Transcriptomic profile of host response in Japanese encephalitis virus infection
© Gupta and Rao; licensee BioMed Central Ltd. 2011
Received: 14 February 2011
Accepted: 4 March 2011
Published: 4 March 2011
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.
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.
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.
The host response to infection is central to the effective control and ultimate clearance of invading pathogens or removal of infected cells. Infection of host with a viral pathogen marks the onset of changes in the host cell's microenvironment. Such changes in host gene expression could be a cellular antivirus response, a virus induced response that facilitate its own replication and spread or a non-specific response that neither promotes nor prevents virus infection. This alteration of expression of many cellular genes can be identified using cDNA microarray .
Defining the transcriptional regulation of host genes on virus infection can be used as a tool to obtain an elaborate insight into mechanisms of host-virus interactions and to unravel the molecular basis of disease pathogenesis. Viruses from several families can infect neurons in the CNS (Central Nervous System) and the study of gene expression changes in the CNS during virus infection can lead to identification of new genes whose function is essential either for the promotion or prevention of virus infection [2, 3].
Japanese Encephalitis is one of the most dreaded mosquito borne encephalitis virus causing acute encephalitis in humans. Among the medically important flaviviruses, JEV infection has the highest mortality rate of 30-50% [4, 5] and remains as a major public health problem in several parts of Asia. The main concern is the constant spreading of JE to new geographical areas . Better understanding of JEV pathogenesis is required to identify risk factors for progression of disease and viral persistence, which may help in the development of differential diagnostics and new therapeutic interventions.
In a previous study, employing cDNA microarray, we identified various antiviral genes along with the innate immune response related chemokine expression at very early phase of infection in mouse neuronal cells . However, there is no information available on genome wide host gene expression changes induced by JEV in the CNS and in a natural route of infection. There is a requirement to understand the molecular events responsible for disease progression, viral persistence and complex biological processes of host response during the complete course of JEV infection, starting from peripheral route to CNS, neuroinflammation, disease severity and death.
Thus, we employed cDNA microarray for the systematic analysis of global host transcriptional responses in CNS of JEV infected mice. In the recent epidemics, it has been observed that the mortality rate in JEV infected patients is higher in 1-5 years of age group with immature immune system. So we employed a young mice model to explore the precise molecular events involved in JEV infection of CNS during the disease severity. Subcutaneous challenge of JEV in one-week-old mice provides a natural route of exposure to study molecular mechanism of JEV pathogenesis in CNS. In our earlier report using this animal model we observed a significant regulation of IFN-γ in virus infected mice spleen, which demonstrates a specific but insufficient anti-viral response in the periphery to limit virus spread to brain . Thus this model with immature immune system may provide important information about the role of host response in disease severity. The reproducibility of the infection and diseases symptoms was verified with a significant number of experimental repetitions in newly established animal model and there was no variation in the disease symptoms of the individual animals at any time point of infection and the mortality rate was also 100% at 6 DPI (Day post-infection).
It has already been reported that cerebral cortex is an important site of virus replication, inflammation, injury and is associated with encephalitis in the JEV-infected host [9, 10]. Thus we employed brain cerebral cortex for the investigation of transcriptomic profile of host response in JEV infection. Our results thus provide a genome-wide investigation of an animal model of JEV infection and a genomic view of systemic host-virus interactions during infection.
Evaluation of viral load and histopathological analysis in mice CNS after subcutaneous infection with JEV
Host transcription profile after JEV infection
Differentially expressed genes in mouse brain at different days after JEV infection.
[Day post infection]
Representation of statistically significant genes categorised into genetic pathways by the KEGG classification system, in mouse brain after JEV infection.
Number of genes
Intracellular signaling cascade
Negative regulation of biological process
Antigen processing and presentation
Integrin-mediated signaling pathway
G-protein-coupled receptor binding
Response to virus
Cytokine and chemokine mediated signaling pathway
Chemokine receptor binding
Leukocyte mediated immunity
MHC protein complex
Immunoglobulin mediated immune response
Expression profile of cell surface molecules in brain after JEV infection
Differential expression of cell surface molecules in brain after JEV infection.
