Dengue Virus Genome Research Articles

Discovering recent selection forces shaping the evolution of dengue viruses based on polymorphism data across geographic scales.

Incomplete selection makes it challenging to infer selection on genes at short time scales, especially for microorganisms, due to stronger linkage between loci. However, in many cases, the selective force changes with environment, time, or other factors, and it is of great interest to understand selective forces at this level to answer relevant biological questions. We developed a new method that uses the change in dN /dS , instead of the absolute value of dN /dS , to infer the dominating selective force based on sequence data across geographical scales. If a gene was under positive selection, dN /dS was expected to increase through time, whereas if a gene was under negative selection, dN /dS was expected to decrease through time. Assuming that the migration rate decreased and the divergence time between samples increased from between-continent, within-continent different-country, to within-country level, dN /dS of a gene dominated by positive selection was expected to increase with increasing geographical scales, and the opposite trend was expected in the case of negative selection. Motivated by the McDonald-Kreitman (MK) test, we developed a pairwise MK test to assess the statistical significance of detected trends in dN /dS . Application of the method to a global sample of dengue virus genomes identified multiple significant signatures of selection in both the structural and non-structural proteins. Because this method does not require allele frequency estimates and uses synonymous mutations for comparison, it is less prone to sampling error, providing a way to infer selection forces within species using publicly available genomic data from locations over broad geographical scales.

Effect of Human microRNA on Dengue Virus Genome: a cross-specie approach

Dengue virus infection being a menace worldwide particularly in West Bengal demanded investigation of a new therapeutic approach. In our work we have performed in silico analysis of target specificity of miRNA to dengue virus genome. Several human miRNAs like hsa-miR-4758-3p_MIMAT0019904, hsa-miR-6760-3p_MIMAT0027421, hsa-miR-6839-3p_MIMAT0027581, hsa-miR-6842-3p_MIMAT0027587 are found to be directly interacting with dengue virus proteins like Envelope, NS1, NS3 and 3’UTR region. The target proteins of respective dengue proteins were analysed using stringDB and Cytoscape. Analysis of the target protein interaction network revealed ACTB, AKT1, TLR4 as few of the hub genes that can be indirectly regulated by miRNA and dengue genome interaction. Thus our work focuses on finding novel miRNA that are not known to regulate dengue infection till date and proposes a novel therapeutic strategy.

A new host-targeted antiviral cyclolignan (SAU-22.107) for Dengue Virus infection in cell cultures. Potential action mechanisms based on cell imaging

Dengue virus (DENV) infection is the most arbovirosis in the world. However, medications have not been approved for its treatment. Drug discovery based on the host-targeted antiviral (HTA) constitutes a new promising strategy, considering their high genetic barrier to resistance and the low probability of selecting drug resistance strains. In this study, we have tested fifty-seven podophyllotoxin-related cyclolignans on DENV-2 infected cells and found the most promising compound was S.71. Using cellular and molecular biology experiments, we have discovered that the new lignan altered the distribution of microtubules, induced changes in cell morphology, and caused retraction of the rough endoplasmic reticulum. In addition, the compound alters the viral envelope protein and the double-stranded RNA, while there is a decrease in negative-strand RNA synthesis; especially when the compound was added between 6- and 12-hours post-infection. Altogether, S.71 decreases the viral yield through an HTA-related mechanism of action, possibly altering the DENV genome replication and/or polyprotein translation, through the alteration of microtubule distribution and endoplasmic reticulum deterioration. Finally, pharmacokinetic predictors show that S.71 falls within the standard ranges established for drugs.

Complete genome sequences and phylogenetic analysis of dengue virus in Southern Vietnam during 2014-2015

