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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., Here, the authors provide detailed analyses of viral RNA structure in virions and in infected cells for four dengue virus serotypes and four Zika virus strains, and identify conserved structures that are important for virus replication.

Cyclic GMP-AMP synthetase (cGAS) is a DNA-specific cytosolic sensor, which detects and initiates host defense responses against microbial DNA. It is thus curious that a recent study identified cGAS as playing important roles in inhibiting positive-sense single-stranded RNA (+ssRNA) viral infection, especially since RNA is not known to activate cGAS. Using a dengue virus serotype 2 (DENV-2) vaccine strain (PDK53), we show that infection creates an endogenous source of cytosolic DNA in infected cells through the release of mitochondrial DNA (mtDNA) to drive the production of cGAMP by cGAS. Innate immune responses triggered by cGAMP contribute to limiting the spread of DENV to adjacent uninfected cells through contact dependent gap junctions. Our result thus supports the notion that RNA virus indirectly activates a DNA-specific innate immune signaling pathway and highlights the breadth of the cGAS-induced antiviral response.

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., Author summary Dengue viruses 1–4 (DENV1-4), Zika virus (ZIKV) and Chikungunya virus (CHIKV) are tropical and subtropical viruses that share a common arthropod vector, and have very similar clinical presentations that are difficult to distinguish. With the recent outbreaks of DENV, ZIKV and CHIKV globally, a single methodology able to simultaneously distinguish these viruses and provide full-genome information would greatly increase our capacity to rapidly characterize outbreaks. As a proof of principle, we have applied this methodology to two large cohorts in Sri Lanka and Singapore taken during recent dengue and Zika outbreaks, respectively. Herein, we present the results of this application to these cohorts and provide the tools to replicate these methodologies for other cohorts.

The World Health Origanization has classified dengue as a major international public health concern. The reasons for the resurgence are complex, and are likely a combination of multiple factors, including population growth associated with rapid uncontrolled urbanization, increased movement of viruses in people among countries and regions via international travel, demographic changes, poor vector control, genetic changes in circulating or introduced viruses, and modulating climatic factors. The past decade has seen more investment in vaccine development and novel vector control measures than ever before. Three dengue vaccine candidates are far advanced in development, with CYD-TDV being the first licensed dengue vaccine. However, this vaccine showed serostatus dependent performance. The two other dengue vaccines are currently in Phase 3 efficacy trials.

Dengue and Zika are clinically important members of the Flaviviridae family that utilizes an 11kb positive strand RNA for genome regulation. While structures have been mapped primarily in the UTRs, much remains to be learnt about how the rest of the genome folds to enable function. Here, we performed secondary structure and pair-wise interaction mapping on four dengue serotypes and four Zika strains in their native virus particles and infected cells. Comparative analysis of SHAPE reactivities across serotypes nominated potentially functional regions that are highly structured, show structure conservation, and low synonymous mutation rates, including a structure associated with ribosome pausing. Pair-wise interaction mapping by SPLASH further reveals new pair-wise interactions, in addition to the known circularization sequence. 40% of pair-wise interactions form alternative structures, suggesting extensive structural heterogeneity. Analysis of shared pair-wise interactions between serotypes revealed macro-organization whereby interactions are preserved at their physical locations, beyond their sequence identities. In addition, structure mapping of virus genomes released in solution-as well as inside host cells-showed that other helicases, in addition to the ribosome, play a role in unwinding viral structures inside cells. Mutational experiments that disrupt in cell and in virion pair-wise interactions result in virus attenuation, demonstrating their importance during the virus life-cycle.

Background: Engineered adenoviruses are being increasingly explored as immunoprophylactic or immunotherapeutic vaccine vectors. Encouraging data from preclinical studies using human adenovirus vectors carrying different antigen genes have resulted in many currently ongoing clinical trials. Objective: The article seeks to review the current status of the use of adenoviruses as vaccine vectors. Methods: This review is based on the patent literature since 2000 pertaining to the development of adenovirus vaccine vectors for infectious and non-infectious diseases. Conclusion: Human adenovirus-vectored vaccines have important limitations that stem from their immunogenicity and restrict their utility. This has spurred intensive efforts to find alternative adenovirus vectors and strategies, each with its own advantages and shortcomings.

Mobile Ad hoc Network (MANET) is a network comprises of mobile nodes connected without any centralized administration. Each node in the network has the ability to share a large number of objects with other nodes. The nodes are connected with other nodes to forward a message to other nodes until the search for node that desire meets. This paper represents a new replication method to recover the performance in distributed system. Objects are replicated on different nodes in the network to minimize the search for an object. It is observed that mobility of nodes predicted using SOM (Self Organizing Maps) technique provides less accuracy. This paper represents an algorithm that uses the network parameters as input condition to replicate objects into the nodes. The decision for each node will be taken by gathering the input conditions and feed to the Hopfield Neural Network. It is view that it will provide more accuracy and improve the performance.

Activating Fc gamma receptors (FcγRs) in hematopoietic cells serve to remove antibody-opsonized antigens, including dengue virus (DENV), from systemic circulation. While neutralizing antibody concentrations provide humoral immunity, cross-reactive or sub-neutralizing levels of antibody can result in antibody-dependent enhancement of DENV infection that increases overall viral burden. Recently, it has been suggested that the antibody levels needed for DENV neutralization differs when different FcγR is engaged. If this is true, the threshold titer used to infer immunity should be influenced by FcγR usage. Here, using cells that express both activating and inhibitory FcγRs, we show that the type of FcγR engaged during phagocytosis can influence the antibody concentration requirement for DENV neutralization. We demonstrate that phagocytosis through FcγRI requires significantly less antibody for complete DENV neutralization compared to FcγRIIA. Furthermore, when DENV is opsonized with sub-neutralizing levels of antibody, FcγRI-mediated phagocytosis resulted in significantly reduced DENV titers compared to FcγRIIA. However, while FcγRI may remove antibody-opsonized DENV more efficiently, this receptor is only preferentially engaged by clustering when neutralizing, but not sub-neutralizing antibody concentrations, were used. Collectively, our study demonstrates that activating FcγR usage may influence antibody titers needed for DENV neutralization.

In vivo gene delivery using human adenovirus serotype 5 (AdV5) vectors is being explored for vaccination purposes. The presence of anti-AdV5 antibodies in human serum arising from natural exposure to AdV5 can interfere potentially with and compromise the efficacy of rAdV5-based vaccine vectors. In this report, a collection of 114 sera from healthy adult Indian blood donors was analyzed for the presence of anti-AdV5 antibodies, using an AdV5 vector encoding the green fluorescent protein (GFP) to monitor the presence of anti-AdV5 neutralizing antibodies in human sera based on their ability to block virus entry into HeLa cells which express the Coxsackievirus-and-Adenovirus Receptor (CAR). In this assay all samples tested were positive for anti-AdV5 antibodies, with titers varying over a very wide range. It was also observed that these antibodies facilitated the uptake of the reporter AdV5 vector into the monocytic cell line U937 which does not express CAR, but expresses Fc receptors (FcRs) instead. These observations have implications for rAdV5-based vaccine development. J. Med. Virol. 82:407-414, 2010. (c) 2010 Wiley-Liss, Inc.