Project description:Dengue and Zika are closely related members of the Flaviviridae family of positive, single-stranded RNA viruses and are of global clinical importance. These viruses utilize an 11kb RNA genome for translation and replication, and much remains to be learnt about how the entire genome folds to enable virus function. Here, we performed high throughput RNA secondary structure and pair-wise interaction mapping on four dengue serotypes and four Zika strains within their virus particles. We identified structures that are associated with translation pausing, and are evolutionary conserved by integrating synonymous mutation rates into our analysis. Genome-wide interaction mapping revealed alternative structures, as well as extensive long-range RNA interactions – including the known circularization signals– within the virus particles. Many of these long-range interactions are conserved across the viruses and/or clustered into “hubs” that are shown to be functionally important. This comprehensive structural resource of dengue and Zika viruses reveals that viral genome organization is much more complex than previously appreciated and deepens our understanding of the molecular basis for viral pathogenesis. Overall design: Local RNA structure probing and long-range RNA interactome mapping in 4 dengue serotypes and 4 Zika strains
Project description:Dengue (DENV) and Zika (ZIKV) viruses are clinically important members of the Flaviviridae family with an 11?kb positive strand RNA genome that folds to enable virus function. Here, we perform structure and interaction mapping on four DENV and ZIKV strains inside virions and in infected cells. Comparative analysis of SHAPE reactivities across serotypes nominates potentially functional regions that are highly structured, conserved, and contain low synonymous mutation rates. Interaction mapping by SPLASH identifies many pair-wise interactions, 40% of which form alternative structures, suggesting extensive structural heterogeneity. Analysis of shared interactions between serotypes reveals a conserved macro-organization whereby interactions can be preserved at physical locations beyond sequence identities. We further observe that longer-range interactions are preferentially disrupted inside cells, and show the importance of new interactions in virus fitness. These findings deepen our understanding of Flavivirus genome organization and serve as a resource for designing therapeutics in targeting RNA viruses.
Project description:The frequency of epidemics caused by Dengue viruses 1-4, Zika virus and Chikungunya viruses have been on an upward trend in recent years driven primarily by uncontrolled urbanization, mobility of human populations and geographical spread of their shared vectors, Aedes aegypti and Aedes albopictus. Infections by these viruses present with similar clinical manifestations making them challenging to diagnose; this is especially difficult in regions of the world hyperendemic for these viruses. In this study, we present a targeted-enrichment methodology to simultaneously sequence the complete viral genomes for each of these viruses directly from clinical samples. Additionally, we have also developed a customized computational tool (BaitMaker) to design these enrichment baits. This methodology is robust in its ability to capture diverse sequences and is amenable to large-scale epidemiological studies. We have applied this methodology to two large cohorts: a febrile study based in Colombo, Sri Lanka taken during the 2009-2015 dengue epidemic (n = 170) and another taken during the 2016 outbreak of Zika virus in Singapore (n = 162). Results from these studies indicate that we were able to cover an average of 97.04% ± 0.67% of the full viral genome from samples in these cohorts. We also show detection of one DENV3/ZIKV co-infected patient where we recovered full genomes for both viruses.
Project description:Viral pathogens are an ongoing threat to public health worldwide. Dissecting their dependence on host biosynthetic pathways could lead to effective antiviral therapies. To define how entero- and flaviviruses redirect host ribosomes to synthesize viral proteins and disable host protein production, we performed proteomic analysis of lysates and isolated polysomes from human Huh7 cells infected with either polio, zika or dengue viruses. We find that infection remodels polysome composition along similar principles, without major changes to core ribosome stoichiometry. These viruses use different strategies to evictfrom polysomes a common set of translation initiation and RNA surveillance factors while recruiting host machineries specifically required for viral biogenesis. We also find that both zika and dengue utilize the collagen prolyl-hydroxylation machinery to mediate co-translational modification of conserved prolines in the viral polyprotein. Our findings show how RNA viruses co-opt polysome modularity and establish a powerful strategy to identify targets for selective antiviral interventions.
Project description:Dengue and Zika are two of the most important human viral pathogens worldwide. In both cases, the envelope glycoprotein E is the main target of the antibody response. Recently, new complex quaternary epitopes were identified which are the consequence of the arrangement of the antiparallel E dimers on the viral surface. Such epitopes can be exploited to develop more efficient cross-neutralizing vaccines. Here we describe a successful covalent stabilization of E dimers from Dengue and Zika viruses in mammalian cells. Folding and dimerization of secretory E was found to be strongly dependent on temperature but independent of PrM co-expression. In addition, we found that, due to the close relationship between flaviviruses, Dengue and Zika viruses E proteins can form heterodimers and assemble into mosaic viral particles. Finally, we present new virus-free analytical platforms to study and screen antibody responses against Dengue and Zika, which allow for differentiation of epitopes restricted to specific domains, dimers and higher order arrangements of E.
