Evaluation of abrin induced nephrotoxicity by using novel renal injury markers.

Abrin is a potent plant toxin analogous to ricin that is derived from the seeds of Abrus precatorius plant. It belongs to the family of type II ribosome-inactivating proteins and causes cell death by irreversibly inactivating ribosomes through site-specific depurination. In this study we examined the in vivo nephrotoxicity potential of abrin toxin in terms of oxidative stress, inflammation, histopathological changes and biomarkers of kidney injury. Animals were exposed to 0.5 and 1.0 LD50 dose of abrin by intraperitoneal route and observed for 1, 3, and 7 day post-toxin exposure. Depletion of reduced glutathione and increased lipid peroxidation levels were observed in abrin treated mice. In addition, abrin also induced inflammation in the kidneys as observed through expression of MMP-9 and MMP-9/NGAL complex in abrin treated groups by using zymography method. Nephrotoxicity was also evaluated by western blot analysis of kidney injury biomarkers including Clusterin, Cystatin C and NGAL, and their results indicate severity of kidney injury in abrin treated groups. Kidney histology confirmed inflammatory changes due to abrin. The data generated in the present study clearly prove the nephrotoxicity potential of abrin.

Differential effects of route of T-2 toxin exposure on hepatic oxidative damage in mice.

T-2 toxin is the most toxic among mycotoxins and poses a potential health hazard for both humans and animals. At high doses, T-2 toxin can cause shock-like syndrome that can result in death. We evaluated the effect of time course and route of exposure on hepatic oxidative damage in mice and it is only such study so far to compare the effects of dermal and subcutaneous exposure of T-2 toxin. Mice were exposed to 1 LD50 of T-2 toxin either by percutaneous (5.94 mg/kg body weight) or subcutaneous (1.54 mg/kg body weight) route and sacrificed at 0, 1, 3, and 7 days postexposure. Analysis of a number of serum biochemical variables, antioxidant enzymes activity, gene and protein expression by immunoblot assay showed time and route dependent effects of T-2 induced hepatic oxidative damage. Time dependent increase in protein carbonyl content and protein oxidation was seen in serum and liver. Results of our study may provide possible mechanism for developing medical countermeasures against T-2 toxin.

Rapid detection and differentiation of dengue virus serotypes by NS1 specific reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay in patients presenting to a tertiary care hospital in Hyderabad, India.

Early and rapid detection of dengue virus (DENV) infection during the acute phase of illness is crucial for proper patient management and prevention of the spread of the infection. In the present study, the standardization and validation of a one step, four tube reverse transcription loop-mediated isothermal amplification assay (RT-LAMP) for rapid detection and serotyping of the DENV targeting NS1 gene using the Genie® II flourometer was carried out. The performance of the RT-LAMP was compared to RT-PCR, CDC 1-4 Real time PCR and the NS1 antigen ELISA, IgM and IgG anti DENV antibodies. Acute DENV infection was confirmed in 250/300 patients suspected clinically of DENV infection. RT- LAMP and CDC 1-4 Real time PCR assay was positive in 148/250 patients, while 92/250 patients were positive for anti- Dengue IgM and IgG antibodies. The RT-LAMP assay and the CDC real-time RT-PCR assay showed high concordance (k=1.0). The detection rate of acute DENV infection improved to 96% (240/250) when the results of RT-LAMP were combined with NS1 Ag, IgM and IgG ELISA. The RT-LAMP had a detection limit of 100 copies for DEN-1 and DEN-2, 10 copies for DEN-3 and DEN-4 compared to 1000 copies for DEN-1 and DEN-2, 100 copies for DEN-3 and DEN-4 by the conventional RT-PCR. The assay showed 100% specificity. The RT-LAMP assay developed in this study has potential use for early clinical diagnosis, serotyping and surveillance of DENV infection in endemic countries such as India.

Comparative evaluation of the diagnostic potential of recombinant envelope proteins and native cell culture purified viral antigens of Chikungunya virus.

