Dengue Virus Genome Research Articles

Dengue virus genomic surveillance in the applying Wolbachia to eliminate dengue trial reveals genotypic efficacy and disruption of focal transmission

Release of Aedes aegypti mosquitoes infected with Wolbachia pipientis (wMel strain) is a biocontrol approach against Ae. aegypti-transmitted arboviruses. The Applying Wolbachia to Eliminate Dengue (AWED) cluster-randomised trial was conducted in Yogyakarta, Indonesia in 2018–2020 and provided pivotal evidence for the efficacy of wMel-Ae. aegypti mosquito population replacement in significantly reducing the incidence of virologically-confirmed dengue (VCD) across all four dengue virus (DENV) serotypes. Here, we sequenced the DENV genomes from 318 dengue cases detected in the AWED trial, with the aim of characterising DENV genetic diversity, measuring genotype-specific intervention effects, and inferring DENV transmission dynamics in wMel-treated and untreated areas of Yogyakarta. Phylogenomic analysis of all DENV sequences revealed the co-circulation of five endemic DENV genotypes: DENV-1 genotype I (12.5%) and IV (4.7%), DENV-2 Cosmopolitan (47%), DENV-3 genotype I (8.5%), and DENV-4 genotype II (25.7%), and one recently imported genotype, DENV-4 genotype I (1.6%). The diversity of genotypes detected among AWED trial participants enabled estimation of the genotype-specific protective efficacies of wMel, which were similar (± 10%) to the point estimates of the respective serotype-specific efficacies reported previously. This indicates that wMel afforded protection to all of the six genotypes detected in Yogyakarta. We show that within this substantial overall viral diversity, there was a strong spatial and temporal structure to the DENV genomic relationships, consistent with highly focal DENV transmission around the home in wMel-untreated areas and a near-total disruption of transmission by wMel. These findings can inform long-term monitoring of DENV transmission dynamics in Wolbachia-treated areas including Yogyakarta.

Genomic surveillance reveals a dengue 2 virus epidemic lineage with a marked decrease in sensitivity to Mosnodenvir

Dengue fever is the most important arbovirosis for public health, with more than 5 million cases worldwide in 2023. Mosnodenvir is the first anti-dengue compound with very high preclinical pan-serotype activity, currently undergoing phase 2 clinical evaluation. Here, by analyzing dengue virus (DENV) genomes from the 2023-2024 epidemic in the French Caribbean Islands, we show that they all exhibit mutation NS4B:V91A, previously associated with a marked decrease in sensitivity to mosnodenvir in vitro. Using antiviral activity tests on four clinical and reverse-genetic strains, we confirm a marked decrease in mosnodenvir sensitivity for DENV-2 ( > 1000 fold). Finally, combining phylogenetic analysis and experimental testing for resistance, we find that virus lineages with low sensitivity to mosnodenvir due to the V91A mutation likely emerged multiple times over the last 30 years in DENV-2 and DENV-3. These results call for increased genomic surveillance, in particular to track lineages with resistance mutations. These efforts should allow to better assess the activity profile of DENV treatments in development against circulating strains.

Cryptic transmission and novel introduction of Dengue 1 and 2 genotypes in Colombia.

Dengue fever remains as a public health challenge in Colombia, standing as the most prevalent infectious disease in the country. The cyclic nature of dengue epidemics, occurring approximately every 3years, is intricately linked to meteorological events like El Niño Southern Oscillation (ENSO). Therefore, the Colombian system faces challenges in genomic surveillance. This study aimed to evaluate local dengue virus (DENV) transmission and genetic diversity in four Colombian departments with heterogeneous incidence patterns (department is first-level territorial units in Colombia). For this study, we processed 266 serum samples to identify DENV. Subsequently, we obtained 118 genome sequences by sequencing DENV genomes from serum samples of 134 patients infected with DENV-1 and DENV-2 serotypes. The predominant serotype was DENV-2 (108/143), with the Asian-American (AA) genotype (91/118) being the most prevalent one. Phylogenetic analysis revealed concurrent circulation of two lineages of both DENV-2 AA and DENV-1 V, suggesting ongoing genetic exchange with sequences from Venezuela and Cuba. The continuous migration of Venezuelan citizens into Colombia can contribute to this exchange, emphasizing the need for strengthened prevention measures in border areas. Notably, the time to most recent common ancestor analysis identified cryptic transmission of DENV-2 AA since approximately 2015, leading to the recent epidemic. This challenges the notion that major outbreaks are solely triggered by recent virus introductions, emphasizing the importance of active genomic surveillance. The study also highlighted the contrasting selection pressures on DENV-1 V and DENV-2 AA, with the latter experiencing positive selection, possibly influencing its transmissibility. The presence of a cosmopolitan genotype in Colombia, previously reported in Brazil and Peru, raises concerns about transmission routes, emphasizing the necessity for thorough DENV evolution studies. Despite limitations, the study underscores genomic epidemiology's crucial role in early detection and comprehension of DENV genotypes, recommending the use of advanced sequencing techniques as an early warning system to help prevent and control dengue outbreaks in Colombia and worldwide.