Fold change over mock-infected
C-type lectin domain family 7
C-type lectin domain family 5
Protein binding scavenger
Lectin, galactoside-binding, soluble, 3 binding protein
Toll-like receptor 2
Sialic acid binding Ig-like lectin 1
C-type lectin domain family 4
G protein-coupled receptor 84
Macrophage scavenger receptor 1
Toll-like receptor 3
T cell mediated cytotoxicity
Protein tyrosine phosphatase, receptor
Chemokine (C-C motif) receptor-like 2
Cytokine & Chemokine signaling
Leukocyte immunoglobulin-like receptor
Lectin, galactose binding, soluble 3
Toll-like receptor 1
Fas (TNF receptor superfamily)
Antibody-dependent cellular cytotoxicity
Fc receptor, IgG, high affinity I
Mannan-binding lectin serine peptidase
Toll-like receptor 4
Cytokine & Chemokine signaling
Colony stimulating factor 2 receptor
Intercellular adhesion molecule
Lectin, galactose binding, soluble 9
Cytokine & Chemokine signaling
Colony stimulating factor 2 receptor
Interleukin 18 binding protein
Fc fragment of IgG, low affinity IIIa, receptor
Toll-like receptor 7
Adhesion molecule, interacts with CXADR antigen 1
CD8 antigen, alpha chain
Vascular cell adhesion molecule 1
Fc receptor, IgG, low affinity IIb
Oncostatin M receptor
Interleukin 21 receptor
Lymphocyte antigen 78
C-type lectin domain family 2
Leukocyte-associated Ig-like receptor
Type IIa hypersensitivity
Fc receptor, IgE, high affinity I, gamma polypeptide
Integrin beta 2
Paired immunoglobin-like type 2
Tnf receptor-associated factor 1
Interleukin 15 receptor, alpha chain
Sphingolipid g-protein-coupled receptor,
Interleukin 10 receptor, alpha
Progestin and adipoq receptor family member V
Triggering receptor expressed on myeloid cells-like 4
Integrin alpha M
Type 1 TNF receptor shedding aminopeptidase regulator
Phosphoinositide-3-kinase adaptor protein 1
Tumor necrosis factor receptor superfamily, member 1b
Interleukin 12 receptor, beta 1
Chemokine (C-C motif) receptor 5
Interleukin 2 receptor, gamma chain
Cytokine & Chemokine signaling
Interleukin 2 receptor, beta chain
Differential expression of genes in brain after JEV infection
Down regulated genes expressed in brain after JEV infection.
Fold change over mock-infected
Major histocompability complex Q1b
Phosphatidylethanolamine binding protein 2
Bone morphogenic protein receptor
T cell mediated cytotoxicity
Protein tyrosine phosphatase, receptor type, C
Chemokine (C-C motif) ligand 24
Leukotriene metabolic process
Mus musculus leukotriene C4 synthase
RAR-related orphan receptor beta
Mannose receptor, C type 1
Leukotriene metabolic process
Macrophage scavenger receptor 2
Transient receptor potential cation channel
Kinesin family member 1B
Chemokine (C-X-C motif) ligand 4
Eukaryotic translation initiation factor
Zinc finger protein 101
Iroquois related homeobox 6
Nerve growth factor receptor
Validation of microarray data by real-time qRT-PCR analysis
The clinical outcome of a viral infection is largely dependent on the balance between host response and viral replication rates. The interaction of the virus with cell and evasion of the host immune response is crucial to the development of disease in a susceptible host. Japanese encephalitis (JE) is, at present, the single most important cause of viral encephalitis in Asia. In addition to causing acute illness with a high mortality rate, the disease may leave survivors with major mental and physical disabilities. Despite the fact that Japanese encephalitis is a major disease affecting the tropical world, little is known of its pathogenesis due, partly, to the lack of a suitable animal model and the complex cell interactions in infected individuals. Understanding the events that occur within the central nervous system after viral exposure is necessary if effective therapeutic interventions against viral encephalitis are to be developed.
The current investigation shows global transcription profile in the brain of JEV-infected animal in a natural route of exposure. In this study, after subcutaneous infection, animals started showing JE specific symptoms from 3-day post-infection. The tremendous increase in viral load was observed from 4 DPI in this animal model, which may be because of the completion of virus dissemination and release from brain cells. In addition to this, another mechanism behind this robust increase in viral load may be the increase in viral spread through a haematogenous route via endothelium , because at this time point of infection there was an increase in BBB permeability also. However, the detailed study is required to explore the possible mechanism of JEV entry in to the CNS. The greatest difference in the gene expression was observed when the viral load peaked in the brain. However, viral titre remains constant after 5 day until time to death; this confirms the role of host response in the severity of disease rather than virus itself. As reported earlier the pooled RNA samples were used for microarray analysis to reduce the effects of biological variation and to easily find the substantive differences . Genes that were differentially expressed during infection can potentially provide insights into the complex regulatory phenomena in response to JEV infection.