Despite the fact that Chikungunya resurgence is associated with epidemic of unprecedented magnitude, there are challenges in the field of its clinical diagnosis. However, serological tests in an ELISA format provide a rapid tool for the diagnosis of Chikungunya infection. Indeed, ELISAs based on recombinant proteins hold a great promise as these methods are cost effective and are free from the risk of handling biohazardous material. In this study, the performance of recombinant CHIKV antigens was compared in various ELISA formats for the diagnosis of Chikungunya. Two recombinant antigens derived from the envelope proteins of Chikungunya virus were prepared and evaluated by comparing their competence for detecting circulating antibodies in serum samples of patients infected with CHIKV using MAC-ELISA and indirect IgM-ELISA. The efficacy of the recombinant antigens was also compared with the native antigen. The indirect antibody capture IgM microplate ELISA revealed ≥90% concordance with the native antigen in detecting the CHIKV specific IgM antibodies whereas the recombinant antigen based MAC-ELISA showed 100% specificity. The recombinant antigens used in this study were effective and reliable targets for the diagnosis of CHIKV infection and also provide an alternative for native antigen use which is potentially biohazardous.

Complete genome sequencing and evolutionary analysis of Indian isolates of Dengue virus type 2.

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.

Molecular and virological investigation of a focal chikungunya outbreak in northern India.

Chikungunya (CHIK) fever is one of the most important arboviral infections of medical significance. The objective of the present study is to identify and characterize the etiology of a focal febrile arthritis outbreak from Gwalior, northern India, during October-November 2010. A detailed virological (isolation) and molecular (end-point RT-PCR, quantitative RT-PCR, and nucleotide sequencing) investigation of this outbreak was carried out by collecting and studying 52 clinical samples and 15 mosquito pools from the affected region. The investigation revealed the presence of CHIK viral RNA in 29% of clinical samples and 13% mosquito pool by RT-PCR. The quantification of CHIK viral RNA in samples varied from 10(2.50) to 10(6.67) copies/mL, as demonstrated through quantitative RT-PCR. In addition, six CHIK viruses were isolated from RT-PCR positive samples. The nucleotide sequences of partial E1 gene of five representative CHIK viruses were deciphered, which revealed that all the viral strains from this outbreak belong to the recently emerging ECS African genotype. Identification of Chikungunya virus ECSA African genotype as the etiology of the present outbreak confirms the continued circulation of the novel genotype, since 2006, in India. The identification of CHIK virus in Aedes aegypti also confirmed it as the major vector in northern India.

Transcriptomic profile of host response in mouse brain after exposure to plant toxin abrin.

Abrin toxin is a plant glycoprotein, which is similar in structure and properties to ricin and is obtained from the seeds of Abrus precatorius (jequirity bean). Abrin is highly toxic, with an estimated human fatal dose of 0.1-1 µg/kg, and has caused death after accidental and intentional poisoning. Abrin is a potent biological toxin warfare agent. There are no chemical antidotes available against the toxin. Neurological symptoms like delirium, hallucinations, reduced consciousness and generalized seizures were reported in human poisoning cases. Death of a patient with symptoms of acute demyelinating encephalopathy with gastrointestinal bleeding due to ingestion of abrin seeds was reported in India. The aim of this study was to examine both dose and time-dependent transcriptional responses induced by abrin in the adult mouse brain. Mice (n=6) were exposed to 1 and 2 LD50 (2.83 and 5.66 µg/kg respectively) dose of abrin by intraperitoneal route and observed over 3 days. A subset of animals (n=3) were sacrificed at 1 and 2 day intervals for microarray and histopathology analysis. None of the 2 LD50 exposed animals survived till 3 days. The histopathological analysis showed the severe damage in brain and the infiltration of inflammatory cells in a dose and time dependent manner. The abrin exposure resulted in the induction of rapid immune and inflammatory response in brain. Clinical biochemistry parameters like lactate dehydrogenase, aspartate aminotransferase, urea and creatinine showed significant increase at 2-day 2 LD50 exposure. The whole genome microarray data revealed the significant regulation of various pathways like MAPK pathway, cytokine-cytokine receptor interaction, calcium signaling pathway, Jak-STAT signaling pathway and natural killer cell mediated toxicity. The comparison of differential gene expression at both the doses showed dose dependent effects of abrin toxicity. The real-time qRT-PCR analysis of selected genes supported the microarray data. This is the first report on host-gene response using whole genome microarray in an animal model after abrin exposure. The data generated provides leads for developing suitable medical counter measures against abrin poisoning.

