologous dengue virus (DENV) serotype. During ADE of infection, cross-reactive non- or poorly-neutralizing antibodies form infectious virus-Ab complexes with the newly infecting serotype and enhance virus infection by binding to the Fcγ receptors (FcγR) on FcγR-bearing cells. In this study, we determined that molecular determinants of DENV2 envelope protein critical for virus entry during non-ADE infection are also required for ADE infection mediated by FcγRIIA, and binding of virus-Ab complexes with FcγRIIA alone is not sufficient for ADE of infection. The FcγRIIA mainly plays an auxiliary role in concentrating the virus–Ab complex to the cell surface, and other primary cellular receptors are required for virus entry. Understanding the viral entry pathway in ADE of DENV infection will greatly facilitate rational designs of anti-viral therapeutics against severe dengue disease associated with ADE. [Copyright &y& Elsevier]

Abstract: Viremia kinetics directly influence the clinical course and transmission dynamics of DENV, but many aspects of viral dynamics remain unknown. Non-human primates (NHP) have been used as a model system for DENV infection for decades. Here, we identify papers with experimentally-infected NHP and estimate the time to- and duration of viremia as well as estimate associations between these and serotype, inoculating dose, viremia assay, and species of NHP. We estimate the time to viremia in rhesus macaques to range from 2.63 to 3.32days for DENV-2 and -1 and the duration to range from 3.13 to 5.13days for DENV-4 and -2. We find no differences between non-human primates for time to viremia or duration, and a significant negative relationship between inoculating dose and duration of viremia. These results aid in understanding the transmission dynamics of sylvatic DENV non-human primates, an issue of growing importance as dengue vaccines become available. [Copyright &y& Elsevier]