Dengue is an infectious disease that causes a worldwide health and economic burden despite the efforts to eradicate the disease. From 2013 to 2015, dengue epidemic significantly increased from 33,626 to 50,205 cases in Vietnam. This study aims to determine the genotype variations of dengue virus (DENV) circulating in Southern Vietnam during 2014-2015. C6/36 cells were infected with twenty-four strains of dengue virus isolated in 2014-2015 and kept frozen. The complete nucleotide sequence of dengue virus genomes was obtained by polymerase chain reaction (PCR). The genome was sequenced in the MiSeq system and analyzed by the basic local alignment search tool (BLAST) program. Data from GeneBank was used to create the phylogenetic trees. Among the 17 analyzed strains from 8 southern provinces, four (23.53%) were DENV-1, three (17.65%) were DENV-2, five (29.41%) were DENV-3, and five (29.41%) DENV-4 were isolated. Four DENV-1 isolates belong to Asia genotype. Three DENV-2 strains were concentrated in a subgroup of Asian 1 genotype. Five DENV-3 isolates were identified as belonged to Asian 2 genotype and five DENV-4 isolates were found as belong to Asia 1 genotype. There were no amino acid mutations and the transition capacity between the nucleotide among four types of DENV serotypes suggested that the probability of conversion from C to T was the highest conversion rate. These DENV isolates were genetically close to other previous strains isolated from Vietnam and its neighboring countries, including Thailand, China, Cambodia, and Singapore, Brazil, Sri Lanka due to dynamic transmission.

A Serotype-Specific and Multiplex PCR Method for Whole-Genome Sequencing of Dengue Virus Directly from Clinical Samples.

ABSTRACTDengue virus (DENV) is the most globally prevalent member of the genus Flavivirus in the family Flaviviridae, which can be classified into four serotypes. Historically, molecular epidemiological studies of DENV depended on E gene sequencing. The development of next-generation sequencing (NGS) allowed its application to viral whole-genome sequencing (WGS). In this study, we report the improvement of the existing WGS process for DENV by optimizing the primer design procedure, designing serotype-specific primer panels and reducing the sizes of amplicons. A total of 31 DENV-positive serum samples belonging to 4 serotypes and 9 genotypes of DENV were involved in the validation of the primer panels. The threshold cycle (CT) values of these samples ranged from 23.91 to 35.11. The validation results showed that the length of consensus sequences generated at a coverage depth of 20× or more ranged from 10,370 to 10,672 bp, with 100.00% coverage of the open reading frames and 97.34% to 99.52% coverage of the DENV genome. The amplification efficiency varied across amplicons, genotypes, and serotypes of DENVs. These results indicate that the serotype-specific primer panels allow users to obtain the whole genome of DENV directly from clinical samples, providing a universal, rapid, and effective tool for the integration of genomics with dengue surveillance.IMPORTANCE Dengue virus (DENV) is becoming the most globally prevalent arbovirus. The number of people living under the threat of DENV is increasing year by year. With the development of next-generation sequencing (NGS) technology, whole-genome sequencing (WGS) has been more and more widely used in infectious disease surveillance and molecular epidemiological studies. DENV population sequencing by NGS can increase our understanding of the changing epidemiology and evolution of the DENV genome at the molecular level, which demands universal primer panels and combination with NGS platforms. Multiplex PCR with a short-amplicon approach proved superior for amplifying viral genomes from clinical samples, particularly when the viral RNA was present at low concentrations. Additionally, DENV are known for their genetic diversity within serotype groups and geographical regions, so the primer panels we designed focused on universality, which would be useful in future local DENV outbreaks.

Towards the diagnosis of dengue virus and its serotypes using designed CRISPR/Cas13 gRNAs.

Dengue Virus (DENV) is a mosquito-borne virus that is prevalent in the world's tropical and subtropical regions. Therefore, early detection and surveillance can help in the management of this disease. Current diagnostic methods rely primarily on ELISA, PCR, and RT-PCR, among others, which can only be performed in specialized laboratories and require sophisticated instruments and technical expertise. CRISPR-based technologies on the other hand have field-deployable viral diagnostics capabilities that could be used in the development of point-of-care molecular diagnostics. The first step in the field of CRISPR-based virus diagnosis is to design and screen gRNAs for high efficiency and specificity. In the present study, we employed a bioinformatics approach to design and screen DENV CRISPR/Cas13 gRNAs for conserved and serotype-specific variable genomic regions in the DENV genome. We identified one gRNA sequence specific for each of the lncRNA and NS5 regions and identified one gRNA against each of DENV1, DENV2, DENV3, and DENV4 to distinguish the four DENV serotypes. These CRISPR/Cas13 gRNA sequences will be useful in diagnosing the dengue virus and its serotypes for in vitro validation and diagnostics.