Development of a pilot-scale production process and characterization of a recombinant Japanese encephalitis virus envelope domain III protein expressed in Escherichia coli.

Japanese encephalitis virus (JEV) is the most important cause of encephalitis in most Asian regions. JEV envelope domain III (JEV EDIII) protein is involved in binding to host receptors, and it contains specific epitopes that elicit virus-neutralizing antibodies. A highly immunogenic, recombinant JEV EDIII protein was expressed in Escherichia coli. In order to take this vaccine candidate for further studies, recombinant JEV EDIII protein was produced employing a pilot-scale fermentation process. Recombinant JEV EDIII protein expressed as inclusion bodies (IBs) was solubilized in 8 M urea and renatured by on-column refolding protocol in the presence of glycerol. A three-step purification process comprising of affinity chromatography, ion-exchange chromatography (IEX) based on salt, and IEX based on pH was developed. About ~124 mg of highly purified and biologically active EDIII protein was obtained from 100 g of biomass. Biological function of the purified EDIII protein was confirmed by their ability to generate EDIII-specific antibodies in mice that could neutralize the virus. These findings suggest that recombinant JEV EDIII protein in combination with compatible adjuvant is highly immunogenic and elicit high-titer neutralizing antibodies. Thus, recombinant JEV EDIII protein produced at large scale can be a potential vaccine candidate.

Expression, purification and evaluation of diagnostic potential and immunogenicity of dengue virus type 3 domain III protein.

Dengue hemorrhagic fever and dengue shock syndrome are the severe manifestations of dengue infection. The quest for reliable dengue diagnostics and a dengue vaccine remained elusive for decades. Domain III of dengue virus envelope contains multiple conformation dependant neutralizing epitopes, thus making it an attractive diagnostic and vaccine candidate. In this report we show the expression of dengue virus type 3 envelope domain III protein (D3EDIII) and demonstrate its potential as a diagnostic and vaccine candidate. Accordingly, D3EDIII was expressed to high levels in Escherichia coli and purified by Ni-NTA affinity chromatography. The purified protein was used to develop an in-house plate ELISA and was further tested with a panel of 40 dengue infected serum samples previously characterized by commercially available serological tests. The in-house results were in excellent agreement with the commercial kits. D3EDIII was refolded by rapid dilution method and the refolded monomer protein was purified by Ion exchange chromatography. Further, the recombinant protein was biologically functional and found to inhibit dengue virus type 3 plaque formation on LLC-MK2 cells demonstrating its function of receptor interaction. Furthermore, D3EDIII in combination with Freund's complete adjuvant induced high antibody titers in BALB/c mice and these antibodies efficiently neutralized dengue 3 virus. Additionally, D3EDIII induced expression of Th1 cytokines that can inhibit the intracellular viral infections. Thus, our results demonstrate that D3EDIII protein has tremendous potential both in diagnosis of dengue infections and in vaccine development.

Production of recombinant nonstructural 1 protein in Escherichia coli for early detection of Japanese encephalitis virus infection.