Climate, demography, immunology, and virology combine to drive two decades of dengue virus dynamics in Cambodia

The incidence of dengue virus disease has increased globally across the past half-century, with highest number of cases ever reported in 2019 and again in 2023. We analyzed climatological, epidemiological, and phylogenomic data to investigate drivers of two decades of dengue in Cambodia, an understudied endemic setting. Using epidemiological models fit to a 19-y dataset, we first demonstrate that climate-driven transmission alone is insufficient to explain three epidemics across the time series. We then use wavelet decomposition to highlight enhanced annual and multiannual synchronicity in dengue cycles between provinces in epidemic years, suggesting a role for climate in homogenizing dynamics across space and time. Assuming reported cases correspond to symptomatic secondary infections, we next use an age-structured catalytic model to estimate a declining force of infection for dengue through time, which elevates the mean age of reported cases in Cambodia. Reported cases in >70-y-old individuals in the 2019 epidemic are best explained when also allowing for waning multitypic immunity and repeat symptomatic infections in older patients. We support this work with phylogenetic analysis of 192 dengue virus (DENV) genomes that we sequenced between 2019 and 2022, which document emergence of DENV-2 Cosmopolitan Genotype-II into Cambodia. This lineage demonstrates phylogenetic homogeneity across wide geographic areas, consistent with invasion behavior and in contrast to high phylogenetic diversity exhibited by endemic DENV-1. Finally, we simulate an age-structured, mechanistic model of dengue dynamics to demonstrate how expansion of an antigenically distinct lineage that evades preexisting multitypic immunity effectively reproduces the older-age infections witnessed in our data.

Deep learning guided prediction modeling of dengue virus evolving serotype

Evolution remains an incessant process in viruses, allowing them to elude the host immune response and induce severe diseases, impacting the diagnostic and vaccine effectiveness. Emerging and re-emerging diseases are among the significant public health concerns globally. The revival of dengue is mainly due to the potential for naturally arising mutations to induce genotypic alterations in serotypes. These transformations could lead to future outbreaks, underscoring the significance of studying DENV evolution in endemic regions. Predicting the emerging Dengue Virus (DENV) genome is crucial as the virus disrupts host cells, leading to fatal outcomes. Deep learning has been applied to predict dengue fever cases; there has been relatively less emphasis on its significance in forecasting emerging DENV serotypes. While Recurrent Neural Networks (RNN) were initially designed for modeling temporal sequences, our proposed DL-DVE generative and classification model, trained on complete genome data of DENV, transcends traditional approaches by learning semantic relationships between nucleotides in a continuous vector space instead of representing the contextual meaning of nucleotide characters. Leveraging 2000 publicly available DENV complete genome sequences, our Long Short-Term Memory (LSTM) based generative and Feedforward Neural Network (FNN) based classification DL-DVE model showcases proficiency in learning intricate patterns and generating sequences for emerging serotype of DENV. The generated sequences were analyzed along with available DENV serotype sequences to find conserved motifs in the genome through MEME Suite (version 5.5.5). The generative model showed an accuracy of 93 %, and the classification model provided insight into the specific serotype label, corroborated by BLAST search verification. Evaluation metrics such as ROC-AUC value 0.818, accuracy, precision, recall and F1 score, all to be around 99.00 %, demonstrating the classification model's reliability. Our model classified the generated sequences as DENV-4, exhibiting 65.99 % similarity to DENV-4 and around 63–65 % similarity with other serotypes, indicating notable distinction from other serotypes. Moreover, the intra-serotype divergence of sequences with a minimum of 90 % similarity underscored their uniqueness.