Dengue virus NS4B protein as a target for developing antivirals.

Dengue virus is an important pathogen affecting global population while no specific treatment is available against this virus. Effort has been made to develop inhibitors through targeting viral nonstructural proteins such as NS3 and NS5 with enzymatic activities. No potent inhibitors entering clinical studies have been developed so far due to many challenges. The genome of dengue virus encodes four membrane-bound nonstructural proteins which do not possess any enzymatic activities. Studies have shown that the membrane protein-NS4B is a validated target for drug discovery and several NS4B inhibitors exhibited antiviral activities in various assays and entered preclinical studies.. Here, we summarize the recent studies on dengue NS4B protein. The structure and membrane topology of dengue NS4B derived from biochemical and biophysical studies are described. Function of NS4B through protein-protein interactions and some available NS4B inhibitors are summarized. Accumulated studies demonstrated that cell-based assays play important roles in developing NS4B inhibitors. Although the atomic structure of NS4B is not obtained, target-based drug discovery approach become feasible to develop NS4B inhibitors as recombinant NS4B protein is available.

Diagnosis and serotyping of dengue using cost-effective high-resolution melting curve based real time PCR

Dengue virus (DENV) is classified into four serotypes: DENV-1, DENV-2, DENV-3, and DENV-4 which are genetically-related but antigenically distinct types. Although primary infection with a serotype of DENV gives lifelong immunity to that serotype, the secondary infection with other serotype leads to the development of dengue hemorrhagic fever and dengue shock syndrome. Serological tests that can detect NS1 antigen on the day one of illness is nonspecific and less sensitive to diagnose dengue in the first week of illness. IgM and IgG antibody detection may not be positive until the later part of first week. Hence it is necessary to develop a nucleic acid-based test kit for detection of DENV in the early phase of the disease such that the complications can be prevented. Simultaneous detection of serotype of DENV helps in understanding the epidemiology of the disease. In the present study, a single primer pair was designed to 3′ untranslated region of DENV genome. After amplification, the melting temperature (Tm) for DENV-1, DENV-2, DENV-3 and DENV-4 was found to be 83.96 ± 0.58 ˚C, 81.99± 0.26 ˚C, 84.54± 0.15 ˚C, and 83.28 ± 0.12 ˚C, respectively. 

Transcriptional and Translational Dynamics of Zika and Dengue Virus Infection.

Zika virus (ZIKV) and dengue virus (DENV) are members of the Flaviviridae family of RNA viruses and cause severe disease in humans. ZIKV and DENV share over 90% of their genome sequences, however, the clinical features of Zika and dengue infections are very different reflecting tropism and cellular effects. Here, we used simultaneous RNA sequencing and ribosome footprinting to define the transcriptional and translational dynamics of ZIKV and DENV infection in human neuronal progenitor cells (hNPCs). The gene expression data showed induction of aminoacyl tRNA synthetases (ARS) and the translation activating PIM1 kinase, indicating an increase in RNA translation capacity. The data also reveal activation of different cell stress responses, with ZIKV triggering a BACH1/2 redox program, and DENV activating the ATF/CHOP endoplasmic reticulum (ER) stress program. The RNA translation data highlight activation of polyamine metabolism through changes in key enzymes and their regulators. This pathway is needed for eIF5A hypusination and has been implicated in viral translation and replication. Concerning the viral RNA genomes, ribosome occupancy readily identified highly translated open reading frames and a novel upstream ORF (uORF) in the DENV genome. Together, our data highlight both the cellular stress response and the activation of RNA translation and polyamine metabolism during DENV and ZIKV infection.

N6-methyladenosine modification of the Aedes aegypti transcriptome and its alteration upon dengue virus infection in Aag2 cell line