Japanese encephalitis is a major public health problem in South-East Asia and Western Pacific countries. The recombinant nonstructural 1 (rNS1) protein of Japanese encephalitis virus is a potential diagnostic as well as vaccine candidate. Developments of cost-effective and simple culture media as well as appropriate culture conditions are generally favourable for large-scale production of recombinant proteins. The effects of medium composition and cultivation conditions on the production of rNS1 protein were investigated in shake flask culture as well as batch cultivation of Escherichia coli. Further, the fed-batch process was also carried out for high cell density cultivation (HCDC) of E. coli expressing rNS1 protein. Isopropyl-ß-d-thiogalactopyranoside (IPTG) was used to induce the expression of rNS1 protein at ∼ 13 g dry cell weight per litre of culture. The final dry cell weight after fed-batch cultivation was ∼ 17 g l(-1) . The Inclusion bodies were isolated and purified through affinity chromatography to give a final product yield of ∼ 142 mg l(-1) . The reactivity of purified protein was confirmed by Western blotting and Enzyme linked immunosorbent assay. These results show that rNS1 protein may be used as a diagnostic reagent or for further prophylactic studies. This approach of producing rNS1 protein in E. coli with high yield may also offer promising method for production of other viral recombinant proteins.

Yeast expressed recombinant Hemagglutinin protein of novel H1N1 elicits neutralising antibodies in rabbits and mice.

Currently available vaccines for the pandemic Influenza A (H1N1) 2009 produced in chicken eggs have serious impediments viz limited availability, risk of allergic reactions and the possible selection of sub-populations differing from the naturally occurring virus, whereas the cell culture derived vaccines are time consuming and may not meet the demands of rapid global vaccination required to combat the present/future pandemic. Hemagglutinin (HA) based subunit vaccine for H1N1 requires the HA protein in glycosylated form, which is impossible with the commonly used bacterial expression platform. Additionally, bacterial derived protein requires extensive purification and refolding steps for vaccine applications. For these reasons an alternative heterologous system for rapid, easy and economical production of Hemagglutinin protein in its glycosylated form is required. The HA gene of novel H1N1 A/California/04/2009 was engineered for expression in Pichia pastoris as a soluble secreted protein. The full length HA- synthetic gene having α-secretory tag was integrated into P. pastoris genome through homologous recombination. The resultant Pichia clones having multiple copy integrants of the transgene expressed full length HA protein in the culture supernatant. The Recombinant yeast derived H1N1 HA protein elicited neutralising antibodies both in mice and rabbits. The sera from immunised animals also exhibited Hemagglutination Inhibition (HI) activity. Considering the safety, reliability and also economic potential of Pichia expression platform, our preliminary data indicates the feasibility of using this system as an alternative for large-scale production of recombinant influenza HA protein in the face of influenza pandemic threat.

Recombinant dengue virus type 3 envelope domain III protein from Escherichia coli.

Dengue is a public health problem of global significance for which there is neither an effective antiviral therapy nor a preventive vaccine. The envelope protein of dengue virus is the major antigen to elicit neutralizing antibody response and protective immunity in hosts. Optimization of culture media was carried out for enhanced production of recombinant dengue virus type 3 envelope domain III (rDen 3 EDIII) protein in E. coli. Further, batch and fed-batch cultivation process were also developed in optimized medium. After fed-batch cultivation, the dry cell weight was about 22.80 g/L of culture. The rDen 3 EDIII protein was purified using immobilized metal affinity chromatography. This process produced ∼649 mg of purified rDen 3 EDIII protein per liter of culture. The purity of the protein was determined by SDS-PAGE analysis and the reactivity was checked by Western blotting as well as ELISA. These results show that the purified protein may be used for the dengue diagnosis or further prophylactic studies for dengue infection.

Monoclonal antibody-based antigen capture immunoassay for detection of circulating non-structural protein NS1: implications for early diagnosis of Japanese encephalitis virus infection.