The global transcription profile of the mouse brain infected with JEV revealed the activation of a variety of antiviral host defenses early after infection: activation of innate immune responses, protein processing, interferons and interferon-stimulated genes, complement system, activation of natural killer cells, macrophages and leukocyte infiltration into brain. These indicated the induction of an important local inflammatory response by the host after the infection of CNS. The major set of genes/pathways with increased expression observed in present study with JEV are similar to those reported for other neuroinvasive viruses like West Nile virus (WNV). These results suggest that some pathways are commonly activated during neurotropic viral infection of the CNS, and the gene products like Ifn-γ, Cxcl10/IP-10 etc. involved in protective roles at early phase of infection may also contribute to the pathogenesis of the disease at later stage [2, 12].
In Flaviviral encephalitis both macrophage and T-cell infiltration appear to play an important role in the virus entry to CNS . The extravasations of these inflammatory cells and generation of host response may require the activation of different mediators involving, respectively, selectins and their ligands, chemokines and chemokine receptors, proinflammatory cytokines, integrins and cell adhesion molecules (CAMs) and matrix metalloproteinases (MMPs).
Chemokines are now recognized as critical regulators of leukocyte trafficking into the CNS. Numerous studies have revealed that resident cell populations of the CNS are able to synthesize and secrete a variety of chemokines. Astrocytes and microglia are the primary source of chemokines following infection with a wide range of neurotropic viruses. Neurons are also capable of secreting chemokines during JEV and WNV infection [7, 14]. The early increased expression of cytokine Tnf-α and T-cell activation chemokines, such as Cxcl10/IP-10, Cxcl11/I-TAC, and Cxcl9/Mig-1 suggests their role in the infiltration of leucocytes at an early phase of host response to highly neuroinvasive JEV infection. These chemokines were also up regulated in the brain of animals infected with highly neuroinvasive strains of WNV . Various reports suggest that the interferon and interferon-regulated genes are playing important role in the activation of leukocytes and their infiltration to CNS. In this study, the earliest consistent transcriptional response was an increase in transcript cluster of Ifn-associated genes. This cluster includes Ifn-α, Ifn-β, and Ifn-γ regulated genes. These genes were also found to be involved in the pathogenesis of West Nile virus encephalitis [15, 16].
Interestingly, chemokines which are responsible for infiltration of monocytes and macrophage like Ccl5/RANTES and Ccl4/MIP-1β showed increased expression at an acute phase of infection and increases the infiltration of monocyte and macrophages. At this phase of infection before severity of disease, there was an increase in expression of various proinflammatory mediators also like Tnf-α, Il1α, Il1β, Il-12 and Ccl2/MIP-1α along with chemoattractants. These immune mediators may be exacerbating the infiltration of inflammatory cells by increasing the expression of various integrins, cell adhesion molecules, selectins and increased blood-brain barrier permeability. A study with neural stem/progenitor cells infected with JEV also suggested the possible role of these cytokines in the cellular infiltration . Thus, according to the present data it can be hypothesized that the uncontrolled expression of these mediators may be detrimental leading to the disease severity.
Cell-cell adhesion mediated by various genes is critical for interaction of lymphocytes and antigen presenting cells with endothelium and recruitment to the inflammation site. Recent report suggests that Icam1 plays an important role in WNV neuroinvasion and this is also involved in the blood-brain barrier permeability and the progression of neuroinflammation [18–20]. The combination of IFN-γ with TNF-α or IL-1 can strikingly up regulate the expression of these adhesion molecules  and our results also confirm the similar induction of these genes. Though adhesion molecules showed important role in neuroinflammation, a more thorough understanding will help to develop effective anti-inflammation strategies. The reports also suggested the importance of integrins in virus entry and a prominent endothelial cell receptor was also identified as the functional receptor for WNV and JEV in vertebrate cells .
