Japanese encephalitis virus infection induces changes of mRNA profile of mouse spleen and brain

A mouse whole gene array was used to perform a systematic analysis of mRNA expression profile of spleen and brain tissues of JEV P3-infected mice which were sacrificed at day 3 and day 6 post-inoculation respectively. Genes that had ≥ |2|-fold change were identified as significantly differential expression. Of 41174 genes represented on the chip, 437 genes were differentially expressed in mouse spleens and 1119 genes were differentially regulated in brains in response to JEV infection (change fold ≥ 2.0, p value < 0.05). Unsupervised clustering (Figure 1) analysis of the expression profiles showed a distinct mRNA signature in both spleens and brains during JEV infection. To elucidate the correlation between gene expression pattern and JEV infection-induced biological processes, functional classification of mRNA transcripts and pathway analysis were performed. Differentially regulated genes in spleens of JEV-infected mice are involved in the biological processes such as cellular process, biological regulation and immune system process, etc (Figure 2A). And the significant pathways of differentially expressed genes are known to be involved in cytokine-cytokine receptor interaction, natural killer cell mediated cytotoxicity, antigen processing and presentation, and chemokine signaling, etc (Table 1). While biological processes which showed differentially regulated genes in brains of JEV infected mice are cellular process, metabolic process, and biological regulation, etc (Figure 2B). And the significant pathways of differentially expressed genes in mouse brain are cytokine-cytokine receptor interaction, MAPK signaling, neuroactive ligand-receptor interaction, and toll-like receptor signaling, etc. (Table 2).

In spleens of mice with JEV infection, 263 genes were detected to be significantly upregulated and 174 genes were downregulated (Table 3). Genes with increased expression in spleens of JEV infected mice mainly fell into the function of immune response to viral infections. These include pro-inflammatory cytokine IFN-γ, IFN response transcription factor IRF7, IFN-induced proteins like IFIT1, IFITM3 and IFITM7, protein degradation gene ubiquitin-specific protease Usp29 and Usp18, apoptosis related genes granzymA, granzymeB, Porferin, and IMNB2, killer cell lectin-like receptors KLRC1, KLRC2 and KLRC3, and chemokines such as CXCL10, CXCL11, CCL12, CCL2 and CCL9. The marked increase in expression of these genes implies the occurrence of a strong antiviral protective response to JEV infection. The significantly downregulated genes are mainly involved in cell adhesion molecules such as monocyte/macrophage-lineage cell marker CD163, transmembrane cell surface receptor of Langerhans cells CD207, and ligand for myeloid cells receptor CD200. Evidence was also found for decreased expression of interferon transcription factor IRF6 and interleukin 7 receptor, which may contribute to JEV pathogenesis as well.

Compared to the expression after mock infection, 551 genes were detected to be significantly upregulated and 568 genes were downregulated in brains of JEV-infected mice (Table 4). Consistent to the results of spleen, chemokines CCL2, CCL3, CCL4 and CXCL10, IFN response transcription factor IRF7, IFN-induced proteins like Ifit1, Ifit2 and Ifit3, protein degradation gene Usp18 were obviously upregulated, and CD163 and IRF6 showed decreased expression upon JEV infection. In addition, increased expression of IFN-inducible transcription factors STAT1 and STAT2, TNF-α induced protein TNFAIP3, apoptosis-related proteins caspase3 and caspase4, suppressors of cytokine signaling Socs1 and Socs3, pro-inflammatory cytokines IL-1 and IL-6, TLR7, as well as IFN response antiviral genes of OAS family were also observed in microarray analysis. These results suggested the occurrence of a strong inflammatory response in mouse brain.

To confirm the microarray hybridization results, quantitative RT-PCR was performed on eight selected differentially expressed mRNAs in mouse spleen and brain respectively. As shown in the RT-qPCR result of spleen, granzymA, granzymeB, Porferin, IRF7, IFN-γ, CXCL10, and ILIR2 were significantly upregulated, while CD163 was downregulated (Table 5). Out of eight tested mRNAs in brains of mice infected with JEV, CCL2, CCL4, CXCL10, Casp3, Casp4, SOCS1 and SOCS3 showed increased expression, and CD163 was also found to have an obviously decreased expression (Table 5). Although absolute values are not identical due to the different sensitivity between the techniques, all genes showed a well comparative expression pattern with microarray data.

JEV wide type strain P3 was propagated in brains of suckling mice and titered in BHK-21 cells which was grown and maintained in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% heated-inactivated fetal bovine serum (FBS, Hyclone, Logan, UT, USA), 100 g/ml streptomycin and 100 IU/ml penicillin (Sigma-Aldrich, MO, USA) at 37°C with 5% CO2.

Four-weeks-old naive female BALB/c mice were purchased from Wuhan Institute of Biologic Products (Hubei Province, China) and inoculated subcutaneously (s.c.) with 5 × 10⁶ PFU of JEV P3 strain. Dilutions were performed in serum-free DMED and experimental controls were mock injected with diluent. A part of JEV-infected mice were sacrificed at day 3 post-inoculation (the day before the neurological symptoms appeared), and spleens were harvested. The left mice were sacrificed at day 6 post-inoculation (the day before the mice started to die), and brains of mice were harvested. Spleen and brain homogenate was made in DMEM for RNA extraction.

The total RNA was isolated from mouse spleens and brains respectively with trizol reagent (Invitrogen) for mRNA Microarray. mRNA hybridization was performed by shanghaiBio Corporation (shanghai, China) with the use of 4 × 44 K Agilent Whole Mouse Genome Oligo Microarray (total 41,174 oligo probes from 41,174 mouse genes). For each sample pair, the experiments were done with two independent hybridizations (Cy3 and Cy5 interchanging labeling). Hybridized arrays were scanned at 5 μm resolution on an Agilent DNA Microarray Scanner (Model G2565BA). Data extraction from images was done by using Agilent Feature Extraction software. Hierarchical cluster, gene ontology and pathway analysis were analyzed by using SAS (ShanghaiBio Analysis System).

For selected mRNA RT-qPCR, total RNA from the same samples used in microarray analysis was tested by using ABI 7500 FAST real-time PCR system. PCR primers were designed with Primer Express 2.0 software (Invitrogen). Results are shown as fold change. For mRNA RT-qPCR, experiments were carried out with the PrimerScript RT reagent Kit (TaKaRa) and SYBR Green Realtim PCR Master Mix (TaKaRa) according to manufacture's instruction. The housekeeping gene GHDAP was used for standardization of the initial RNA content of a sample. Relative changes of gene expression were calculated by the following formula, and the data are represented as fold upregulation/downregulation. fold change = 2^-ΔΔCt, whereΔΔCt =(Ct of gene of interest, treated -Ct of HK gene, treated)-(Ct of gene of interest, control-Ct of HK gene, control), Ct is the threshold cycle number and HK is the house keeping gene.