Development and evaluation of a real-time one step Reverse-Transcriptase PCR for quantitation of Chandipura Virus

<p>Abstract</p> <p>Background</p> <p>Chandipura virus (CHPV), a member of family <it>Rhabdoviridae </it>was attributed to an explosive outbreak of acute encephalitis in children in Andhra Pradesh, India in 2003 and a small outbreak among tribal children from Gujarat, Western India in 2004. The case-fatality rate ranged from 55–75%. Considering the rapid progression of the disease and high mortality, a highly sensitive method for quantifying CHPV RNA by real-time one step reverse transcriptase PCR (real-time one step RT-PCR) using TaqMan technology was developed for rapid diagnosis.</p> <p>Methods</p> <p>Primers and probe for P gene were designed and used to standardize real-time one step RT-PCR assay for CHPV RNA quantitation. Standard RNA was prepared by PCR amplification, TA cloning and run off transcription. The optimized real-time one step RT-PCR assay was compared with the diagnostic nested RT-PCR and different virus isolation systems [<it>in vivo </it>(mice) <it>in ovo </it>(eggs), <it>in vitro </it>(Vero E6, PS, RD and Sand fly cell line)] for the detection of CHPV. Sensitivity and specificity of real-time one step RT-PCR assay was evaluated with diagnostic nested RT-PCR, which is considered as a gold standard.</p> <p>Results</p> <p>Real-time one step RT-PCR was optimized using <it>in vitro </it>transcribed (IVT) RNA. Standard curve showed linear relationship for wide range of 10²-10¹⁰(r²= 0.99) with maximum Coefficient of variation (CV = 5.91%) for IVT RNA. The newly developed real-time RT-PCR was at par with nested RT-PCR in sensitivity and superior to cell lines and other living systems (embryonated eggs and infant mice) used for the isolation of the virus. Detection limit of real-time one step RT-PCR and nested RT-PCR was found to be 1.2 × 10⁰PFU/ml. RD cells, sand fly cells, infant mice, and embryonated eggs showed almost equal sensitivity (1.2 × 10²PFU/ml). Vero and PS cell-lines (1.2 × 10³PFU/ml) were least sensitive to CHPV infection. Specificity of the assay was found to be 100% when RNA from other viruses or healthy individual was used.</p> <p>Conclusion</p> <p>On account of the high sensitivity, reproducibility and specificity, the assay can be used for the rapid detection and quantitation of CHPV RNA from clinical samples during epidemics and from endemic areas. The assay may also find application in screening of antiviral compounds, understanding of pathogenesis as well as evaluation of vaccine.</p

Neurological and neuropsychological sequelae of Zika virus infection in children in León, Nicaragua

Objectives. To describe the presence and persistence of neurological and neuropsychological sequelae among children with acquired Zika virus infection and assess whether those sequelae were more common in children infected with Zika virus compared to uninfected children. Methods. We conducted a prospective cohort study of children with and without Zika virus infection in León, Nicaragua, using a standard clinical assessment tool and questionnaire to collect data on symptoms at three visits, about 6 months apart, and a battery of standardized instruments to evaluate neurocognitive function, behavior, depression, and anxiety at the last two visits. Results. Sixty-two children were enrolled, with no significant differences in demographics by infection group. Children infected with Zika virus had a range of neurological symptoms, some of which persisted for 6 to 12 months; however, no consistent pattern of symptoms was observed. At baseline a small percentage of children infected with Zika virus had an abnormal finger-to-nose test (13%), cold touch response (13%), and vibration response (15%) versus 0% in the uninfected group. Neurocognitive deficits and behavioral problems were common in both groups, with no significant differences between the groups. Children infected with Zika virus had lower cognitive efficiency scores at the 6-month visit. Anxiety and depression were infrequent in both groups. Conclusions. Larger studies are needed to definitively investigate the relationship between Zika virus infection and neurological symptoms and neurocognitive problems, with adjustment for factors affecting cognition and behavior, including mood and sleep disorders, home learning environment, history of neuroinvasive infections, and detailed family history of neuropsychological problems

Detection, Isolation and Confirmation of Crimean-Congo Hemorrhagic Fever Virus in Human, Ticks and Animals in Ahmadabad, India, 2010–2011

A nosocomial outbreak of CCHFV occurred in January 2011, in a tertiary care hospital in Ahmadabad, Gujarat State in western India. Out of a total five cases reported, contact transmission occurred to three treating medical professionals, all of whom succumbed to the disease. The only survivor was the husband of the index case. These results highlight the importance of considering CCHFV as a potential aetiology for Hemorrhagic fever (HF) cases in India. This also underlines the need for strict barrier nursing and patient isolation while managing these patients. During the investigation presence of CCHFV RNA in Hyalomma anatolicum ticks and livestock were detected in the village from where the primary case (case A) was reported. Further retrospective investigation confirmed two CCHF human cases in Rajkot village 20 kilometres to the west of Ahmadabad in 2010, and CCHFV presence in the livestock 200 kilometres to the north in the neighbouring State Rajasthan. This report shows the presence of CCHFV in human, ticks and animals in Gujarat, India. The fact of concern is the spread of this disease from one state to another due to trading of livestock

Human antibody response to Zika targets type-specific quaternary structure epitopes

