Position Paper on Road Map for RNA Virus Research in India

The Indian subcontinent with its population density, climatic conditions, means of subsistence, socioeconomic factors as well as travel and tourism presents a fertile ground for thriving of RNA viruses. Despite being pathogens of huge significance, there is very little focus on research into the biology and pathogenesis of RNA viruses in India. Studies on epidemiology and disease burden, risk factors, the immune response to RNA viruses, circulating virus strains and virus evolution, animal models of disease, antivirals and vaccines are strikingly absent. Emerging RNA viruses such as Zika virus, Nipah virus and Crimean-Congo haemorrhagic fever virus are a matter of grave concern to India. Here we summarize the outcome of the India|EMBO symposium on “RNA viruses: immunology, pathogenesis and translational opportunities” organized at Faridabad, National Capital Region, India, on March 28–30, 2018. The meeting focused on RNA viruses (non-HIV), and both national and international experts on RNA viruses covered topics ranging from epidemiology, immune response, virus evolution and vaccine trials concerning RNA viruses. The aim of the symposium was to create a road map for RNA virus research in India. Both concrete and tentative ideas pointing towards short-term and long-term goals were presented with recommendations for follow-up at government level

Dengue virus capsid interacts with DDX3X-a potential mechanism for suppression of antiviral functions in dengue infection

Dengue virus is a pathogen of global concern and has a huge impact on public health system in low- and middle-income countries. The capsid protein of dengue virus is least conserved among related flavivirus and there is very limited information on the role of cytosolic proteins that interact with dengue virus capsid. We identified DEAD (Asp-Glu-Ala-Asp) Box Helicase 3, an X-Linked (DDX3X), cytosolic ATP-dependent RNA helicase as a dengue virus capsid-interacting protein. We show that the N-terminal region of capsid is important for interaction with DDX3X, while the N-terminal domain of DDX3X seems to be involved in interaction with dengue capsid. DDX3X was down-regulated in dengue virus infected cells at later stages of infection. Our results show that DDX3X is an antiviral protein as suppression of DDX3X expression by siRNA led to an increase in viral titers and overexpression of DDX3X led to inhibition of viral replication. Knock-down of DDX3X did not affect induction of type I interferon response upon infection suggesting that the effect of DDX3X knock-down is independent of the interferon-dependent pathways that DDX3X modulates under normal conditions. Thus, our study identifies DDX3X as a dengue virus capsid interacting protein and indicates a potential link between the antiviral functions of DDX3X and dengue capsid at later stages of dengue infection

Role of human GRP75 in miRNA mediated regulation of dengue virus replication

In recent times, RNAi has emerged as an important defence system that regulates replication of pathogens in host cells. Many RNAi related host factors especially the host miRNAs play important roles in all intrinsic cellular functions, including viral infection. We have been working on identification of mammalian host factors involved in Dengue virus infection. In the present study, we identified Glucose Regulated Protein 75kDa (GRP75), as a host factor that is associated with dicer complex, in particular with HADHA (trifunctional enzyme subunit alpha, mitochondrial), an auxiliary component of dicer complex. Knockdown of GRP75 by respective siRNAs in Huh-7 cells resulted in the accumulation of dengue viral genomic RNA suggesting a role of GRP75 in regulating dengue virus replication in human cell lines. To elucidate the mode of action of GRP75, we over expressed the protein in Huh-7 cells and analysed the host miRNAs processing. The results revealed that, GRP75 is involved in processing of host miRNA, hsa-mir-126, that down regulates dengue virus replication. These findings suggest a regulatory role of human miRNA pathway especially GRP75 protein and hsa-mir-126 in dengue virus replication. These results thus provide insights into the role of miRNAs and RNAi machinery in dengue life cycle

Role of RNA interference (RNAi) in dengue virus replication and identification of NS4B as an RNAi suppressor