Toll-like receptors signal transduction leads to the expression of several proteins that have important roles in the inflammation and immune response to virus. Recent report suggests that Tlr3 is involved in the WNV replication in brain and induction of neuronal injury, may be through inflammation induced cell death in the brain . The present data also showed the increased expression of Tlr3 along with TNF-α at an early phase of JEV infection and it may be involved in virus entry and resultant encephalitis. Studies showed that the induction of Tlr2-mediated cytokine response in the brain contributes to the death of the animal . Tlr2 and Tlr3 cooperation leads to the expression of the macrophage chemoattractants Ccl2 and Ccl5 , which may be the case in JEV infection also. Other receptors like Tlr1, Tlr4 and Tlr7 are known to be involved in the exacerbation of virus-induced inflammation and defense response [26, 27]. The TLR mediated pathway has been supported with various other important signaling receptors like Lilrb3, Ptprc [28, 29]. The transcription profile of these receptors during disease course suggest a cumulative effect of these genes in JEV pathogenesis.
The innate immune response of multicellular organisms is initiated by the binding of soluble and membrane-bound host molecules including lectins to the surface of pathogenic organism. These pattern recognition receptors (PRRs) are required for signal transduction during host response. Lectin receptors like dendritic cell receptor Clec7a, Clec4e are playing important role in the activation of macrophage and innate immune response [30, 31]. Recent reports suggest the critical role of macrophage receptor Clec5a in dengue and Japanese encephalitis severity [8, 32]. Other macrophage receptors like Masp2 and Siglec1 plays important role in inflammation [33, 34]. Taken together, it can be hypothesized that, the JEV activates signal transduction through these receptors, exacerbates the inflammation, and disease severity. These are the newly explored pattern-recognizing C-type lectin receptors on dendritic cells and macrophages and targeting these molecules may provide improved therapeutic options.
The data also presents an interesting set of genes in JEV pathogenesis. These are the caspases, which are not only playing an essential role during apoptotic cell death, but a subfamily of them--the inflammatory caspase, are associated with immune responses to microbial pathogens . These include caspase-1, 4, 5, 11 and 12. Activation of inflammatory caspases, such as caspase-1 and caspase-5, occurs upon assembly of an intracellular complex, designated the inflammasome . The activation of various pattern recognition receptors like Tlr, C-type lectins and up regulation of Casapse-1, Caspase-4, Pycard, Cathepsin, Il1β and Il18bp indicates the possible generation of inflammasomes during JEV infection. The critical role of Cryopyrin/Nalp3 inflammasomes in case of virus infection has already been discussed . These inflammasomes may be playing important role in JEV pathogenesis and further research is required to identify the role of these inflammasomes in JEV infection.
The present study also provides various important mediators of host anti-viral response at an early phase of infection, those with increased expression included Mx1 and Mx2 (myxovirus resistance 1 and 2), antiviral GTPases, as well as various members of the Oas family, Guanylate nucleotide binding proteins and TRIM protein family. Oas has shown involvement in west-nile virus infection also . Some of these genes like, Oasl2, Oas1a, Oasl1, G1p2, Ifit3 and Iigp2 showed significant regulation at an early phase of infection and these results were consistent with our earlier report on host response in neuronal cell infected with JEV . The significant regulation of these genes along with Trim proteins like Trim 30, 25 and Trim 34 was also observed in the brain of animals infected with highly neuroinvasive strains of WNV . The genes of complement system like C1r, C2, C3, and C4b are known to recognize pathogen-associated molecular patterns (PAMPs) and their involvement in WNV infection is also reported recently . Various hematopoietic cell surface molecules had increased expression, including some Ly6 (lymphocyte) antigens, a group of molecules that are involved in signal transduction and cell activation .
These data illustrate that the activation of various PRRs, provides a first line of defense by inducing interferons, proinflammatory cytokines and chemokines [41, 42]. These mediators can promote tissue damage if not dampened in a timely manner, such as the increased leukocytic infiltration, which has aggravated the CNS inflammation and causes fatal encephalomyelitis during JEV infection. Recent studies also supported this notion of immunopathogenesis in JEV infection [43, 44]. The role of chemokines can not be ignored in this neuroinflammation as they are critical mediators of neuropathology during JEV infection either by attracting pathogenic inflammatory cells or directly mediating neurotoxicity and cell death. The involvement of this aggravated inflammation in disease severity has already been studied with other viral diseases [45–47].
