Dengue virus replication enhances labile zinc pools by modulation of ZIP8.

Zinc-dependent viral proteins rely on intracellular zinc homeostasis for successful completion of infectious life-cycle. Here, we report that the intracellular labile zinc levels were elevated at early stages of dengue virus (DENV) infection in hepatic cells and this increase in free zinc was abolished in cells infected with UV-inactivated virus or with a DENV replication inhibitor implicating a role for zinc homeostasis in viral RNA replication. This change in free zinc was mediated by zinc transporter, ZIP8, as siRNA-mediated knockdown of ZIP8 resulted in abrogation of increase in free zinc levels leading to significant reduction in DENV titers suggesting a crucial role for ZIP8 in early stages of DENV replication. Furthermore, elevated free zinc levels correlated with high copy numbers of dengue genome in peripheral blood leukocytes obtained from dengue patients compared to healthy controls suggesting a critical role for zinc homeostasis in dengue infection. TAKE AWAYS: Dengue virus utilises cellular zinc homeostasis during replication of its RNA. ZIP8 upregulates free zinc levels during dengue virus replication. Enhanced viremia associates with elevated intracellular free zinc in dengue.

The coevolution of large and small terminases of bacteriophages is a result of purifying selection leading to phenotypic stabilization.

Genome packaging in many dsDNA phages requires a series of precisely coordinated actions of two phage-coded proteins, namely, large terminase (TerL) and small terminase (TerS) with DNA and ATP, and with each other. Despite the strict functional conservation, TerL and TerS homologs exhibit large sequence variations. We investigated the sequence variability across eight phage types and observed a coevolutionary framework wherein the genealogy of TerL homologs mirrored that of the corresponding TerS homologs. Furthermore, a high purifying selection observed (dN/dS«1) indicated strong structural constraints on both TerL and TerS, and identify coevolving residues in TerL and TerS of phage T4 and lambda. Using the highly coevolving (correlation coefficient of 0.99) TerL and TerS of phage N4, we show that their biochemical features are similar to the phylogenetically divergent phage λ terminases. We also demonstrate using the Surface Plasma Resonance (SPR) technique that phage N4 TerL transiently interacts with TerS.

Label-Free Detection of Escherichia coli from Mixed Bacterial Cultures Using Bacteriophage T4 on Plasmonic Fiber-Optic Sensor.

Consumption of water contaminated with pathogenic bacteria is a major cause of water-borne diseases. To address this challenge, we have developed a novel and sensitive sensing scheme for the rapid detection of bacteria (Escherichia coliB40) on a fiber-optic platform using bacteriophage (T4) as a bio-recognition element. The novelty of our sensing scheme is that instead of bacteriophages, bacteria (analyte) were first captured on the sensing surface and then the sensing surface was subjected to bacteriophages for specific detection of bacteria. The sensor was subjected to 100 to 107 cfu/mL of E. coliB40 spiked in a lake water matrix, and the least concentration of bacteria that could be easily detected was found to be 1000 cfu/mL. The control studies were performed with nonhost bacteria Pseudomonas aeruginosa. Bacteriophage T4, being specific to its host E. coliB40, did not interact with P. aeruginosa captured on the sensing probe, giving a negligible nonspecific response. Due to the specificity of bacteriophages to its host bacteria, it is possible to use this scheme to carry out the detection of specific bacteria in a mixed sample (containing a combination of bacteria) using bacteriophages specific to it. The sensor was able to detect E. coliB40 (target bacteria) even in the presence of a very high concentration (1000 times higher) of P. aeruginosa (nontarget bacteria).

Oxidative stress specifically inhibits replication of dengue virus.

Reactive oxygen species (ROS) are chemically active species which are involved in maintaining cellular and signalling processes at physiological concentrations. Therefore, cellular components that regulate redox balance are likely to play a crucial role in viral life-cycle either as promoters of viral replication or with antiviral functions. Zinc is an essential micronutrient associated with anti-oxidative systems and helps in maintaining a balanced cellular redox state. Here, we show that zinc chelation leads to induction of reactive oxygen species (ROS) in epithelial cells and addition of zinc restores ROS levels to basal state. Addition of ROS (H2O2) inhibited dengue virus (DENV) infection in a dose-dependent manner indicating that oxidative stress has adverse effects on DENV infection. ROS affects early stages of DENV replication as observed by quantitation of positive and negative strand viral RNA. We observed that addition of ROS specifically affected viral titres of positive strand RNA viruses. We further demonstrate that ROS specifically altered SEC31A expression at the ER suggesting a role for SEC31A-mediated pathways in the life-cycle of positive strand RNA viruses and provides an opportunity to identify drug targets regulating oxidative stress responses for antiviral development.

The genera Erhaia and Tricula (Gastropoda, Rissooidea, Amnicolidae and Pomatiopsidae) in Bhutan and elsewhere in the eastern Himalaya.

Shells of the Rissooidea species that are known from Bhutan are characterized. Tricula montana is reported from that country for the first time. Two Erhaia species from Bhutan are described as new to science, viz. E. jannei sp. nov., and E. pelkiae sp. nov., The holotypes of the Erhaia species that were described from Nepal are figured with photographs for the first time and compared with the congeneric taxa from Bhutan and India. Erhaia nainitalensis is considered a senior synonym of E. chandeshwariensis. An identification key is presented for the Erhaia species of the Himalayan foothills.

Bacteriophage N4 large terminase: expression, purification and X-ray crystallographic analysis.

Genome packaging is a critical step in the assembly of dsDNA bacteriophages and is carried out by a powerful molecular motor known as the large terminase. To date, wild-type structures of only two large terminase proteins are available, and more structural information is needed to understand the genome-packaging mechanism. Towards this goal, the large and small terminase proteins from bacteriophage N4, which infects the Escherichia coli K12 strain, have been cloned, expressed and purified. The purified putative large terminase protein hydrolyzes ATP, and this is enhanced in the presence of the small terminase. The large terminase protein was crystallized using the sitting-drop vapour-diffusion method and the crystal diffracted to 2.8 Šresolution using a home X-ray source. Analysis of the X-ray diffraction data showed that the crystal belonged to space group P212121, with unit-cell parameters a = 53.7, b = 93.6, c = 124.9 Å, α = ß = γ = 90°. The crystal had a solvent content of 50.2% and contained one molecule in the asymmetric unit.