RNA interference (RNAi) is an important antiviral defense response in plants and invertebrates; however, evidences for its contribution to mammalian antiviral defense are few. In the present study, we demonstrate the anti-dengue virus role of RNAi in mammalian cells. Dengue virus infection of Huh 7 cells decreased the mRNA levels of host RNAi factors, namely, Dicer, Drosha, Ago1, and Ago2, and in corollary, silencing of these genes in virus-infected cells enhanced dengue virus replication. In addition, we observed downregulation of many known human microRNAs (miRNAs) in response to viral infection. Using reversion-of-silencing assays, we further showed that NS4B of all four dengue virus serotypes is a potent RNAi suppressor. We generated a series of deletion mutants and demonstrated that NS4B mediates RNAi suppression via its middle and C-terminal domains, namely, transmembrane domain 3 (TMD3) and TMD5. Importantly, the NS4B N-terminal region, including the signal sequence 2K, which has been implicated in interferon (IFN)-antagonistic properties, was not involved in mediating RNAi suppressor activity. Site-directed mutagenesis of conserved residues revealed that a Phe-to-Ala (F112A) mutation in the TMD3 region resulted in a significant reduction of the RNAi suppression activity. The green fluorescent protein (GFP)-small interfering RNA (siRNA) biogenesis of the GFP-silenced line was considerably reduced by wild-type NS4B, while the F112A mutant abrogated this reduction. These results were further confirmed by in vitro dicer assays. Together, our results suggest the involvement of miRNA/RNAi pathways in dengue virus establishment and that dengue virus NS4B protein plays an important role in the modulation of the host RNAi/miRNA pathway to favor dengue virus replication

Immune Response to the West Nile Virus in Aged Non-Human Primates

Background: Risk of encephalitis from West Nile virus (WNV) infection increases dramatically with age. Understanding the basis of this susceptibility requires development of suitable animal models. Here, we investigated the immune response to WNV in old non-human primates. Methodology/Principal Findings: We investigated clinical, immunological and virological correlates of WNV infection in aging non-human primates. Aged (17-30yrs) and adult (6-9yrs) Rhesus macaques (RM) were challenged with WNV in the presence or the absence of the mosquito salivary gland extract (SGE) to approximate natural infection. None of the 26 animals exhibited clinical signs of the disease. Quantitative PCR suggested discrete and short-lived viremia, but infectious virus was never isolated. There was markedly increased, age-independent, proliferation of CD3- non-B cells, followed by Bcell proliferation, which correlated to the loss of detectable WNV genomes. Moreover, animals primed with mosquito salivary gland extract exhibited reduced circulating WNV RNA. While we found the expected age-associated reduction in T cell proliferation, adaptive immunity did not correlate with infection outcome. That was further confirmed in a cohort of thymectomized and/or CD8 T-cell depleted Cynomolgus macaques (CM; N = 15), who also failed to develop WNV disease. Conclusions/significance: Results are consistent with strong and age-independent innate resistance of macaques against WNV challenge. This animal model is therefore not suitable for vaccine and therapeutic testing against WNV. However, understanding the basis of their innate resistance against WNV in macaques could provide helpful clues to improve anti- WNV protection of older adults. © 2010 Wertheimer et al

Autorregulación y trabajo autónomo del estudiante en una actividad de aprendizaje basada en TIC

Este estudio analiza las estrategias de autorregulación en una práctica basada en las TIC con trabajo autónomo del estudiante (TAE). Participaron 53 estudiantes del primer curso de psicología de la Universidad Autónoma de Barcelona que realizaron prácticas de atención y percepción. Los objetivos, contenidos, materiales y evaluación de la práctica se diseñaron y desarrollaron en un entorno en línea. Posteriormente, se aplicaron varios cuestionarios con la finalidad de identificar las autovaloraciones que sobre la actividad autorregulatoria y el nivel de participación manifestaron los estudiantes. Los resultados muestran que los componentes de la dimensión cognitiva de la autorregulación (concepciones de aprendizaje y estrategias metacognitivas) se relacionan significativamente entre sí; sin embargo, no explican las diferencias encontradas en la valoración que los estudiantes realizan de su participación activa. Los factores del componente motivacional (orientación y autoeficacia) se encuentran, igualmente, relacionados entre sí, pero sólo la autoeficacia explica las diferencias observadas en la valoración de la participación activaThis work analyses a self-regulation strategies in autonomous task in an ICT-based learning activity. Participants were 53 psychology undergraduates at the Universidad Autónoma de Barcelona who made a practice in Attention and Perception Psychology. The aims, contents, materials and assessment of the practice were implemented in a virtual environment. We applied several questionnaires in order to identify the students' self-assessments about their self-regulatory activity and participation level. The results showed that cognitive dimension components in the self-regulation construct (conception of learning and metacognitive strategies) are significantly interrelated. However, this dimension does not account for the differences found in self-assessments students' active participation. With regard to motivational dimension factors (goal orientation and self-efficacy), a significant relation between them is observed, but only self-efficacy explains the differences found in self-assessments' active participatio