Inhibition of NS2B-NS3 protease from all four serotypes of dengue virus by punicalagin, punicalin and ellagic acid identified from Punica granatum

Despite a major threat to the public health in tropical and subtropical regions, dengue virus (DENV) infections are untreatable. Therefore, efforts are needed to investigate cost-effective therapeutic agents that could cure DENV infections in future. The NS2B-NS3 protease encoded by the genome of DENV is considered a critical target for the development of anti-dengue drugs. The objective of the current study was to find out a specific inhibitor of the NS2B-NS3 proteases from all four serotypes of DENV. To begin with, nine plant extracts with a medicinal history were evaluated for their role in inhibiting the NS2B-NS3 proteases by Fluorescence Resonance Energy Transfer (FRET) assay. Among the tested extracts, Punica granatum was found to be the most effective one. The metabolic profiling of this extract revealed the presence of several active compounds, including ellagic acid, punicalin and punicalagin, which are well-established antiviral agents. Further evaluation of IC50 values of these three antiviral molecules revealed punicalagin as the most potent anti-NS2B-NS3 protease drug with IC50 of 0.91 ± 0.10, 0.75 ± 0.05, 0.42 ± 0.03, 1.80 ± 0.16 µM against proteases from serotypes 1, 2, 3 and 4, respectively. The docking studies demonstrated that these compounds interacted at the active site of the enzyme, mainly with His and Ser residues. Molecular dynamics simulations analysis also showed the structural stability of the NS2B-NS3 proteases in the presence of punicalagin. In summary, this study concludes that the punicalagin can act as an effective inhibitor against NS2B-NS3 proteases from all four serotypes of DENV. Communicated by Ramaswamy H. Sarma

Direct Dengue Virus Genome Sequencing from Antigen NS1 Rapid Diagnostic Tests: A Proof-of-Concept with the Standard Q Dengue Duo Assay.

With nearly half of the world's population being at risk of infection, dengue virus represents a major global health issue. The use of dengue antigen rapid diagnostic tests (Ag-RDTs) represents an alternative to PCR methods for the diagnosis of acute infections since they display excellent sensitivities and specificities and can be performed outside the laboratory. The high genetic diversity of the dengue virus genome represents a challenge for vaccine development, and the progressive expansion of this virus into previously nonendemic regions justifies the implementation of a genomic surveillance program. In this proof-of-concept study, we show the feasibility of sequencing dengue virus genomes directly from positive Ag-RDT (Standard Q Dengue Duo Test assay, n = 7) cassettes stored up to 31 days at room temperature after testing. For 5 of the 7 samples, a high number of reads were obtained allowing phylogenetic analyses to be carried out to determine not only the serotypes (dengue 1, 2, 3 and 4 were detected) but also the genotypes. Furthermore, in one sample, our unbiased metagenomic next-generation sequencing approach made it possible to detect epizootic hemorrhagic disease virus sequences, an arthropod-transmitted virus in ruminants. To conclude, as such an approach requires no cold storage or freezing of samples, dengue Ag-RDTs represent a very pragmatic and robust alternative for the genomic surveillance of dengue virus.

Novel mutations in structural proteins of dengue virus genomes

BackgroundGenomic characterization of the dengue virus (DENV) is useful for understanding its molecular evolution, transmission, pathogenicity and infectivity. The DENV genomic RNA encodes three structural proteins, capsid (C) envelope (E) and membrane (M) proteins mediating viral entry and assembly during host infection. The current study aims to explore the DENV serotypes and mutations in the E and M proteins. MethodsTwenty-three samples of DENV-positive patients were processed and selected for whole genome sequencing (WGS) from the Punjab Province of Pakistan. ResultsAmong the 23 WGS, 19 samples showed numerous mutations (BioProject ID PRJNA943555). DENV1 and DENV2 are the most prevalent serotypes. A total of 179 mutations were detected in the E protein, in which K203E, T88A, I114L, and I293T are novel. The I270L, T272A, S273L, and T277A were found in the “kl” β-hairpin (aa 270–279). The M protein harbors 74 mutations, of which 24 were novel. Three prominent complementary regions in the prM and E protein complex formations include R6, E46, D47, D63, and D65 on ‘pr’ peptide, and E84, K64, and H244, K247 on E, remain conserved except R6C. To our knowledge, it is the first comprehensive study of mutations in structural proteins. ConclusionGenomic epidemiology is critical for analyzing emerging mutations and designing new policies therapeutic efforts for future outbreaks.

