Comprehensive analysis of dengue virus-specific responses supports an HLA-linked protective role for CD8+ T cells.

The role of CD8(+) T cells in dengue virus infection and subsequent disease manifestations is not fully understood. According to the original antigenic sin theory, skewing of T-cell responses induced by primary infection with one serotype causes less effective response upon secondary infection with a different serotype, predisposing individuals to severe disease. A comprehensive analysis of CD8(+) responses in the general population from the Sri Lankan hyperendemic area, involving the measurement of ex vivo IFNγ responses associated with more than 400 epitopes, challenges the original antigenic sin theory. Although skewing of responses toward primary infecting viruses was detected, this was not associated with impairment of responses either qualitatively or quantitatively. Furthermore, we demonstrate higher magnitude and more polyfunctional responses for HLA alleles associated with decreased susceptibility to severe disease, suggesting that a vigorous response by multifunctional CD8(+) T cells is associated with protection from dengue virus disease.

EVALUATION OF A COMMERCIAL RAPID TEST KIT FOR DETECTION OF ACUTE DENGUE INFECTION.

Early diagnosis is important for clinical management of dengue disease. While classic laboratory tests are often tedious and time consuming, point of care devices offer a rapid, cost-effective and user-friendly alternative provided their accuracy is acceptable. This study evaluated the sensitivity, specificity and efficiency of SD BIOLINE Dengue Duo® rapid NS1, IgM and IgG test kit for diagnosis of acute dengue virus infection. Standard laboratory diagnostics, RT-PCR, IgM and IgG capture ELISAs were carried out on 143 suspected dengue patient samples obtained from a Sri Lankan population. Using the results of these standard laboratory tests as reference, the sensitivity and specificity of the SD Dengue Duo® NS1 test was 57% and 87%, respectively, and those of the IgM test was 50% and 84%, respectively. The combined sensitivity and specificity of the SD Dengue Duo® NS1/ IgM test was 72% and 80%, respectively. The SD Dengue Duo® NS1 test detected NS1 for up to 9 days from onset of fever. Primary and secondary dengue cases were classified according to the IgG test, of which the kit identified 88% and 26% of primary and of secondary infection, respectively. Although the SD Dengue Duo® kit was not as accurate as the standard tests, it still can serve the useful reference for initial screening of suspected dengue cases, especially in poor resource hospital settings and aid in clinical disease management of dengue infection.

Associative and structural properties of the region of complement factor H encompassing the Tyr402His disease-related polymorphism and its interactions with heparin.

Factor H (FH) is a major complement control protein in serum. The seventh short complement regulator (SCR-7) domain of the 20 in FH is associated with age-related macular degeneration through a Tyr402His polymorphism. The recombinant SCR-6/8 domains containing either His402 or Tyr402 and their complexes with a heparin decasaccharide were studied by analytical ultracentrifugation and X-ray scattering. The sedimentation coefficient is concentration dependent, giving a value of 2.0 S at zero concentration and a frictional ratio f/f(o) of 1.2 for both allotypes. The His402 allotype showed a slightly greater self-association than the Tyr402 allotype, and small amounts of dimeric SCR-6/8 were found for both allotypes in 50 mM, 137 mM and 250 mM NaCl buffers. Sedimentation equilibrium data were interpreted in terms of a monomer-dimer equilibrium with a dissociation constant of 40 microM for the His402 form. The Guinier radius of gyration R(G) of 3.1-3.3 nm and the R(G)/R(O) ratio of 2.0-2.1 showed that SCR-6/8 is relatively extended in solution. The distance distribution function P(r) showed a maximum dimension of 10 nm, which is less than the length expected for a linear domain arrangement. The constrained scattering and sedimentation modelling of FH SCR-6/8 showed that bent SCR arrangements fit the data better than linear arrangements. Previously identified heparin-binding residues were exposed on the outside curvature of this bent domain structure. Heparin caused the formation of a more linear structure, possibly by binding to residues in the linker. It was concluded that the His402 allotype may self-associate more readily than the Tyr402 allotype, SCR-6/8 is partly responsible for the folded-back structure of intact FH, and SCR-6/8 changes conformation upon heparin binding.