An early diagnosis of Japanese encephalitis (JE) is important for timely clinical management and epidemiological control in areas where multiple flaviviruses are endemic. The NS1 antigen has an advantage over IgM enzyme-linked immunosorbent assay (ELISA) for early confirmatory diagnosis of Japanese encephalitis virus (JEV) infection due to its proliferation on the surface of the host cells in the acute phase of infection. In this study, the development and evaluation of JE-specific NS1 antigen capture ELISA is described using high-affinity monoclonal antibody specific to the recombinant NS1 protein for early diagnosis of JE. The gene encoding NS1 protein was cloned and expressed in the pQE30UA expression vector followed by purification of the recombinant protein by affinity chromatography. A sandwich ELISA for antigen detection was developed using purified rabbit IgG antibody and mouse monoclonal antibody as the capture and detector antibody, respectively. The application of JE NS1 antigen ELISA for early diagnosis was evaluated with 120 acute phase sera and 80 CSF samples. The comparative evaluation of the JE NS1 antigen ELISA by real-time RT-PCR revealed 97% concordance with a sensitivity and specificity of 97% and 98%, respectively. The JE NS1 antigen was detectable in the blood from the first day up to day 9 after the onset of symptoms. These findings suggest that the JEV NS1 antigen capture ELISA may help early diagnosis of JE infection.

Transcriptomic profile of host response in Japanese encephalitis virus infection.

BACKGROUND: Japanese encephalitis (JE) is one of the leading causes of acute encephalopathy with the highest mortality rate of 30-50%. The purpose of this study was to understand complex biological processes of host response during the progression of the disease. Virus was subcutaneously administered in mice and brain was used for whole genome expression profiling by cDNA microarray. RESULTS: The comparison between viral replication efficiency and disease progression confirms the active role of host response in immunopathology and disease severity. The histopathological analysis confirms the severe damage in the brain in a time dependent manner. Interestingly, the transcription profile reveals significant and differential expression of various pattern recognition receptors, chemotactic genes and the activation of inflammasome. The increased leukocyte infiltration and aggravated CNS inflammation may be the cause of disease severity. CONCLUSION: This is the first report that provides a detailed picture of the host transcriptional response in a natural route of exposure and opens up new avenues for potential therapeutic and prophylactic strategies against Japanese encephalitis virus.

Alteration of blood brain barrier permeability by T-2 toxin: Role of MMP-9 and inflammatory cytokines.

T-2 toxin is a cytotoxic fungal secondary metabolite produced by different species of Fusarium such as F. sporotichioides, F. poae, F. equiseti, F. acuminatum etc. This class of mycotoxins causes a number of pathologies including nervous disorders, cardiovascular alterations, immunodepression and hemostatic derangements. In the present study, mechanism of T-2 toxin induced alteration of blood-brain barrier (BBB) permeability was assessed in terms of oxidative stress, gene expression of MMP-9, MMP-2 and their inhibitors TIMP-1 and TIMP-2, activation of inflammatory cytokines in both brain and peripheral tissue spleen. Gel zymography was used to show the activity of MMP-9 and MMP-2. The percutaneous exposure of 1 LD50 T2 toxin caused a reversible alteration in BBB permeability as observed by extravasation of Evans blue dye. Maximum dye level was observed on day 3 and reduced by day 7. A significant GSH depletion was observed on days 1 and 3. Brain ROS and lipid peroxidation levels increased significantly on 1 and 3 days and decreased by day 7. The SOD levels in brain showed significantly higher activity on 3 days (4-fold) and 7 days (5-fold) of toxin exposure compared to control. A similar trend was observed with catalase enzyme levels. The gene expression analysis of cNOS and iNOS showed varying levels of expression on different time points of post exposure. MMP-9 expression was significantly high on days 3 and 7 in brain with corresponding alteration in TIMP-1. MMP-2 and TIMP-2 showed no effect. Gene expression analysis of the inflammatory cytokines, IL-1α, IL-1ß, IL-6 and TNF-α showed elevated levels on day 7 in brain. As spleen plays an important role in inflammatory response we analyzed MMP-9, MMP-2 and inflammatory cytokines in spleen. The MMP-9 was activated on day 7. MMP-2 activity was found to be elevated on 3 and 7 days and TIMP-2 mRNA level increased on 1 and 3 days in spleen. Inflammatory cytokines, IL-1 α, IL-1ß, IL-6 and TNF-α showed elevated levels on days 1 and 3 in spleen indicating an early effect in spleen than in brain. In summary, the results of the study showed that the T-2 induced alteration in BBB permeability is mediated through oxidative stress, activation of MMP-9, and proinflammatory cytokines in brain as well as contribution from peripheral tissue spleen.