In conclusion, there is clear evidence for an immunopathological mechanism in the pathogenesis of Japanese encephalitis in mice and may be of use in determining a role for anti-inflammatory agents in the disease management. One of the most interesting aspects of our results is the information on various receptors that may be involved in the complex interaction between host and virus. Resident brain cells appeared to be the source of early immune mediators while infiltrating leukocyte are playing important role in the severity of the disease. This will aid further attempts to control the inflammatory conditions during JEV infection. The further elucidation of significantly regulated receptor-ligand interaction and resulted signal transduction processes may demonstrate the complexity of the interplay between the virus and the host, and may open new ways for therapeutic strategies for diseases which has inflammation as the major cause of disease severity.
Materials and methods
The JaOArS982 strain of Japanese encephalitis virus was employed throughout this study. The virus was propagated in suckling BALB/c mice. The brain tissue was harvested when clinical signs of sickness became apparent. A 10% suspension of the brain tissue was made by homogenization in the minimum essential medium (MEM). It was then centrifuged at 10,000 × g to remove cellular debris and filtered through 0.22 μm sterile filters. The mouse brain tissue-derived virus was stored at -70°C in small aliquots and was used as the source of virus for all the experiments.
The infectivity of the virus stock (PFU JEV/ml) was assessed by a quantitative plaque-forming assay on the monolayers of porcine stable (PS) kidney cells. Monolayers of cells were inoculated with 10-fold dilutions of virus sample made in MEM containing 1% FBS and incubated for 1 h at 37°C with occasional shaking. The inoculum was removed by aspiration and the monolayers were further overlaid with 1.25% methylcellulose containing MEM with 1% FBS. After incubation for 4 days the overlay medium was removed, the cells were fixed with methanol and stained with 0.5% crystal violet, and the end-point titre was determined by macroscopic counting of plaques.
We have adopted a new mice animal model for Japanese encephalitis virus infection with modification in age and the route of infection, as reported by us earlier . Briefly, one-week-old BALB/c mice of either sex were injected subcutaneously with approximately 103 PFU (in 50 μl of PBS) of JEV. Control animals received the same volume of PBS as the experimental group. All experiments were performed according to the protocol approved by the Institutional Animal Ethics Committee.
Viral load in the CNS of infected mice
Mice were sacrificed each day post-infection (DPI) from day 1 to day 6 to harvest brain tissue. The animals were perfused with cold PBS before harvesting the brain tissue. The cerebral cortex of brain tissue was homogenized in the minimum essential medium (MEM). It was then centrifuged at 10,000 × g and filtered through 0.22 μm sterile filters. Presence and growth kinetics of virus was confirmed and titrated in the prepared sample by plaque formation on the monolayers of porcine stable (PS) kidney cells.
Mock- and JEV-infected animals were sacrificed after specific time points. Tissue sample of brain was dissected out and fixed in Bouin's solution. After fixation, small pieces were processed by automated tissue processor (Leica TP1020) dehydrated and embedded in paraffin wax. Multiple sections of 12-μm thickness were prepared using automatic microtome (Microm HM360) and stained with hematoxylin and eosin in Leica Autostain-XL. Microscopic observation was performed on sections of cerebral cortex under LEICA DMLB microscope and photographs were taken using Leica DC 500 camera.
Sample Acquisition, RNA Isolation and Quality Control
Brain tissues were collected from mock- and JEV-infected group of three mice at different days post infection. The sections of cerebral cortex of brain tissue were stored in RNA later (Qiagen, Hilden, Germany) at -70°C until processed for RNA extraction. Total RNA was extracted using the Qiagen (GmbH, Hilden) RNAEasy Mini kit according to the instructions of the manufacturer. RNA quality and integrity was assessed using RNA 6000 Nano Lab Chip on the 2100 Bioanalyzer (Agilent, Germany) following the manufacturer's protocol. RNA samples with RIN (RNA Integrity Number) ≥ 8 were used in all experiments. Equal concentration of total RNA from three animals of each mock- and JEV-infected group were pooled and used for microarray analysis. Validation of microarray data of relevant genes was carried out by qRT-PCR with three biological replicates without pooling of RNA.