Polymorphisms of Transporter Associated with Antigen Presentation, Tumor Necrosis Factor-α and Interleukin-10 and their Implications for Protection and Susceptibility to Severe Forms of Dengue Fever in Patients in Sri Lanka.

CONTEXT: To date, a clear understanding of dengue disease pathogenesis remains elusive. Some infected individuals display no symptoms while others develop severe life-threatening forms of the disease. It is widely believed that host genetic factors influence dengue severity. AIMS: This study evaluates the relationship between certain polymorphisms and dengue severity in Sri Lankan patients. SETTINGS AND DESIGN: Polymorphism studies are carried out on genes for; transporter associated with antigen presentation (TAP), promoter of tumor necrosis factor-α (TNF-α), and promoter of interleukin-10 (IL-10). In other populations, TAP1 (333), TAP2 (379), TNF-α (-308), and IL-10 (-1082, -819, -592) have been associated with dengue and a number of different diseases. Data have not been collected previously for these polymorphisms for dengue patients in Sri Lanka. MATERIALS AND METHODS: The polymorphisms were typed by amplification refractory mutation system polymerase chain reaction in 107 dengue hemorrhagic fever (DHF) patients together with 62 healthy controls. STATISTICAL ANALYSIS USED: Pearson's Chi-square contingency table analysis with Yates' correction. RESULTS: Neither the TAP nor the IL-10 polymorphisms considered individually can define dengue disease outcome with regard to severity. However, the genotype combination, IL-10 (-592/-819/-1082) CCA/ATA was significantly associated with development of severe dengue in these patients, suggesting a risk factor to developing DHF. Also, identified is the genotype combination IL-10 (-592/-819/-1082) ATA/ATG which suggested a possibility for protection from DHF. The TNF-α (-308) GG genotype was also significantly associated with severe dengue, suggesting a significant risk factor. CONCLUSIONS: The results reported here are specific to the Sri Lankan population. Comparisons with previous reports imply that data may vary from population to population.

Phylogeography and molecular epidemiology of an epidemic strain of dengue virus type 1 in Sri Lanka.

In 2009, a severe epidemic of dengue disease occurred in Sri Lanka, with higher mortality and morbidity than any previously recorded epidemic in the country. It corresponded to a shift to dengue virus 1 as the major disease-causing serotype in Sri Lanka. Dengue disease reached epidemic levels in the next 3 years. We report phylogenetic evidence that the 2009 epidemic DENV-1 strain continued to circulate within the population and caused severe disease in the epidemic of 2012. Bayesian phylogeographic analyses suggest that the 2009 Sri Lankan epidemic DENV-1 strain may have traveled directly or indirectly from Thailand through China to Sri Lanka, and after spreading within the Sri Lankan population, it traveled to Pakistan and Singapore. Our findings delineate the dissemination route of a virulent DENV-1 strain in Asia. Understanding such routes will be of particular importance to global control efforts.

X-ray and neutron scattering data and their constrained molecular modeling.

X-ray and neutron solution scattering methods provide multiparameter structural and compositional information on proteins that complements high-resolution protein crystallography and NMR studies. We describe the procedures required to (1) obtain validated X-ray and neutron scattering data, (2) perform Guinier analyses of the scattering data to extract the radius of gyration R(G) and intensity parameters, and (3) calculate the distance distribution function P(r). Constrained modeling is important because this confirms the experimental data analysis and produces families of best-fit molecular models for comparison with crystallography and NMR structures. The modeling procedures are described in terms of (4) generating appropriate starting models, (5) randomizing these for trial-and-error scattering fits, (6) identifying the final best-fit models, and (7) applying analytical ultracentrifugation (AUC) data to validate the scattering modeling. These procedures and pitfalls in them will be illustrated using work performed in the authors' laboratory on antibodies and the complement proteins of the human immune defense system. Four different types of modeling procedures are distinguished, depending on the number and type of domains in the protein. Examples when comparisons with crystallography and NMR structures are important are described. For multidomain proteins, it is often found that scattering provides essential evidence to validate or disprove a crystal structure. If a large protein cannot be crystallized, scattering provides the only means to obtain a structure.