Supersensitive detection of T-2 toxin by the in situ synthesized π-conjugated molecularly imprinted nanopatterns. An in situ investigation by surface plasmon resonance combined with electrochemistry.

A π-conjugated molecularly imprinted polymer (MIP) with nanopatterns for T-2 toxin (T-2) was prepared on SPR chip by in situ electropolymerization of 3-aminophenylboronicacid (3-APBA) with T-2. The complete removal of T-2 from polymer was confirmed in situ by SPR and EIS and also ex situ by SEM, EDAX, fluorescence microscopy and Raman spectroscopy. SEM image of T-2 MIP exhibited nanopatterns due to imprinting of T-2. The MIP of T-2 showed a linear response for T-2 from 2.1 fM to 33.6 fM with a detection limit of 0.1 fM (0.05 pg/mL). In this study, thermodynamic parameters such as change in Gibb's free energy (ΔG), change in enthalpy (ΔH) and change in entropy (ΔS) were determined and the values revealed that the interaction between T-2 and T-2 MIP as spontaneous, endothermic and entropy driven one. Moreover, interactions of very high concentration of interferents with T-2 MIP showed very less response due to the presence of nanopatterns of T-2 in the T-2 MIP. Equilibrium constant (12.7 fM) obtained in this study indicates the super binding affinity of T-2 with T-2 MIP. Moreover, the present methodology provides an outline to develop field-detection equipment capable of detecting T-2 toxin at or well below the guideline concentrations recommended by American subcommittee on military field drinking water.

Brain oxidative stress after dermal and subcutaneous exposure of T-2 toxin in mice.

T-2 toxin belongs to group of mycotoxins and is found as a natural contaminant in cereals, feed and vegetables. In the present study we evaluated acute toxicity of dermal and subcutaneous exposure of T-2 toxin on brain oxidative stress in mice. Mice were exposed to 1 LD50 of T-2 toxin either by dermal (5.94 mg/kg) or subcutaneous (1.54 mg/kg body weight) route and sacrificed at 1, 3 and 7 days post-exposure. T-2 toxin treated animals showed time dependent increase in reactive oxygen species generation, glutathione depletion, lipid peroxidation and protein carbonyl content in brain in both the routes of exposure. Gene expression profile of antioxidant enzymes showed significant increase in superoxide dismutase and catalase in percutaneous route and glutathione reductase and glutathione peroxidase in subcutaneous route. Immunoblot analysis of antioxidant enzymes correlated with gene expression profile. T-2 toxin exposure resulted in down regulation of transcription factor Nrf2 and its downstream target genes of phase II detoxifying enzymes NQO1, Gclc, Gclm and hemeoxygenase-1. Results of our study show that percutaneously and subcutaneously applied T-2 toxin can cause brain oxidative damage possibly after crossing blood-brain barrier by altering its permeability.

Expression profile of Japanese encephalitis virus induced neuroinflammation and its implication in disease severity.