The N6-methyladenosine (m6A) modification of RNA has been reported to affect viral infections. Studies have confirmed the role of m6A in replication of several vector-borne flaviviruses, including dengue virus (DENV), in mammalian cells. Here, we explored the role of m6A in DENV replication in the mosquito Aedes aegypti Aag2 cell line. We first determined the presence of m6A on the RNAs from mosquito cells and using methylated RNA immunoprecipitation and sequencing (MeRIP-Seq) identified m6A modification of the mosquito transcriptome and those that changed upon DENV infection. Depletion of m6A methyltransferases and the m6A binding protein YTHDF3 RNAs decreased the replication of DENV. In particular, we found that the Ae. aegypti ubiquitin carrier protein 9 (Ubc9) is m6A modified and its expression increases after DENV infection. Silencing of the gene and ectopic expression of Ubc9 led to reduced and increased DENV replication, respectively. The abundance of Ubc9 mRNA and its stability were reduced with the inhibition of m6A modification, implying that m6A modification of Ubc9 might enhance expression of the gene. We also show that the genome of DENV is m6A modified at five sites in mosquito cells. Altogether, this work reveals the involvement of m6A modification in Ae. aegypti-DENV interaction.

Retrospective Genomic Characterization of a 2017 Dengue Virus Outbreak, Burkina Faso

Knowledge of contemporary genetic composition of dengue virus (DENV) in Africa is lacking. By using next-generation sequencing of samples from the 2017 DENV outbreak in Burkina Faso, we isolated 29 DENV genomes (5 serotype 1, 16 serotype 2 [DENV-2], and 8 serotype 3). Phylogenetic analysis demonstrated the endemic nature of DENV-2 in Burkina Faso. We noted discordant diagnostic results, probably related to genetic divergence between these genomes and the Trioplex PCR. Forward and reverse1 primers had a single mismatch when mapped to the DENV-2 genomes, probably explaining the insensitivity of the molecular test. Although we observed considerable homogeneity between the Dengvaxia and TetraVax-DV-TV003 vaccine strains as well as B cell epitopes compared with these genomes, we noted unique divergence. Continual surveillance of dengue virus in Africa is needed to clarify the ongoing novel evolutionary dynamics of circulating virus populations and support the development of effective diagnostic, therapeutic, and preventive countermeasures.

The helper oligonucleotides enable detection of folded single-stranded DNA by lateral flow immunoassay after HCR signal amplification

A combination of Hybridization Chain Reaction (HCR) and Lateral Flow Immunoassay (LFIA) is an attractive strategy for a simple signal amplification DNA/RNA detection. The present study aimed to report a strategy used to solve a problem encountered when the target DNA contained folded secondary structure during HCR, enabling HCR hairpin probes to easily access the target site. The 24-nt conserved sequence within 3′-UTR, present only in dengue virus genome but not in other species, is an ideal target to use as a probe binding site for pan-dengue virus detection. Thus, the 105-nt target containing the 24-nt target sequence was chosen as a target with secondary structures.The 24-nucleotide (nt) synthetic target DNA successfully induced HCR reaction within 5 min at room temperature. However, the HCR detection of the 105-nt synthetic target DNA with secondary structures was problematic. The probe hybridization was prevented by the secondary structures of the target, resulting in a failure to generate HCR product. To solve this problem, two helper oligonucleotides (helper1 and helper2) were designed to linearize the folded structure of the 105-nt target through strand-displacement mechanism, allowing the HCR hairpin probes to easily access the target site. The HCR product with the labeled helper oligonucleotides and the labeled probes were successfully detected by LFIA. With this strategy, the combination of the helper-enhanced HCR and LFIA exhibited a limit of detection (LOD) in a nanomolar range of the 105-nt DENV synthetic target DNA. Our study demonstrated that signal amplification by the combination of HCR and LFIA could successfully detect the target DNA with secondary structure, but not target RNA with secondary structure.In summary, this work provided a proof of concept of two main issues including probe hybridization enhancement by helper oligonucleotide for the target with complicated secondary structure and the advantage of a combination of labeled helper and HCR probes design for LFIA to overcome the false positive result from HCR probe leakage. Our findings on the use of helper oligonucleotides may be beneficial for the development of other isothermal amplification, since the secondary structure of the target is one of the major obstacles among hybridization-based methods.

