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Dash P.K.,Institute Pasteur Paris | Boutonnier A.,Institute Pasteur Paris | Prina E.,Institute Pasteur Paris | Sharma S.,Defence R and D Establishment DRDE | Reiter P.,Institute Pasteur Paris
Virology Journal | Year: 2012

Background. Yellow Fever virus (YFV) is an important arboviral pathogen in much of sub-Saharan Africa and the tropical Americas. It is the prototype member of the genus Flavivirus and is transmitted primarily by Aedes (Stegomyia) mosquitoes. The incidence of human infections in endemic areas has risen in recent years. Prompt and dependable identification of YFV is a critical component of response to suspect cases. Results. We developed a one-step SYBR Green I-based real-time quantitative RT-PCR (qRT-PCR) assay targeting the 5'NTR and capsid-gene junction - for rapid detection and quantification of YFV. The detection limit was 1 PFU/mL, 10-fold more sensitive than conventional RT-PCR, and there was no cross-reactivity with closely related flaviviruses or with alphaviruses. Viral load in samples was determined by standard curve plotted from cycle threshold (Ct) values and virus concentration. The efficacy of the assay in mosquitoes was assessed with spiked samples. The utility of the assay for screening of pooled mosquitoes was also confirmed. Replication of a Cameroon isolate of YFV in Ae. aegypti revealed a marked variation in susceptibility among different colonies at different days post infection (pi). Conclusions. The SYBR Green-1 based qRT-PCR assay is a faster, simpler, more sensitive and less expensive procedure for detection and quantification of YFV than other currently used methods. © 2011 Dash et al; licensee BioMed Central Ltd.

Parashar A.,KLE University | Parashar S.,Defence R and D Establishment DRDE | Zingade A.,KLE University | Gupta S.,Peoples University | Sanikop S.,KLE University
Oral Science International | Year: 2015

The diversity of signalling opportunities within microbial communities, and the significant role of these molecules in coordinating gene expression and promoting biofilm formation, has provided the impetus to investigate the potential of inhibitory analogues to disrupt these networks, thereby providing mechanisms to control or influence the development of dental plaque. Within the oral biofilms, resident bacterial cells interact with one another and exchange messages in the form of signalling molecules and metabolites. In this review article, our aim is to elaborate the role of this quorum sensing and their involvement in pathogenesis to decipher information that can be useful to target pathways to control diseases. © 2015 Japanese Stomatological Society.

Dash P.K.,Defence R and D Establishment DRDE | Sharma S.,Defence R and D Establishment DRDE | Soni M.,Defence R and D Establishment DRDE | Agarwal A.,Defence R and D Establishment DRDE | And 3 more authors.
Virus Research | Year: 2015

Dengue is now hyper-endemic in most parts of south and southeast Asia including India. The northern India particularly national capital New Delhi witnessed major Dengue outbreaks with Dengue virus type 1 (DENV-1) as the dominant serotype since last five years. This study was initiated to decipher the complete genome information of recently circulating DENV-1 (2009-2011) along with the prototype Indian DENV-1, isolated in 1956. Further extensive ML phylogenetic and Bayesian phylogeography analysis was carried out to investigate the evolution of this virus and understand its spatiotemporal diffusion across the globe. The complete genome analysis revealed deletion of a unique 21-nucleotide stretch in the 3' un-translated region of recent Indian DENV-1. The north Indian DENV-1 revealed up to 5.2% nucleotide sequence difference compared to recent isolates from southern India. Selection pressure analysis revealed positive selection in few amino acid sites of both structural and non-structural proteins. The molecular phylogeny classified the Indian DENV-1 into genotype III, which is also known as cosmopolitan genotype. The northern and southern Indian DENV-1 were grouped into distinct clades. The molecular clock analysis estimated a mean evolutionary rate of 7.08. ×. 10(-4) substitutions/site/year for cosmopolitan genotype. The phylogeography analysis revealed that the cosmopolitan genotype DENV-1 originated ~1938 in India and subsequently spread globally. The diffusion of virus from India to Caribbean and South America was confirmed through SPREAD analysis. This study also confirmed the temporal displacement of different clades of DENV-1 in India over last five decades. © 2014 Elsevier B.V.