Project description:Clinical manifestations of Zika virus, chikungunya virus, and dengue virus infections can be similar. To improve virus detection, streamline molecular workflow, and decrease test costs, we developed and evaluated a multiplex real-time reverse transcription PCR for these viruses.
Project description:Dengue virus (DENV) and Zika virus (ZIKV) are two mosquito-borne flaviviruses afflicting nearly half of the world population. Human infection by these viruses can either be asymptomatic or manifest as clinical diseases from mild to severe. Despite more cases are presented as self-limiting febrile illness, severe dengue disease can be manifested as hemorrhagic fever and hemorrhagic shock syndrome, and ZIKV infection has been linked to increased incidence of peripheral neuropathy Guillain-Barre syndrome and central neural disease such as microcephaly. The current prevention and treatment of these infectious diseases are either non-satisfactory or entirely lacking. Because DENV and ZIKV have much similarities in genomic and structural features, almost identical mode of mosquito-mediated transmission, and probably the same pattern of host innate and adaptive immunity toward them, it is reasonable and often desirable to investigate these two viruses side-by-side, and thereby devise common countermeasures against both. Here, we review the existing knowledge on DENV and ZIKV regarding epidemiology, molecular virology, protective immunity and vaccine development, discuss recent new discoveries on the functions of flavivirus NS1 protein in viral pathogenesis and transmission, and propose a one-two punch strategy using vaccine and vector blockade to overcome antibody-dependent enhancement and defeat Dengue and Zika viruses.
Project description:Current multiplexed diagnostics for Zika, dengue, and chikungunya viruses are situated outside the intersection of affordability, high performance, and suitability for use at the point-of-care in resource-limited settings. Consequently, insufficient diagnostic capabilities are a key limitation facing current Zika outbreak management strategies. Here we demonstrate highly sensitive and specific detection of Zika, chikungunya, and dengue viruses by coupling reverse-transcription loop-mediated isothermal amplification (RT-LAMP) with our recently developed quenching of unincorporated amplification signal reporters (QUASR) technique. We conduct reactions in a simple, inexpensive and portable "LAMP box" supplemented with a consumer class smartphone. The entire assembly can be powered by a 5?V USB source such as a USB power bank or solar panel. Our smartphone employs a novel algorithm utilizing chromaticity to analyze fluorescence signals, which improves the discrimination of positive/negative signals by 5-fold when compared to detection with traditional RGB intensity sensors or the naked eye. The ability to detect ZIKV directly from crude human sample matrices (blood, urine, and saliva) demonstrates our device's utility for widespread clinical deployment. Together, these advances enable our system to host the key components necessary to expand the use of nucleic acid amplification-based detection assays towards point-of-care settings where they are needed most.
Project description:The emergence and spread of Zika virus (ZIKV) presented a challenge to the diagnosis of ZIKV infections in areas with transmission of dengue (DENV) and chikungunya (CHIKV) viruses. To facilitate detection of ZIKV infections, and differentiate these infections from DENV and CHIKV, we developed the Trioplex real-time RT-PCR assay (Trioplex assay). Here, we describe the optimization of multiplex and singleplex formats of the assay for a variety of chemistries and instruments to facilitate global standardization and implementation. We evaluated the analytical performance of all Trioplex modalities for detection of these three pathogens in serum and whole blood, and for ZIKV in urine. The limit of detection for the three viruses and in different RNA-extraction modalities is near 103 genome copy equivalents per milliliter (GCE/mL). Simultaneous testing of more than one specimen type from each patient provides a 6.4% additional diagnostic sensitivity. Overall, the high sensitivity of the Trioplex assay demonstrates the utility of this assay ascertaining Zika cases.
Project description:Current and reoccurring viral epidemic outbreaks such as those caused by the Zika virus illustrate the need for rapid development of antivirals. Such development would be facilitated by computational approaches that can provide experimentally testable predictions for possible antiviral strategies. To this end, we focus here on the fact that viruses are directly dependent on their host metabolism for reproduction. We develop a stoichiometric, genome-scale metabolic model that integrates human macrophage cell metabolism with the biochemical demands arising from virus production and use it to determine the virus impact on host metabolism and vice versa. While this approach applies to any host-virus pair, we first apply it to currently epidemic viruses Chikungunya, Dengue and Zika in this study. We find that each of these viruses causes specific alterations in the host metabolic flux towards fulfilling their biochemical demands as predicted by their genome and capsid structure. Subsequent analysis of this integrated model allows us to predict a set of host reactions, which, when constrained, inhibit virus production. We show that this prediction recovers known targets of existing antiviral drugs, specifically those targeting nucleotide production, while highlighting a set of hitherto unexplored reactions involving both amino acid and nucleotide metabolic pathways, with either broad or virus-specific antiviral potential. Thus, this computational approach allows rapid generation of experimentally testable hypotheses for novel antiviral targets within a host.