Emergence of human West Nile Virus infection in Sri Lanka.

Background: West Nile virus (WNV) has emerged as one of the most common causes of epidemic meningoencephalitis worldwide. Most human infections are asymptomatic. However, neuroinvasive disease characterized by meningitis, encephalitis and/or acute flaccid paralysis is associated with significant morbidity and mortality.

Viral RNA intermediates as targets for detection and discovery of novel and emerging mosquito-borne viruses.

Mosquito-borne viruses encompass a range of virus families, comprising a number of significant human pathogens (e.g., dengue viruses, West Nile virus, Chikungunya virus).

Plasmodium vivax: restricted tropism and rapid remodeling of CD71-positive reticulocytes.

Plasmodium vivax merozoites only invade reticulocytes, a minor though heterogeneous population of red blood cell precursors that can be graded by levels of transferrin receptor (CD71) expression. The development of a protocol that allows sorting reticulocytes into defined developmental stages and a robust ex vivo P vivax invasion assay has made it possible for the first time to investigate the fine-scale invasion preference of P vivax merozoites. Surprisingly, it was the immature reticulocytes (CD71(+)) that are generally restricted to the bone marrow that were preferentially invaded, whereas older reticulocytes (CD71(-)), principally found in the peripheral blood, were rarely invaded.

The combination of type I IFN, TNF-α, and cell surface receptor engagement with dendritic cells enables NK cells to overcome immune evasion by dengue virus.

Clinical studies have suggested the importance of the NK cell response against dengue virus (DenV), an arboviral infection that afflicts >50 million individuals each year. However, a comprehensive understanding of the NK cell response against dengue-infected cells is lacking. To characterize cell-contact mechanisms and soluble factors that contribute to the antidengue response, primary human NK cells were cocultured with autologous DenV-infected monocyte-derived dendritic cells (DC).

Microscopy techniques in flavivirus research.

The Flavivirus genus is composed of many medically important viruses that cause high morbidity and mortality, which include Dengue and West Nile viruses. Various molecular and biochemical techniques have been developed in the endeavour to study flaviviruses. However, microscopy techniques still have irreplaceable roles in the identification of novel virus pathogens and characterization of morphological changes in virus-infected cells.

Chikungunya virus nsP3 & nsP4 interacts with HSP-90 to promote virus replication: HSP-90 inhibitors reduce CHIKV infection and inflammation in vivo.

The global emergence of Chikungunya virus (CHIKV) infection is alarming and currently there is no licensed vaccine or antiviral treatment available to mitigate this disease. CHIKV infection typically results in high viral load with an outcome of high fever, skin rashes, muscle pain, and sequelae of prolonged arthritis, which occurs in >90% of the infected cases. In this study, using biochemical pull-downs, mass-spectrometry, and microscopic imaging techniques, we have identified novel interactions between CHIKV nsP3 or nsP4 proteins with the host stress-pathway chaperone HSP-90 protein.

Significant biochemical, biophysical and metabolic diversity in circulating human cord blood reticulocytes.

Background: The transition from enucleated reticulocytes to mature normocytes is marked by substantial remodeling of the erythrocytic cytoplasm and membrane. Despite conspicuous changes, most studies describe the maturing reticulocyte as a homogenous erythropoietic cell type. While reticulocyte staging based on fluorescent RNA stains such as thiazole orange have been useful in a clinical setting; these 'sub-vital' stains may confound delicate studies on reticulocyte biology and may preclude their use in heamoparasite invasion studies.

Differential regulation of angiopoietin 1 and angiopoietin 2 during dengue virus infection of human umbilical vein endothelial cells: implications for endothelial hyperpermeability.

Infection with dengue virus (DV) can result in dengue hemorrhagic fever and dengue shock syndrome, where patients suffer from bleeding and plasma leakage involving endothelial cells. Angiopoietins (Ang) 1 and 2 are important angiogenic factors that affect endothelial barrier integrity. In this study, DV was observed to induce endothelial leakage at multiplicity of infection of 10 in primary human umbilical vein endothelial cells (HUVEC) with interendothelial gap formation.

A highly divergent Encephalomyocarditis virus isolated from nonhuman primates in Singapore.

Background: In 2001 and 2002, fatal myocarditis resulted in the sudden deaths of four, two adult and two juvenile, orang utans out of a cohort of 26 in the Singapore Zoological Gardens.