Funktion von Adaptorproteinen in der MPR-Sortierung

Mannose 6-phosphat Rezeptoren transportieren lösliche lysosomale Proteine und sind daher essentiel für die Biogenese des Lysosomes. Sie binden die Enzyme im trans-Golgi Netzwerk und transportieren sie in Endosomen, wo sie aufgrund des niedrigeren pH-Wertes abdissoziieren. Die Rezeptoren kehren zurück zum trans-Golgi Netzwerk und stehen für einen weiteren Transport zur Verfügung. Der Transport der Rezeptoren erfordert die monomeren Adaptor-Proteine GGA und den heterotetrameren Adaptor-Protein Komplex AP-1. Wir generierten und analysierten chimere Proteine der Rezeptor-bindenden Untereinheit µ1 des AP-1 und der homologen µ2 Untereinheit des AP-2 Komplexes der Plasmamembran und identifizierten so eine neue funktionelle Domäne in µ1 für die Sortierung der Rezeptoren. Wir analysierten auch den Endosomen - trans-Golgi Netzwerk Transport der Rezeptoren in vitro und identifizierten die beteiligten SNARE und Rab Proteine. Desweiteren erzeugten wir Zelllinien, die defizient waren für AP-1 und GGA um die Funktion der beiden Adaptoren für den trans-Golgi Netzwerk - Endosomen Transport der Rezeptoren näher zu untersuchen

JEV capsid alone alters permeability barrier functions.

<p>(A) Lysates were prepared from two caco-2 clones expressing JEV-C and analyzed by western blot for expression of JEV-C (anti-His antibody) or β-actin. (B) TER was measured in caco-2 capsid clones grown on trans-wells as described above. (C) <b>%</b> TER levels in the indicated cells at day 7 post-seeding is shown. (D) Control or JEV-C caco-2 clones were incubated with soluble fluorescein and the amount of fluorescein passing from apical to basolateral side was measured as described in materials and methods. (E) Caco-2 and capsid clone-19 cells were infected with JEV (5 pfu/cell) and supernatants were collected at indicated time post-infection. Viral titer in the supernatant was measured by plaque assay. The figures are representative of two experiments performed with three replicates. Error bars indicate mean ± s.d. *** p<0.0001 and ** p<0.002 as determined by two-tailed t-test.</p

Japanese Encephalitis Virus Disrupts Cell-Cell Junctions and Affects the Epithelial Permeability Barrier Functions

<div><p>Japanese encephalitis virus (JEV) is a neurotropic flavivirus, which causes viral encephalitis leading to death in about 20–30% of severely-infected people. Although JEV is known to be a neurotropic virus its replication in non-neuronal cells in peripheral tissues is likely to play a key role in viral dissemination and pathogenesis. We have investigated the effect of JEV infection on cellular junctions in a number of non-neuronal cells. We show that JEV affects the permeability barrier functions in polarized epithelial cells at later stages of infection. The levels of some of the tight and adherens junction proteins were reduced in epithelial and endothelial cells and also in hepatocytes. Despite the induction of antiviral response, barrier disruption was not mediated by secreted factors from the infected cells. Localization of tight junction protein claudin-1 was severely perturbed in JEV-infected cells and claudin-1 partially colocalized with JEV in intracellular compartments and targeted for lysosomal degradation. Expression of JEV-capsid alone significantly affected the permeability barrier functions in these cells. Our results suggest that JEV infection modulates cellular junctions in non-neuronal cells and compromises the permeability barrier of epithelial and endothelial cells which may play a role in viral dissemination in peripheral tissues.</p></div

Image_1_Position Paper on Road Map for RNA Virus Research in India.PDF

<p>The Indian subcontinent with its population density, climatic conditions, means of subsistence, socioeconomic factors as well as travel and tourism presents a fertile ground for thriving of RNA viruses. Despite being pathogens of huge significance, there is very little focus on research into the biology and pathogenesis of RNA viruses in India. Studies on epidemiology and disease burden, risk factors, the immune response to RNA viruses, circulating virus strains and virus evolution, animal models of disease, antivirals and vaccines are strikingly absent. Emerging RNA viruses such as Zika virus, Nipah virus and Crimean-Congo haemorrhagic fever virus are a matter of grave concern to India. Here we summarize the outcome of the India|EMBO symposium on “RNA viruses: immunology, pathogenesis and translational opportunities” organized at Faridabad, National Capital Region, India, on March 28–30, 2018. The meeting focused on RNA viruses (non-HIV), and both national and international experts on RNA viruses covered topics ranging from epidemiology, immune response, virus evolution and vaccine trials concerning RNA viruses. The aim of the symposium was to create a road map for RNA virus research in India. Both concrete and tentative ideas pointing towards short-term and long-term goals were presented with recommendations for follow-up at government level.</p