Convergent trends and spatiotemporal patterns of Aedes-borne arboviruses in Mexico and Central America.

Aedes-borne arboviruses cause both seasonal epidemics and emerging outbreaks with a significant impact on global health. These viruses share mosquito vector species, often infecting the same host population within overlapping geographic regions. Thus, comparative analyses of the virus evolutionary and epidemiological dynamics across spatial and temporal scales could reveal convergent trends. Focusing on Mexico as a case study, we generated novel chikungunya and dengue (CHIKV, DENV-1 and DENV-2) virus genomes from an epidemiological surveillance-derived historical sample collection, and analysed them together with longitudinally-collected genome and epidemiological data from the Americas. Aedes-borne arboviruses endemically circulating within the country were found to be introduced multiple times from lineages predominantly sampled from the Caribbean and Central America. For CHIKV, at least thirteen introductions were inferred over a year, with six of these leading to persistent transmission chains. For both DENV-1 and DENV-2, at least seven introductions were inferred over a decade. Our results suggest that CHIKV, DENV-1 and DENV-2 in Mexico share evolutionary and epidemiological trajectories. The southwest region of the country was determined to be the most likely location for viral introductions from abroad, with a subsequent spread into the Pacific coast towards the north of Mexico. Virus diffusion patterns observed across the country are likely driven by multiple factors, including mobility linked to human migration from Central towards North America. Considering Mexico's geographic positioning displaying a high human mobility across borders, our results prompt the need to better understand the role of anthropogenic factors in the transmission dynamics of Aedes-borne arboviruses, particularly linked to land-based human migration.

Dengue Fever Surveillance in Mato Grosso do Sul: Insights from Genomic Analysis and Implications for Public Health Strategies.

Since its discovery in early 1916, dengue fever, a common vector-borne illness in Brazil, has resulted in extensive urban outbreaks and poses a serious threat to the public's health. Understanding the dynamics of Dengue Virus (DENV) serotypes circulating in different regions of Brazil is essential for implementing effective disease control and prevention measures. In response to this urgent need, we conducted an on-site training program in genomic surveillance in collaboration with the Central Laboratory of Health and the Secretary of Health of the Mato Grosso do Sul state. This initiative resulted in the generation of 177 DENV genome sequences collected between May 2021 and May 2022, a period during which over 11,391 dengue fever cases were reported in the state. Through this approach, we were able to identify the co-circulation of two different dengue serotypes (DENV1 and DENV2) as well as the existence of diverse viral lineages within each genotype, suggesting that multiple introduction events of different viral strains occurred in the region. By integrating epidemiological data, our findings unveiled temporal fluctuations in the relative abundance of different serotypes throughout various epidemic seasons, highlighting the complex and changing dynamics of DENV transmission throughout time. These findings demonstrate the value of ongoing surveillance activities in tracking viral transmission patterns, monitoring viral evolution, and informing public health actions.

Structures of dengue virus RNA replicase complexes

Dengue is a mosquito-borne viral infection caused by dengue virus (DENV), a member of the flaviviruses. The DENV genome is a 5'-capped positive-sense RNA with a unique 5'-stem-loop structure (SLA), which is essential for RNA replication and 5' capping. The virus-encoded proteins NS5 and NS3 are responsible for viral genome replication, but the structural basis by which they cooperatively conduct the required tasks has remained unclear. Here, we report the cryoelectron microscopy (cryo-EM) structures of SLA-bound NS5 (PC), NS3-bound PC (PC-NS3), and an RNA-elongating NS5-NS3 complex (EC). While SLA bridges the NS5 methyltransferase and RNA-dependent RNA polymerase domains in PC, the NS3 helicase domain displaces it in elongation complex (EC). The SLA- and NS3-binding sites overlap with that of human STAT2. These structures illuminate the key steps in DENV genome replication, namely, SLA-dependent replication initiation, processive RNA elongation, and 5' capping of the nascent genomic RNA, thereby providing foundations to combat flaviviruses.

Higher-temperature-adapted dengue virus serotype 2 strain exhibits enhanced virulence in AG129 mouse model.