Intra-genomic heterogeneity in CpG dinucleotide composition in dengue virus

PurposeDengue virus is a life-threatening virus and cases of dengue infection have been increasing steadily in the past decades causing millions of deaths every year. So far, there is no vaccine that works effectively on all serotypes. Recently, CpG-recoded vaccines have proved to be effective against few viruses. MethodsIn this study, evaluation and interpretation of more than 4547 Dengue virus genome sequences were included for analyzing novel CpG dinucleotides rich regions which are shared amid all serotypes. Genomic regions of DENV were synonymously CpG recoded using in silico methods and analyzed for adaptation in both human and Aedes spp. hosts based on CAI scores. ResultsThe analysis mirrored that serotypes 1, 3, and 4 shared CpG islands present in common regions. DENV-2 CpG islands showed no similarity with any of the CpG islands present in other serotypes. While DENV-3 sequences were found to possess the maximum number of conserved CpG islands stretches; DENV-2 was found to possess the lowest number. We found that all serotypes (with an exception of serotype 2) have CpG island in their 3′ UTR. In silico CpG recoding of DENV genomic regions resulted in ∼ 3 fold increase of CpG dinucleotide frequency and comparative analysis based on CAI scores showed decreased adaptive fitness of CpG recoded DENV inside human host. ConclusionThese CG-dinucleotide-enriched RNA sequences can be targeted by ZAP (zinc-finger antiviral protein) which can differentiate between host mRNA and viral mRNA. Our in silico findings can further be exploited for CpG-recoding of DENV genomes which can evoke cellular and humoral immune responses by recruiting ZAP-induced RNA degradation machinery and hence providing a promising approach for vaccine development.

A novel strategy for developing a tetravalent vaccine (dvac) against dengue utilizing conserved regions from all DENV proteins

Dengue fever is a global health issue which is infecting millions of people each year and number of reported infections are constantly increasing. Though the only commercialized vaccine i.e. dengvaxia has banned in several countries due to its potential health risk, overall vaccine holds promising potential against viruses. In this study, we have developed a novel formulation of multi-epitope peptide vaccine (dvac), which utilizes peptides from each dengue protein with >80% sequence conservancy within each serotype and their respective genotypes. Simultaneous utilization of all dengue proteins and their conservancy among dengue virus genome is targeted to evoke balanced immunity against dengue serotypes without eliciting antibody-dependent enhancement and antigenic sin like response, which are primarily responsible for severe dengue fever. Immunoinformatic approaches are used to identify the potential of dvac in inducing cytotoxic T-lymphocytes, helper T-lymphocytes, Interleukin-4, Interferon-gamma and B-cell immune responses without inducing allergic responses. Cross-reactivity of dvac with human cellular machinery is also taken into consideration to avoid any cross-reactive pathogenicity. Furthermore, interaction of dvac with immune receptors i.e. toll-like receptors (TLR3 and TLR4) using molecular docking studies revealed favorable interaction between synthetic peptide and immune receptors. Our findings suggest that designed multi-epitope peptide holds great potential to evoke balanced immunity against all dengue serotypes without eliciting any significant harmful side-effects.

Magnetic Field-Enhanced Agglutination Readout Combined With Isothermal Reverse Transcription Recombinase Polymerase Amplification for Rapid and Sensitive Molecular Detection of Dengue Virus.

Among the numerous molecular diagnostic methods, isothermal reverse transcription recombinase polymerase amplification (RT-RPA) is a simple method that has high sensitivity and avoids the use of expensive instruments. However, detection of amplified genomes often requires a fluorescence readout on costly readers or migration on a lateral flow strip with a subjective visual reading. Aiming to establish a new approach to rapidly and sensitively detect viruses, we combined RT-RPA with a magnetic field-enhanced agglutination (MFEA) assay and assessed the ability of this method to detect the dengue virus (DENV). Magnetization cycles accelerated the capture of amplified DENV genomes between functionalized magnetic nanoparticles by a fast chaining process to less than 5 min; the agglutination was quantified by simple turbidimetry. A total of 37 DENV RNA+ and 30 DENV RNA− samples were evaluated with this combined method. The sensitivity and specificity were 89.19% (95% CI, 72.75–100.00%) and 100% (95% CI, 81.74–100.00%), respectively. This approach provides a solution for developing innovative diagnostic assays for the molecular detection of emerging infections.