Joshi G.,Defence R and D Establishment DRDE | Dash P.K.,Defence R and D Establishment DRDE | Agarwal A.,Defence R and D Establishment DRDE | Sharma S.,Defence R and D Establishment DRDE | Parida M.,Defence R and D Establishment DRDE
Current Gene Therapy | Year: 2015

Rationale: The first influenza pandemic of 21st century was attributed to a novel quadruple reassortant H1N1 virus that emerged in 2009. Currently available therapies for influenza have drugresistant. Therefore, there is a need to develop new generation immunotherapeutic antiviral strategy. This study described the efficacy of a novel bifunctional immunostimulatory siRNAs against H1N1pdm swine flu virus by targeting the Nucleocapsid (NP) gene. Methods & Findings: Small interfering RNAs (siRNA) targeting conserved region of NP were screened for antiviral efficacy in human lung epithelial cells (A549). Further, a bifunctional siRNA was synthesized by combining immunostimulatory sequence (5’-UGUGU-3’) with NP specific siRNA. This immunostimulatory siRNA (NP-1-is) revealed strong antiviral effect through reduction in mRNA copies (99.58%), reduction in virus associated cell apoptosis and inhibition of nucleocapsid protein in western blot. This immunostimulatory siRNA was found more effective than nontagged siRNA. Further studies including dose dependent and time course kinetics revealed that the NP-1-is siRNA is more effective at 20-80 nM with significant protection upto 48 hpi. Besides, the qRT-PCR and western blot analysis confirmed higher antiviral response of immunostimulatory siRNA was due to upregulation of TLR-7 MyD88, IRF-7 and IFN-_. Conclusions: This study paves the way for broad-spectrum RNAi-based therapeutics using immunostimulatory motif towards improved antiviral effect. Hence this approach will be useful to confront the sudden emergence of pandemic strains. © 2015 Bentham Science Publishers.

Dash P.K.,Defence R and D Establishment DRDE | Sharma S.,Defence R and D Establishment DRDE | Soni M.,Defence R and D Establishment DRDE | Agarwal A.,Defence R and D Establishment DRDE | And 2 more authors.
Biochemical and Biophysical Research Communications | Year: 2013

Dengue is the most important arboviral infection of global public health significance. It is now endemic in most parts of the South East Asia including India. Though Dengue virus type 2 (DENV-2) is predominantly associated with major outbreaks in India, complete genome information of Indian DENV-2 is not available. In this study, the full-length genome of five DENV-2 isolates (four from 2001 to 2011 and one from 1960), from different parts of India was determined. The complete genome of the Indian DENV-2 was found to be 10,670 bases long with an open reading frame coding for 3391 amino acids. The recent Indian DENV-2 (2001-2011) revealed a nucleotide sequence identity of around 90% and 97% with an older Indian DENV-2 (1960) and closely related Sri Lankan and Chinese DENV-2 respectively. Presence of unique amino acid residues and non-conservative substitutions in critical amino acid residues of major structural and non-structural proteins was observed in recent Indian DENV-2. Selection pressure analysis revealed positive selection in few amino acid sites of the genes encoding for structural and non-structural proteins. The molecular phylogenetic analysis based on comparison of both complete coding region and envelope protein gene with globally diverse DENV-2 viruses classified the recent Indian isolates into a unique South Asian clade within Cosmopolitan genotype. A shift of genotype from American to Cosmopolitan in 1970s characterized the evolution of DENV-2 in India. Present study is the first report on complete genome characterization of emerging DENV-2 isolates from India and highlights the circulation of a unique clade in South Asia. © 2013 Elsevier Inc.

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