The factors that drive dengue virus (DENV) evolution, and selection of virulent variants are yet not clear. Higher environmental temperature shortens DENV extrinsic incubation period in mosquitoes, increases human transmission, and plays a critical role in outbreak dynamics. In the present study, we looked at the effect of temperature in altering the virus virulence. We found that DENV cultured at a higher temperature in C6/36 mosquito cells was significantly more virulent than the virus grown at a lower temperature. In a mouse model, the virulent strain induced enhanced viremia and aggressive disease with a short course, hemorrhage, severe vascular permeability, and death. Higher inflammatory cytokine response, thrombocytopenia, and severe histopathological changes in vital organs such as heart, liver, and kidney were hallmarks of the disease. Importantly, it required only a few passages for the virus to acquire a quasi-species population harboring virulence-imparting mutations. Whole genome comparison with a lower temperature passaged strain identified key genomic changes in the structural protein-coding regions as well as in the 3'UTR of the viral genome. Our results point out that virulence-enhancing genetic changes could occur in the dengue virus genome under enhanced growth temperature conditions in mosquito cells.

Comparative phylogenetic analysis and transcriptomic profiling of Dengue (DENV-3 genotype I) outbreak in 2021 in Bangladesh

BackgroundThe next-generation sequencing (NGS) technology facilitates in-depth study of host-pathogen metatranscriptome. We, therefore, implicated phylodynamic and transcriptomic approaches through NGS technology to know/understand the dengue virus (DENV) origin and host response with dengue fever.MethodsIn this study, blood serum RNA was extracted from 21 dengue patients and 3 healthy individuals. Total transcriptomic data were analyzed for phylogenetic, phylodynamic, differential express gene (DEG), and gene ontology (GO) using respective bioinformatics tools.ResultsThe viral genome sequence revealed dengue viral genome size ranges 10647 to 10707 nucleotide. Phylogenetic and phylodynamic analysis showed that the 2021 epidemic isolates were DENV-3 genotype-I and maintained as a new clade in compared to 2019 epidemic. Transcriptome analysis showed a total of 2686 genes were DEG in dengue patients compared to control with a q-value < 0.05. DESeq2 plot counts function of the top 24 genes with the smallest q-values of differential gene expression of RNA-seq data showed that 11 genes were upregulated, whereas 13 genes were downregulated. GO analysis showed a significant upregulation (p = < 0.001) in a process of multicellular organismal, nervous system, sensory perception of chemical stimulus, and G protein-coupled receptor signaling pathways in the dengue patients. However, there were a significant downregulation (p = < 0.001) of intracellular component, cellular anatomical entity, and protein-containing complex in dengue patients. Most importantly, there was a significant increase of a class of immunoregulatory proteins in dengue patients in compared to the controls, with increased GO of immune system process. In addition, upregulation of toll receptor (TLR) signaling pathways were found in dengue patients. These TLR pathways were particularly involved for the activation of innate system coupled with adaptive immune system that probably involved the rapid elimination of dengue virus infected cells. These differentially expressed genes could be further investigated for target based prophylactic interventions for dengue.ConclusionThis is a first report describing DENV complete genomic features and differentially expressed genes in patients in Bangladesh. These genes may have diagnostic and therapeutic values for dengue infection. Continual genomic surveillance is required to further investigate the shift in dominant genotypes in relation to viral pathogenesis.

A REVIEW OF DENGUE VIRUS GENOME, STRUCTURAL AND NON-STRUCTURAL PROTEINS, AND LIFE CYCLE

The dengue virus-infected Aedes mosquito bites that cause dengue fever and propagate the potentially fatal disease. Dengue virus infection poses a risk to over 3.9 billion individuals worldwide. Its widespread incidence is currently a significant health issue. A pathogenic creature with a unique nucleotide sequence in its genome provides instructions for RNA or DNA synthesis, and protein expression, also for the organism’s survival and evolution. New species or strains that are potentially more virulent than their parent strains can emerge as a result of mutations or changes in the nucleotide sequence. In this review, we have discussed the structural organization, genome, proteins, and life cycle of dengue virus. We describe in detail the structural and non-structural proteins and their functions. We describe the organization of viral RNA; it consists of one open reading frame (encodes a single polyprotein), 5’ UTRs with 5’ capping, and 3’ UTR without poly ‘A’ tail. We have described in detail the life cycle of the dengue virus. This will aid in a better understanding of dengue virus organization, and life cycle. Keywords: Dengue virus, genomics, proteomics, structural proteins, non-structural proteins,

