No description available.

Dengue fever and dengue hemorrhagic fever (DF /DHF) are caused by the dengue viruses, which belong to family Flaviviridae. DPff incidence has been increasing globally in the last 40 years. Over half the world's population lives in areas potentially at risk for dengue transmission, making dengue the most important human viral disease transmitted by arthropod vectors in terms of morbidity and mortality. Recently, the age distribution of Dfff cases in Asia has changed. The numbeT of adults with DHF has increased since the 1990s in Southeast Asian countries including Viet Nam. A series of studies were conducted in the Hospital for Tropical Diseases, Ho Chi Minh City, Viet Nam on clinical manifestations, cytokine production in acute infection and T cell responses in adult dengue patients.

Clinical experience suggests that adults with dengue manifest a pattern of complications different from those observed in children, but direct comparisons among populations experiencing the same exposure have rarely been published. I conducted a large prospective descriptive study of dengue across all age-groups presenting to a single institution in an endemic country during a defined time-period. Vascular leakage was more severe in the paediatric patients and DSS developed much more frequently in this age-group. In contrast haemorrhagic manifestations and severe organ involvement were more common in adults. Similar to the established findings in children, typical coagulation abnormalities were apparent in the adults .. - i.e. prolonged APTT with reduced fibrinogen levels but without evidence of true disseminated intravascular coagulation. However thrombocytopenia was significantly worse among the adults throughout the evolution of the disease, even after adjusting for the higher rate of secondary infections in this group, and platelet counts after recovery remained lower than in the children. Clinically severe liver involvement was seen only in adults and was infrequent but usually resulted in I severe bleeding. Chronic hepatitis B co-infection was associated with modestly but significantly increased levels of alanine aminotransferase, but did not otherwise impact the clinical picture. To investigate the mechanisms underlying the increase in APTT I carried out APTT Mixing Studies confirming that deficiency of coagulation factors is a major contributory factor. Since there is little evidence for procoagulant activation the most likely mechanism for this would be leakage of coagulation proteins, many of which are of a similar size to albumin. An additional explanation for the increased APTT could be the presence of a circulating anticoagulant - I found very high levels of heparan sulfate (HS) in the dengue plasma, but was not able to show that the HS exerts an anticoagulant effect. I also used FACS analysis to demonstrate that circulating endothelial cells (CECs) are increased during dengue infections and that percentage CECs correlate with the severity of the coagulopathy and with bleeding. Parallel. increases in both CECs and HS levels support the theory that disruption of the endothelial cell/glycocalyx complex occurs during dengue infections - i.e. CECs appear to be shed from the endothelial layer while HS may be shed from the surface glycocalyx. These disruptions likely affect the function of the complex and could contribute to the pathogenesis of the systemic vascular leak syndrome.

The four serotypes of dengue virus (DENV 1-4) cause the most important arthropod-borne viral disease of humans. The DENV RNA genome is translated into a polyprotein that is cleaved into three structural and seven nonstructural proteins. Nonstructural protein 5 (NSS) has enzymatic activities required for capping and replication of the viral genome. NSS is phosphorylated and with some serotypes can accumulate in the nucleus of infected cells. However the role of NSS phosphorylation or nuclear trafficking in the virus lifecycle, is not well understood. Using a combination of proteomic and bioinformatic approaches, potential NSS phosphorylation sites were identified and analysed for their role in the virus lifecycle using a DENV reverse genetic system. Mutation of the NSS amino acid Thr-39S was found to decrease NSS nuclear localisation and viral replication in a cell specific manner. Mutations preventing and mimicking phosphorylation at Thr-39S had similar effects on NSS localisation and viral growth, showing that the effects were not caused by phosphorylation. Mutation of Thr-39S did not influence NSS mediated suppression of interferon signalling. For the first time this thesis revealed that there are serotype specific differences in NSS localisation. DENV-1 and DENV-4 NSS are predominantly localised to the cytoplasm, whereas DENV-2 and DENV-3 NSS accumulate in the nucleus. The role of the NSS nuclear localisation signal (NLS) in mediating these differences was investigated. NSS of all DENV serotypes bound to importin-a, the protein responsible for active nuclear import, but exchanging the NLSs of DENV-2 and 4 NSS didn't fully confer the properties of the NLS to the background protein, suggesting that protein context was also important for NSS nuclear localisation. Chimeric DENV-2 containing sequences encoding the DENV-3 NSS or polymerase subdomain were produced, whilst DENV-2 containing the DENV-4 NSS sequence failed to replicate. The two DENV-2/3 chimeric viruses showed decreased replication especially in the insect cell line C6/36; continued passaging resulted in the accumulation of mutations in NS1 and NS2B that increased viral replication.