BACKGROUND: Host immune response particularly through the induction of proinflammatory cytokines and chemokines in Japanese encephalitis virus infection has not been clearly understood in relation with pathogenicity and disease severity. The newly identified host mediators of pathogenesis could be the future target for diagnostic and therapeutics purpose. OBJECTIVES: We investigated the mechanism of JE virus induced pathogenesis in terms of proinflammatory cytokine and chemokine secretion at molecular level in young one-week-old BALB/c mouse after subcutaneous administration of JEV. STUDY DESIGN: Histopathology of brain was done to observe the morphological changes after JEV infection and genes relevant to macrophage activation, chemokine secretion, inflammatory cell infiltration, and blood-brain barrier permeability were examined at their gene and protein expression level for various time points after infection. RESULTS: At 6-day post-infection 100% mortality was observed. At 5-day post-infection, there was a robust expression of proinflammatory cytokines and chemokines with increased number of infiltrating inflammatory cells into the brain. Histopathology data confirms the infiltration of leucocytes and there was a marked upregulation in expression of genes relevant to infiltration. The expression pattern of macrophage receptor CLEC5A/DAP-12 signaling has shown the involvement in this robust neuroinflammation. CONCLUSIONS: This is the first report that shows the involvement of monocyte and macrophage receptor CLEC5A in severe inflammatory response in JEV infection of brain. This study at gene expression level provides a hypothesis of neuroinflammation, a new lead in development of appropriate therapeutic, and prophylactics against Japanese encephalitis.

Development of a quantitative competitive reverse transcription polymerase chain reaction (QC-RT-PCR) for detection and quantitation of Chikungunya virus.

Chikungunya is one of the most important emerging arboviral infections of public health significance. Due to lack of a licensed vaccine, rapid diagnosis plays an important role in early management of patients. In this study, a QC-RT-PCR assay was developed to quantify Chikungunya virus (CHIKV) RNA by targeting the conserved region of E1 gene. A competitor molecule containing an internal insertion was generated, which provided a stringent control of the quantification process. The introduction of 10-fold serially diluted competitor in each reaction was further used to determine sensitivity. The applicability of this assay for quantification of CHIKV RNA was evaluated with human clinical samples, and the results were compared with real-time quantitative RT-PCR. The sensitivity of this assay was estimated to be 100 RNA copies per reaction with a dynamic detection range of 10(2) to 10(10) copies. Specificity was confirmed using closely related alpha and flaviviruses. The comparison of QC-RT-PCR result with real-time RT-PCR revealed 100% concordance for the detection of CHIKV in clinical samples. These findings demonstrated that the reported assay is convenient, sensitive and accurate method and has the potential usefulness for clinical diagnosis due to simultaneous detection and quantification of CHIKV in acute-phase serum samples.

Chemokine profiling of Japanese encephalitis virus-infected mouse neuroblastoma cells by microarray and real-time RT-PCR: implication in neuropathogenesis.

Japanese encephalitis (JE) is one of the leading causes of acute encephalopathy affecting children and adolescents in the tropics. JE virus (JEV) infection causes prominent neurological sequelae in approximately one-third of the survivors. In humans, the inflammatory response of CNS consequent to JEV induced viral encephalitis is mediated through chemokines released by various cells of CNS. In the present study, the chemokine profiles of mouse neuroblastoma cells (N2A) following JEV infection was analyzed by cDNA microarray followed by real-time RT-PCR. Eighty mRNA transcripts belonging to various functional classes exhibited significant alterations in gene expression. There was considerable induction of genes involved in apoptosis and anti-viral response. Modified levels of several transcripts involved in proinflammatory and anti-inflammatory processes exemplified the balance between opposing forces during JEV pathogenesis. Other genes displaying altered transcription included those associated with host translation, cellular metabolism, cell cycle, signal transduction, transcriptional regulation, protein trafficking, neurotransmitters, neuron maturation, protein modulators, ER stress and cytoskeletal proteins. The infection of neurons results in the synthesis of proinflammatory chemokines, which are early important mediators of leukocyte recruitment to sites of viral infection. Our results clearly suggest the implication of chemokines in neuropathogenesis of JEV infection leading to neurological sequelae. Pro- and anti-inflammatory agents targeted against chemokines such as CXCL10 may provide possible therapeutic modalities that can mitigate the morbidity associated with JEV infection of the CNS.