A REVIEW OF DENGUE VIRUS GENOME, STRUCTURAL AND NON-STRUCTURAL PROTEINS, AND LIFE CYCLE

The dengue virus-infected Aedes mosquito bites that cause dengue fever and propagate the potentially fatal disease. Dengue virus infection poses a risk to over 3.9 billion individuals worldwide. Its widespread incidence is currently a significant health issue. A pathogenic creature with a unique nucleotide sequence in its genome provides instructions for RNA or DNA synthesis, and protein expression, also for the organism‟s survival and evolution. New species or strains that are potentially more virulent than their parent strains can emerge as a result of mutations or changes in the nucleotide sequence. In this review, we have discussed the structural organization, genome, proteins, and life cycle of dengue virus. We describe in detail the structural and non-structural proteins and their functions. We describe the organization of viral RNA; it consists of one open reading frame (encodes a single polyprotein), 5‟ UTRs with 5‟ capping, and 3‟ UTR without poly „A‟ tail. We have described in detail the life cycle of the dengue virus. This will aid in a better understanding of dengue virus organization, and life cycle.

Dengue Virus Serotypes Circulating among Aedes Mosquitoes in the Lucknow District of North India: Molecular Identification and Characterization

Dengue is one of the most critical diseases, caused by Dengue virus (DENV) serotypes (DENV-1 to DENV-4). Study aims to detect DENV in natural A. aegypti from endemic regions of Uttar Pradesh. From 2010- 2013, mosquitoes collected from Uttar Pradesh tested for Dengue virus serotypes by capsid-premembrane gene-based PCR analyzed for genotypes sequencing of the C-prM junction of DENV genome. A total of 4731, 53.54% (n=2671) A. aegypti and 46 % (n=2060) A. albopictus mosquitoes were collected. Of 226 mosquito pools, 10 pools of A. aegypti and 14 pools of A. albopictus were positive for DENV by PCR. All 24 isolates identified as DENV-I; Genotype (G)-III (n=8), G-V (n=1); DENV-2; G-IV (n=4); DENV-3; G-III (n=11). The overall minimum infection rate was much higher in A. albopictus mosquitoes and presence of MIR in male mosquitoes is an indicating natural vertical transmission and important observation in geographical area indicating natural vertical transmission.

Experimental Dengue Virus Type 4 Infection Increases the Expression of MicroRNAs-15/16, Triggering a Caspase-Induced Apoptosis Pathway

The World Health Organization has estimated the annual occurrence of approximately 392 million dengue virus (DENV) infections in more than 100 countries where the virus is endemic, which represents a serious threat to humanity. DENV is a serologic group with four distinct serotypes (DENV-1, DENV-2, DENV-3, and DENV-4) belonging to the genus Flavivirus, in the family Flaviviridae. Dengue is the most widespread mosquito-borne disease in the world. The ~10.7 kb DENV genome encodes three structural proteins (capsid (C), pre-membrane (prM), and envelope (E)) and seven non-structural (NS) proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5). The NS1 protein is a membrane-associated dimer and a secreted, lipid-associated hexamer. Dimeric NS1 is found on membranes both in cellular compartments and cell surfaces. Secreted NS1 (sNS1) is often present in patient serum at very high levels, which correlates with severe dengue symptoms. This study was conducted to discover how the NS1 protein, microRNAs-15/16 (miRNAs-15/16), and apoptosis are related during DENV-4 infection in human liver cell lines. Huh 7.5 and HepG2 cells were infected with DENV-4, and miRNAs-15/16, viral load, NS1 protein, and caspases-3/7 were quantified after different durations of infection. This study demonstrated that miRNAs-15/16 were overexpressed during the infection of HepG2 and Huh 7.5 cells with DENV-4 and had a relationship with NS1 protein expression, viral load, and the activity of caspases-3/7, thus making these miRNAs potential injury markers during DENV infection in human hepatocytes.

Dengue Virus Capsid Protein Facilitates Genome Compaction and Packaging.