Fusion process is known to be the initial step of viral infection and hence targeting the entry process is a promising strategy to design antiviral therapy. The self-inhibitory peptides derived from the envelope (E) proteins function to inhibit the protein-protein interactions in the membrane fusion step mediated by the viral E proteins. Thus, they have the potential to be developed into effective antiviral therapy. We developed a Monte Carlo-based computational method with the aim to identify and optimize potential peptide hits from the E proteins. Some novel peptides may inhibit the protein-protein interaction in the Dengue Virus (DENV) or Herpes Simplex Virus-! (HSV -1) fusion process and serve as starting points for the development of antiviral therapy to treat viral infection. Residue-specific all-atom conditional probability discriminatory function (RAPDF) has been justified effective for designing novel peptide inhibitors targeting E proteins in our computational method, The statistical potential has its universality and specificity which is decided by the reference state and the decoy sets. We built new distance-dependant statistical potentials using E, tetratricopeptide and ankyrin repeat proteins respectively. We demonstrated that the specific statistical potentials outperformed the general statistical potentials. Our computational method for identifying self-inhibitory peptides from three types of E proteins has also illustrated E proteins may have some unique features in common. In order to identify the active peptides from non-active peptides, we applied a support vector machine (SVM) approach for envelope peptide inhibitors activity prediction (EAP) based on the physicochemical properties, amino acid composition and statistical scoring function of peptide inhibitors which target E proteins. The results suggest that the rational connecting between properties of peptide inhibitors derived from E proteins and antivirus activity.

No description available.

Over half of the world's population are at risk of infection from dengue fever (Guha-Sapir 2005). This viral disease is transmitted by the female Aedes aegypti mosquito and is the major source of human death in the world when compared with any other vector borne disease (Gubler1998a). The first important epidemic of dengue haemorrhagic fever (DHF) in America was reported in Cuba in 1981 and subsequently in Venezuela during 1989 and 1990 (Oletta2006, Brightmer1998). There has been a trend of increased incidence in many Central and South American countries since 1990 - Brazil, Venezuela, Honduras and Mexico (SanMartin2010) with Venezuela having the highest number of cases of DHF. The urgent need for more effective public health measures to combat this disease in Venezuela drove the decision to undertake the work described in this dissertation. Spatio-Temporal modelling has been developed for the prediction of the occurrence of dengue fever in Zulia state, Venezuela. A systematic approach has been adopted to validate this tool. At the first stage of the analysis an exploratory study was performed to underline the most significant features of the dynamics of incidence rates of dengue fever from 2002 to 2008. In the second stage a Generalized Linear Model (GLM) approach was used in the form of Negative Binomial Generalized Linear Mixed model (GLMM) to compare Relative Risk (RR) across exposure groups by age and sex, using an epidemiological dataset covering the whole of Zulia State, Venezuela. This approach used both a frequentist and a Bayesian perspective for comparative purposes of both outcomes and methodologies. Finally a Spatio-Temporal model was constructed based on Generalized Additive Mixed model (GAMM) framework because the earlier analysis identified a complex association between covariates and response variables. This GAMM structure was further developed so that it could be used to help predict future outbreaks of the disease in Zulia state with a good degree of accuracy.