Transgenic refractory Aedes aegypti lines are resistant to multiple serotypes of dengue virus

The areas where dengue virus (DENV) is endemic have expanded rapidly, driven in part by the global spread of Aedes species, which act as disease vectors. DENV replicates in the mosquito midgut and is disseminated to the mosquito’s salivary glands for amplification. Thus, blocking virus infection or replication in the tissues of the mosquito may be a viable strategy for reducing the incidence of DENV transmission to humans. Here we used the mariner Mos1 transposase to create an Aedes aegypti line that expresses virus-specific miRNA hairpins capable of blocking DENV replication. These microRNA are driven by the blood-meal-inducible carboxypeptidase A promoter or by the polyubiquitin promoter. The transgenic mosquitoes exhibited significantly lower infection rates and viral titers for most DENV serotypes 7 days after receiving an infectious blood meal. The treatment was also effective at day 14 post infection after a second blood meal had been administered. In viral transmission assay, we found there was significantly reduced transmission in these lines. These transgenic mosquitoes were effective in silencing most of the DENV genome; such an approach may be employed to control a dengue fever epidemic.

Exploiting Dengue Virus Protease as a Therapeutic Target: Current Status, Challenges and Future Avenues.

Dengue, the oldest and the most prevalent mosquito-borne illness, is caused by the dengue virus (DENV), from the family of Flaviviridae. It infects approximately 400 million individuals per annum, with approximately half of the global population residing in high-risk areas. The factors attributed to the geographic expansion of dengue, include urbanization, population density, modern means of transportation, international travels, habit modification, climate change, virus genetics, vector capacity, and poor vector control. Despite the significant progress made in the past against dengue, no effective antiviral therapy is currently available. Among the structural and non-structural proteins encoded by DENV genome, the NS2B-NS3 protease complex is amongst the extensively studied targets for the development of antiviral therapeutics owing to its multiple roles in virus life cycle. Furthermore, protease inhibitors were found to be successful in Hepatitis C Virus (HCV) and Human Immunodeficiency Virus (HIV). Likewise, several peptidic, peptide derived/peptidomimetic, and small molecules inhibitors have been identified as DENV protease inhibitors. Unfortunately, none of them have resulted in a clinically approved drug. Considering all the abovementioned facts, this review descriptively explains the molecular mechanism and therapeutic potential of DENV protease along with an up to date information on various competitive inhibitors reported against DENV protease. This review might be helpful for the researchers working in this area to understand the critical aspects of DENV protease that will help them develop effective and novel inhibitors against DENV to protect lives of millions of people worldwide.

Tracing and tracking the emergence, epidemiology and dispersal of dengue virus to Africa during the 20th century

The four mosquito-borne dengue virus serotypes (DENV1–DENV4) cause a high burden of disease throughout the tropical and sub-tropical regions of the world. Nevertheless, their precise epidemiological history in Africa, including when and where they originated and were distributed during the 20th century, remains unclear stressing the need for One Health focused research. Accordingly, we conducted a time-scaled molecular epidemiological reconstruction using publicly available and newly sequenced dengue virus genomes of African origin representing all four serotypes to deduce the most likely temporal and spatial transmission routes of each DENV serotype from their ancestral regions to, within and from Africa.Our analyses suggest that during the 20th century, serotypes DENV1–DENV3 were introduced to Africa from South East Asia on multiple occasions. The earliest evidence recorded indicates introduction of DENV2 during the early-1940s and of DENV1 during the mid-1940s to Western Africa from South East Asia. The analysis also implies an early introduction of DENV4 during the mid-1940s to Western Africa, alongside DENV1, probably originating in South East Asia. Establishment of DENV3 in Africa appears to have occurred later in the 1960s, apparently originating from South East Asia. However, with the re-establishment of DENV in the Americas, following the cessation of the PAHO mosquito control programme during the mid-20th century, evidence of introductions of DENV1 and DENV2 from the Americas to Western Africa was also observed. The data also identify intra-regional circulation of DENV, but also inter-regional dispersal of all four serotypes within Africa, which has led to a high degree of geographical overlap among serotypes. It is also noteworthy that DENV from both Eastern and Western Africa, have been introduced into Central Africa but there is no support for the converse relationship. For serotypes DENV1–DENV3, we observed probable exports from within established African DENV clusters (≥2 sequences) primarily to Eastern and Southern Asia.Collectively, our findings support the view that all DENV serotypes, apart from DENV4, have been introduced on multiple occasions to Africa, primarily originating from South East Asia, and subsequently to neighbouring regions within Africa.