Dengue virus (DENV) is a single-stranded (+)-sense RNA virus that infects humans and mosquitoes, posing a significant health risk in tropical and subtropical regions. Mature virions are composed of an icosahedral shell of envelope (E) and membrane (M) proteins circumscribing a lipid bilayer, which in turn contains a complex of the approximately 11 kb genomic RNA with capsid (C) proteins. Whereas the structure of the envelope is clearly defined, the structure of the packaged genome in complex with C proteins remains elusive. Here, we investigated the interactions of C proteins with viral RNA, in solution and inside mature virions, via footprinting and cross-linking experiments. We demonstrated that C protein interaction with DENV genomes saturates at an RNA:C protein ratio below 1:250. Moreover, we also showed that the length of the RNA genome interaction sites varies, in a multimodal distribution, consistent with the C protein binding to each RNA site mostly in singlets or pairs (and, in some instances, higher numbers). We showed that interaction sites are preferentially sites with low base pairing, as previously measured by 2'-acetylation analyzed by primer extension (SHAPE) reactivity indicating structuredness. We found a clear association pattern emerged: RNA-C protein binding sites are strongly associated with long-range RNA-RNA interaction sites, particularly inside virions. This, in turn, explains the need for C protein in viral genome packaging: the protein has a chief role in coordinating these key interactions, promoting proper packaging of viral RNA. Such sites are, thus, highly consequential for viral assembly, and, as such, may be targeted in future drug development strategies against these and related viruses.

Structure-first identification of RNA elements that regulate dengue virus genome architecture and replication

The genomes of RNA viruses encode the information required for replication in host cells both in their linear sequence and in complex higher-order structures. A subset of these RNA genome structures show clear sequence conservation, and have been extensively described for well-characterized viruses. However, the extent to which viral RNA genomes contain functional structural elements-unable to be detected by sequence alone-that nonetheless are critical to viral fitness is largely unknown. Here, we devise a structure-first experimental strategy and use it to identify 22 structure-similar motifs across the coding sequences of the RNA genomes for the four dengue virus serotypes. At least 10 of these motifs modulate viral fitness, revealing a significant unnoticed extent of RNA structure-mediated regulation within viral coding sequences. These viral RNA structures promote a compact global genome architecture, interact with proteins, and regulate the viral replication cycle. These motifs are also thus constrained at the levels of both RNA structure and protein sequence and are potential resistance-refractory targets for antivirals and live-attenuated vaccines. Structure-first identification of conserved RNA structure enables efficient discovery of pervasive RNA-mediated regulation in viral genomes and, likely, other cellular RNAs.

Recent Advances in Methods Detecting the Dengue Virus

Dengue fever, caused by the Dengue virus (DENV), has emerged as a mild to lethal human infection. Globally, an estimated 400 million patients have been infected with DENV over the past 10 years, and this number is expected to increase in the coming years. The DENV, possessing a single positive- stranded RNA with five serotypes, is transmitted by mosquitos of the Flaviviridae family—particularly, the Flavivirus genus and Aedes species. The DENV genome encodes three structural and seven non- structural proteins. In the Kingdom of Saudi Arabia (KSA), serotype 2 of the dengue virus (DENV-2) emerged in 1994 and caused a major epidemic in Jeddah, KSA. Dengue outbreaks first appeared in Makkah in 2004, according to the characteristics studied in hospitalized patients. Major factors causing the wide and rapid distribution of the virus include increased urbanization, migration, global commerce, weather variation, inadequate mosquito control, the development of pesticide resistance in mosquitos, irregular use of insecticides, and shifting climatic circumstances. Detection methods currently used for DENV include the detection of viral antigens (Ag) (virus extraction and purification, immunofluorescence test, and NS1 detection assay), serological assays (plaque reduction neutralization titers (PRNT), IgM/IgG immunological assays), and RNA detection using RT-PCR. Low sensitivity, specificity, and accessibility of the detection protocols represent major challenges necessitating the advent of more amenable protocols. The Aedes mosquito is the primary vector for horizontal transmission of DENV. DENV-infected mosquitos infect people, and DENV passes from one human to another through this vector. Once acquired, the virus requires 5–7 days of incubation before the patient exhibits various symptoms of dengue fever; subsequently, uninfected mosquitoes that come in contact with infected patients feed on their blood and become infected. The DENV may also be spread through the mating of male and female Aedes mosquitoes. The reverse transcription loop-mediated isothermal application (RT-LAMP) has emerged as one of the most adaptable viral detection procedures. This method could prove to be an excellent pathogen detection tool because it is cheap, simple, sensitive, cost-effective, accessible, and fast. The method relies on the use of 4–6 primers to recognize eight different loci in the target sequence contained in the DENV clinical isolates with a 100% success rate and a sensitivity of about 93%. We strongly recommend the use of LAMP in detecting spots of virus spread, especially in urban regions where accessibility to detection methods is scarce.