Microarrays and Hybridization
Low RNA Input Fluorescent Linear Amplification Kit (Agilent, Santa Clara, CA) was used for labeling. Briefly, both first and second strand cDNA were synthesized by incubating 500 ng of pooled total RNA with 1.2 μl of oligo dT-T7 promoter primer in nuclease-free water at 65°C for 10 min followed by incubation with 4.0 μl of 5× First strand buffer, 2 μl of 0.1 M DTT, 1 μl of 10 mM dNTP mix, 1 μl of 200 U/μl MMLV-RT, and 0.5 μl of 40 U/μl RNaseOUT, at 40 °C for 2 h. Immediately following cDNA synthesis, the reaction mixture was incubated with 2.4 μl of 10 mM Cyanine-3-CTP or 2.4 μl of 10 mM Cyanine-5-CTP (Perkin-Elmer, Boston, MA), 20 μl of 4× Transcription buffer, 8 μl of NTP mixture, 6 μl of 0.1 M DTT, 0.5 μl of RNaseOUT, 0.6 μl of inorganic pyrophosphatase, 0.8 μl of T7 RNA polymerase, and 15.3 μl of nuclease-free water at 40 °C for 2 h. Qiagen's RNeasy mini spin columns were used for purifying amplified cRNA samples. The quantity and specific activity of cRNA was determined by using NanoDrop ND-1000 UV-VIS Spectrophotometer version 3.2.1. Samples with specific activity >8 were used for hybridization. 825 ng of each Cyanine 3 or Cyanine 5 labeled cRNA in a volume of 41.8 μl were combined with 11 μl of 10× Blocking agent and 2.2 μl of 25× Fragmentation buffer (Agilent), and incubated at 60°C for 30 minutes in dark. The fragmented cRNA were mixed with 55 μl of 2× hybridization buffer (Agilent). About 110 μl of the resulting mixture was applied to the Agilent Whole Genome Mouse 4 × 44 k Gene Expression Microarray (AMADID: 14868, Agilent Technologies) and hybridized in a two-color comparative format at 65°C for 17 h in an Agilent Microarray Hybridization Chamber (SureHyb: G2534A) with hybridization oven. After hybridization, slides were washed with Agilent Gene expression wash buffer I for 1 min at room temperature followed by a 1 min wash with Agilent gene expression wash buffer II for 37°C. Slides were finally rinsed with acetonitrile for cleaning up and drying. Hybridized arrays were scanned at 5 μm resolution on an Agilent DNA Microarray Scanner, Model G2565BA. Data extraction from images was done using Feature Extraction software of Agilent.
Microarray Data Analysis
Feature extracted data was analyzed using GeneSpring Gx v 11.0 software from Agilent. Normalization of the data was done using per spot per chip intensity dependent lowess normalization. Further quality control of normalized data was done using correlation based condition tree to eliminate experimental error. Genes that had ≥2 (Up regulated) and ≤-2 (Down regulated) fold change at 5 DPI were filtered from the data, irrespective of their regulation at early time points and selected for further analysis. Differentially regulated genes were clustered using gene tree to identify significant gene expression patterns. Ontology based biological analysis was done using Gene Ontology browser in GeneSpring Gx.
Real Time qRT-PCR
The quantitative real-time RT-PCR was carried out to validate the microarray data with three biological replicates without pooling of RNA, using gene-specific primers from Quanti Tect primer assay kit (Qiagen Germany). Quanti Fast one-step RT-PCR kit (Qiagen Germany) was used for real time PCR and Glyceraldehyde-3-phosphate dehydrogenase (Gapdh) was used as an endogenous reference gene . The relative quantification level of expression was determined using the 2nd derivative maximum analysis with the determination of the crossing points for each transcript. Crossing point values for each gene was normalized to the respective crossing point values for the reference gene Gapdh. Data are presented as normalized ratios of genes along with standard error using the Roche Applied Science E-method .
For microarray, sequential Student's t test (time point versus mock) was used to identify genes differentially expressed (P ≤ 0.05) for each group and the experiment was repeated once. The quantitative real-time RT-PCR data was analyzed by one-way ANOVA followed by Dunnet's test for comparison between mock- and JEV-infected groups. The level of significance was set at P ≤ 0.05. The data were expressed as mean ± SE of three animals per group. The real-time RT-PCR experiments were repeated twice.
We thank Dr. R. Vijayaraghavan, Director, Defence Research and Development Establishment for offering all facilities and support required for this study. Mr. Nimesh Gupta is recipient of DRDO Senior research fellowship. This work was supported by the grant from Ministry of Defence, India.
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