The recent Zika virus (ZIKV) epidemic in the Americas has revealed rare but serious manifestations of infection. ZIKV has emerged in regions endemic for dengue virus (DENV), a closely related mosquito-borne flavivirus. Cross-reactive antibodies confound studies of ZIKV epidemiology and pathogenesis. The immune responses to ZIKV may be different in people, depending on their DENV immune status. Here, we focus on the human B cell and antibody response to ZIKV as a primary flavivirus infection to define the properties of neutralizing and protective antibodies generated in the absence of preexisting immunity to DENV. The plasma antibody and memory B cell response is highly ZIKV type–specific, and ZIKV-neutralizing antibodies mainly target quaternary structure epitopes on the viral envelope. To map viral epitopes targeted by protective antibodies, we isolated 2 type-specific monoclonal antibodies (mAbs) from a ZIKV case. Both mAbs were strongly neutralizing in vitro and protective in vivo. The mAbs recognize distinct epitopes centered on domains I and II of the envelope protein. We also demonstrate that the epitopes of these mAbs define antigenic regions commonly targeted by plasma antibodies in individuals from endemic and nonendemic regions who have recovered from ZIKV infections

Multifunctional smart conducting polymers – silver nanocomposites-modified biocellulose fibers for innovative food packaging applications

In recent decades, food-packaging markets have attracted researchers’ interest in many ways because such industries can directly affect human health. In this framework, the present study emphasizes the interesting and smart properties provided by new nanocomposites based on conducting polymers (CPs), silver nanoparticles (AgNPs), and cellulose fibers (CFs) and their possible applications as active food packaging. Polyaniline and poly(3,4-ethylenedioxythiophene) containing AgNPs were elaborated on via a simple one-step in situ chemical oxidative polymerization on CFs. Spectroscopic and microscopic characterization allowed a full discussion of the morphology and chemical structure of the nanocomposites and confirmed the successful polymerization of the monomer as well as the incorporation of AgNPs into the CP-based formulation. This study aims to demonstrate that it is possible to produce a highly efficient package with enhanced protective properties. Thus, the synthesized nanocomposites were tested as volatile organic compounds, sensors, and antibacterial and antioxidant agents. It is shown that the elaborated materials can, on the one hand, inhibit the development of biofilms and decrease the oxidation reaction rate of foodstuffs and, on the other hand, detect toxic gases generated by spoiled food. The presented method has unlocked massive opportunities for using such formulations as an interesting alternative for classical food containers. The smart and novel properties offered by the synthesized composites can be operated for future industrial applications to prevent any degradation of the packaged products by offering optimum protection and creating an atmosphere that can extend the shelf life of foodstuffs

Location-Dependent Excitatory Synaptic Interactions in Pyramidal Neuron Dendrites

<div><p>Neocortical pyramidal neurons (PNs) receive thousands of excitatory synaptic contacts on their basal dendrites. Some act as classical driver inputs while others are thought to modulate PN responses based on sensory or behavioral context, but the biophysical mechanisms that mediate classical-contextual interactions in these dendrites remain poorly understood. We hypothesized that if two excitatory pathways bias their synaptic projections towards proximal vs. distal ends of the basal branches, the very different local spike thresholds and attenuation factors for inputs near and far from the soma might provide the basis for a classical-contextual functional asymmetry. Supporting this possibility, we found both in compartmental models and electrophysiological recordings in brain slices that the responses of basal dendrites to spatially separated inputs are indeed strongly asymmetric. Distal excitation lowers the local spike threshold for more proximal inputs, while having little effect on peak responses at the soma. In contrast, proximal excitation lowers the threshold, but also substantially increases the gain of distally-driven responses. Our findings support the view that PN basal dendrites possess significant analog computing capabilities, and suggest that the diverse forms of nonlinear response modulation seen in the neocortex, including uni-modal, cross-modal, and attentional effects, could depend in part on pathway-specific biases in the spatial distribution of excitatory synaptic contacts onto PN basal dendritic arbors.</p> </div

A spectrum of possible excitatory driver-modulator (classical-contextual) interactions.

<p>Conceptual curve families illustrate: <b><i>A,</i></b> pure threshold-lowering, <b><i>B,</i></b> pure gain-boosting, and <b><i>C,</i></b> mixed modulatory effects.</p

Experimental results match model predictions.

<p><b><i>A, B,</i></b> Orthogonal views of 3D surface shown in <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1002599#pcbi-1002599-g004" target="_blank">Figure 4D</a> for the 2-compartment model. Curves are grouped into 4 categories (colors) based on modulation strength. Averages within each category are shown in bold. Gray curves in (<b><i>A</i></b>) were excluded from averaging, since corresponding experimental cases were not observed. <b><i>C, D,</i></b> Data from <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1002599#pcbi-1002599-g005" target="_blank">Figure 5F,G</a> was scaled vertically and horizontally using fiducial points for each cell (triangle, square, pentagon, circle; see <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1002599#s4" target="_blank">Materials and Methods</a>) to allow comparison to model results despite different stimulus locations, efficacy, branch input resistances, etc.</p

Proximal-distal interactions in a time-invariant 2-compartmental model are nearly indistinguishable from those produced by the detailed compartmental model.

<p><b><i>A,</i></b> Schematic proximal and distal ‘stimulating electrodes’ are shown activating one highlighted terminal basal dendrite. <b><i>B,</i></b> Peak somatic responses for inputs at 90 and 150 µm as illustrated in (<b><i>A</i></b>). Plot shares color bar with <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1002599#pcbi-1002599-g003" target="_blank">Figure 3A</a>. <b><i>C,</i></b> 2-compartment circuit diagram with proximal and distal NMDA conductances. <b><i>D,</i></b> Time-invariant responses for 2-compartment model. Parameters were hand tuned to resemble (<b><i>C</i></b>): Axial, distal leak and proximal leak conductances were 2.5,0.25, and 4 A.U., respectively. NMDA peak conductance was 0.5 A.U. per synapse. Overall peak response in 2-compartment model was scaled to match overall peak in (<b><i>C</i></b>) (V_soma = 15.2 mV). For details on 2-compartment model see Figure S1 in <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1002599#pcbi.1002599.s001" target="_blank">Text S1</a>.</p