Methods: Of the four orang utans that underwent post-mortem examination, virus isolation was performed from the tissue homogenates of the heart and lung obtained from the two juvenile orang utans in Vero cell cultures. The tissue culture fluid was examined using electron microscopy.

Contributions of mast cells and vasoactive products, leukotrienes and chymase, to dengue virus-induced vascular leakage.

Dengue Virus (DENV), a flavivirus spread by mosquito vectors, can cause vascular leakage and hemorrhaging. However, the processes that underlie increased vascular permeability and pathological plasma leakage during viral hemorrhagic fevers are largely unknown. Mast cells (MCs) are activated in vivo during DENV infection, and we show that this elevates systemic levels of their vasoactive products, including chymase, and promotes vascular leakage.

A novel platform for virus-like particle-display of flaviviral envelope domain III: induction of Dengue and West Nile virus neutralizing antibodies.

CD16-RIgE is a chimeric human membrane glycoprotein consisting of the CD16 ectodomain fused to the transmembrane domain and cytoplasmic tail of the gamma chain of the high affinity receptor of IgE (RIgE). Coexpression of CD16-RIgE and HIV-1 Pr55Gag polyprotein precursor (Pr55GagHIV) in insect cells resulted in the incorporation of CD16-RIgE glycoprotein into the envelope of extracellular virus-like particles (VLPs), a phenomenon known as pseudotyping. Taking advantage of this property, we replaced the CD16 ectodomain of CD16-RIgE by the envelope glycoprotein domain III (DIII) of dengue virus serotype 1 (DENV1) or West Nile virus Kunjin (WNVKun).

West Nile virus and dengue virus capsid protein negates the antiviral activity of human Sec3 protein through the proteasome pathway.

Flavivirus capsid (C) protein is a key structural component of virus particles. The non-structural role of C protein in the pathogenesis of arthropod-borne flaviviruses is not clearly deciphered. This study showed that West Nile virus (WNV) and dengue virus (DENV) utilized C protein to reduce human Sec3p (hSec3p) levels at post-transcriptional level through activation of chymotrypsin-like proteolytic function of 20S proteasome.

Optimized sequential purification protocol for in vivo site-specific biotinylated full-length dengue virus capsid protein.

Dengue virus (DENV) capsid (C) protein is one of the three structural proteins that form a mature virus. The main challenge impeding the study of this protein is to generate pure non-truncated, full-length C proteins for structural and functional studies. This is mainly due to its small molecular weight, highly positively charged, stability and solubility properties.

Differential unfolded protein response during Chikungunya and Sindbis virus infection: CHIKV nsP4 suppresses eIF2α phosphorylation.

Chikungunya (CHIKV) and Sindbis (SINV) are arboviruses belonging to the alphavirus genus within the Togaviridae family. They cause frequent epidemics of febrile illness and long-term arthralgic sequelae that affect millions of people each year. Both viruses replicate prodigiously in infected patients and in vitro in mammalian cells, suggesting some level of control over the host cellular translational machinery that senses and appropriately directs the cell's fate through the unfolded protein response (UPR).

Inhibition of chikungunya virus replication by harringtonine, a novel antiviral that suppresses viral protein expression.

Chikungunya virus (CHIKV) is a mosquito-transmitted virus that has reemerged as a significant public health threat in the last decade. Since the 2005-2006 chikungunya fever epidemic in the Indian Ocean island of La Réunion, millions of people in more than 40 countries have been infected. Despite this, there is currently no antiviral treatment for chikungunya infection.

Dengue virus infection mediates HMGB1 release from monocytes involving PCAF acetylase complex and induces vascular leakage in endothelial cells.

High mobility group box 1 (HMGB1) protein is released from cells as a pro-inflammatory cytokine in response to an injury or infection. During dengue hemorrhagic fever (DHF)/dengue shock syndrome (DSS), a number of pro-inflammatory cytokines are released, contributing to disease pathogenesis. In this study, the release of HMGB1 from human myelogenous leukemia cell line K562 and primary peripheral blood monocytes (PBM) cells was examined during dengue virus (DV)-infection.

Generation of patient-specific induced pluripotent stem cell-derived cardiomyocytes as a cellular model of arrhythmogenic right ventricular cardiomyopathy.