Blood glucose promotes dengue virus infection in the mosquito Aedes aegypti

BackgroundDengue fever is the most rapidly spreading mosquito-borne viral disease globally. More than 2.5 billion people live in dengue-endemic areas. Previous studies suggested an interrelationship between diabetes mellitus (DM) and dengue hemorrhagic fever (DHF). Conversely, glycolysis is a critical metabolic pathway for optimal dengue virus (DENV) replication. However, little is known concerning the effect of glucose on DENV replication in mosquitoes. In this study, we investigated the impact of glucose on DENV replication in mosquitoes Aedes aegypti.MethodsMosquitoes (Ae. aegypti UGAL/Rockefeller strain) were orally infected with DENV (serotype 2, 16681 strain) through infectious blood feeding. The DENV infection and transmission rates were determined by examining mosquito bodies and saliva, respectively, for DENV positivity at different time points after infection. In addition, a reverse genetic approach was applied by introducing double-stranded RNA against genes of interest into the mosquitoes to inhibit gene expression.ResultsOur data revealed a significant increase of DENV genome levels in mosquitoes consuming an infectious blood meal supplemented with glucose, suggesting that blood glucose is an important factor for viral replication. Interestingly, a significant increase of DENV E protein levels was detected in the saliva 4 days faster in mosquitoes that consumed infectious blood meals supplemented with glucose than in those consuming infectious blood meals alone. Furthermore, we perform RNAi to silence AKT or TOR and investigate the molecular mechanism regulating the glucose-mediated enhancement of viral replication. Silencing of AKT or TOR significantly reduced DENV titers in mosquitoes.ConclusionsThis study suggested that blood glucose is beneficial to DENV replication and that it facilitates virus transmission in mosquitoes via AKT and TOR signaling. Therefore, our results strengthen our understanding of dengue fever and DM co-morbidity and possibly reveal new targets for specific antiviral therapies.Graphical abstract

Specific Interaction of DDX6 with an RNA Hairpin in the 3' UTR of the Dengue Virus Genome Mediates G1 Phase Arrest.

The extent to which viral genomic RNAs interact with host factors and contribute to host response and disease pathogenesis is not well known. Here, we report that the human RNA helicase DDX6 specifically binds to the viral most conserved RNA hairpin in the A3 element in the dengue 3' UTR, with nanomolar affinities. DDX6 CLIP confirmed the interaction in HuH-7 cells infected by dengue virus serotype 2. This interaction requires three conserved residues-Lys307, Lys367, and Arg369-as well as the unstructured extension in the C-terminal domain of DDX6. Interestingly, alanine substitution of these three basic residues resulted in RNA-independent ATPase activity, suggesting a mechanism by which RNA-binding and ATPase activities are coupled in DEAD box helicases. Furthermore, we applied a cross-omics gene enrichment approach to suggest that DDX6 is functionally related to cell cycle regulation and viral pathogenicity. Indeed, infected cells exhibited cell cycle arrest in G1 phase and a decrease in the early S phase. Exogenous expression of intact DDX6, but not A3-binding-deficient mutants, alleviated these effects by rescue of the DNA preinitiation complex expression. Disruption of the DDX6-binding site was found in dengue and Zika live-attenuated vaccine strains. Our results suggested that dengue virus has evolved an RNA aptamer against DDX6 to alter host cell states and defined DDX6 as a new regulator of G1/S transition. IMPORTANCE Dengue virus (DENV) is transmitted by mosquitoes to humans, infecting 390 million individuals per year globally. About 20% of infected patients shows a spectrum of clinical manifestation, ranging from a mild flu-like syndrome, to dengue fever, to life-threatening severe dengue diseases, including dengue hemorrhagic fever and dengue shock syndrome. There is currently no specific treatment for dengue diseases, and the molecular mechanism underlying dengue pathogenesis remains poorly understood. In this study, we combined biochemical, bioinformatics, high-content analysis and RNA sequencing approaches to characterize a highly conserved interface of the RNA genome of DENV with a human factor named DDX6 in infected cells. The significance of our research is in identifying the mechanism for a viral strategy to alter host cell fates, which conceivably allows us to generate a model for live-attenuated vaccine and the design of new therapeutic reagent for dengue diseases.