Aims: Arrhythmogenic right ventricular cardiomyopathy (ARVC) is a primary heart muscle disorder associated with sudden cardiac death. Its pathophysiology is still poorly understood. We aimed to produce an in vitro cellular model of ARVC using patient-specific induced pluripotent stem cell (iPSC)-derived cardiomyocytes and determine whether the model could recapitulate key features of the disease phenotype.

The structural basis for serotype-specific neutralization of dengue virus by a human antibody.

Dengue virus (DENV) is a mosquito-borne flavivirus that affects 2.5 billion people worldwide. There are four dengue serotypes (DENV1 to DENV4), and infection with one elicits lifelong immunity to that serotype but offers only transient protection against the other serotypes.

Characterization and separation of Cryptosporidium and Giardia cells using on-chip dielectrophoresis.

Dielectrophoresis (DEP) has been shown to have significant potential for the characterization of cells and could become an efficient tool for rapid identification and assessment of microorganisms. The present work is focused on the trapping, characterization, and separation of two species of Cryptosporidium (C. parvum and C.

Immune surveillance by mast cells during dengue infection promotes natural killer (NK) and NKT-cell recruitment and viral clearance.

A wealth of evidence supports the essential contributions of mast cells (MCs) to immune defense against bacteria and parasites; however, the role of MCs in viral infections has not been defined. We now report that rodent, monkey, and human MCs are able to detect dengue virus (DENV), a lymphotropic, enveloped, single-stranded, positive-sense RNA virus that results in MC activation and degranulation. We observe that the response of MCs to DENV also involves the activation of antiviral intracellular host response pathways, melanoma differentiation-associated gene 5 (MDA5) and retinoic acid inducible gene 1 (RIG-I), and the de novo transcription of cytokines, including TNF-α and IFN-α, and chemokines, such as CCL5, CXCL12, and CX3CL1.

A novel CARD containing splice-isoform of CIITA regulates nitric oxide synthesis in dendritic cells.

MHC class II expression is controlled mainly at transcriptional level by class II transactivator (CIITA), which is a non-DNA binding coactivator and serves as a master control factor for MHC class II genes expression. Here, we describe the function of a novel splice-isoform of CIITA, DC-expressed caspase inhibitory isoform of CIITA (or DC-CASPIC), and we show that the expression of DCCASPIC in DC is upregulated upon lipopolysaccharides (LPS) induction. DC-CASPIC localizes to mitochondria, and protein-protein interaction study demonstrates that DC-CASPIC interacts with caspases and inhibits its activity in DC.

Dephosphorylation of West Nile virus capsid protein enhances the processes of nucleocapsid assembly.

West Nile virus (WNV) capsid (C) protein is one of the three viral structural proteins and it encapsidates the viral RNA to form the nucleocapsid. It is known to be a multifunctional protein involved in assembly and apoptosis. WNV C protein was previously found to be phosphorylated in infected cells and bioinformatic analysis revealed 5 putative phosphorylation sites at serine 26, 36, 83, 99 and threonine 100.

Metabolomics approach for investigation of effects of dengue virus infection using the EA.hy926 cell line.

This paper describes a multiplatform analytical approach combining proton nuclear magnetic resonance ((1)H NMR) spectroscopy and mass spectrometry (MS), together with pattern recognition tools in a metabolomic study used to investigate the effects of dengue virus infection. The four serotypes of dengue, DEN-1, DEN-2, DEN-3, and DEN-4, were inoculated into the EA.hy926 cell line, which was then incubated for various time intervals.

Rapid purification of recombinant dengue and West Nile virus envelope Domain III proteins by metal affinity membrane chromatography.

Arthropod-borne flaviviruses such as dengue virus (DENV) and West Nile virus (WNV) pose significant health threats to the global community. Due to escalating numbers of DENV and WNV infections worldwide, development of an effective vaccine remains a global health priority. As flavivirus envelope Domain III (DIII) protein is highly immunogenic and capable of inducing neutralizing antibodies against wild-type virus, it is both a potential protein subunit vaccine candidate and a suitable diagnostic reagent.

West Nile virus capsid protein interaction with importin and HDM2 protein is regulated by protein kinase C-mediated phosphorylation.

West Nile virus (WNV) capsid (C) protein was shown to enter the nucleus via importin-mediated pathway and induce apoptosis although the precise regulatory mechanisms for such events have remained elusive. In this study, it was shown that WNV C protein was phosphorylated by protein kinase C (PKC). PKC-mediated phosphorylation influenced nuclear trafficking of C protein by modulating the efficiency of C protein-importin-alpha binding.