Tick-borne encephalitis virus induces chemokine RANTES expression via activation of IRF-3 pathway

All experimental procedures were conducted according to the guidance of the Institutional Animal Care and Use Committee of Wuhan Institute of Virology, Chinese Academy of Sciences. All surgeries were performed under sodium pentobarbital anesthesia, and all efforts were made to minimize suffering as well as the number of animals used.

WH2012, a Far Eastern strain of TBEV, was isolated from tick (I. persulcatus) samples and characterized as previously described [18]. The nucleotide acid sequence of WH2012 strain was deposited in the GenBank database (accession number KJ755186). Working stocks of TBEV were routinely propagated on Vero cells. All procedures with infectious materials were performed under biosafety level-3 (BSL-3) conditions.

BHK-21, Vero, SK-N-SH (a human neuroblastoma cell line; ATCC HTB-11), and T98G (a human glioblastoma cell line; ATCC CRL-1690) cells were cultured in Minimum Essential Medium (MEM). CCF-STTG1 (a human astrocytoma cell line; ATCC CRL-1718) and THP-1 (a human monocytic cell line; ATCC TIB-202) were grown in RPMI 1640 medium. A549 (a human alveolar epithelial cell line, ATCC CCL-185) cells were cultured in F-12K medium. All media were supplemented with 10 % fetal bovine serum, 2 mM l-glutamine, 100 U ml⁻¹ penicillin and 100 mg ml⁻¹ streptomycin (Life Technologies, NY). Human progenitor-derived astrocytes (HPDAs) were generated from neural progenitor cells and cultured as previously described [19, 20]. Differentiation of THP-1 (2 × 10⁵ cells/ml) monocytes into macrophage-like cells was achieved using 200 nM phorbol 12-myristate 13-acetate (PMA; Sigma, NY) for 3 days as previously described [21].

Groups of 1-week-old BALB/c mice of either sex were challenged with TBEV WH2012 by intraperitoneal (ip) or intracerebral (ic) inoculation route with a volume of 20 μl of virus suspensions at various concentrations, using Hamilton syringe (Hamilton Co., Switzerland). Mock-infected animals were inoculated with 20 μl of diluent (serum-free DMEM). Groups of female mice aged 7–8 weeks were infected intracerebrally with 10³ median tissue culture infective dose (TCID₅₀)s of TBEV WH2012. Mice were then monitored daily for signs of neurological disease and survival over a period of 12 to 25 days. Mortality rate was assessed at the time points indicated below.

Cohorts of virus-infected mice were sacrificed after ic inoculation with a virus dose containing 10³ TCID₅₀s for the time points indicated below. Whole blood was obtained by cardiac puncture, and sera were separated by centrifugation. Following perfusion with ice-cold phosphate-buffered saline (PBS), the small intestine, hind limb muscle, heart, lung, spleen, liver, and brain were collected, homogenized, and cleared by low-speed centrifugation. Then, sera and tissue samples were immediately stored at −80 °C for further analysis.

Virus infectivity was determined by estimation of the TCID₅₀ using standard cell culture conditions. Briefly, BHK-21 cells were seeded in 96-well plates. When the cells reach 80 % confluence, they were infected with 150 μl of serial decimal dilutions of each sample for 4 days. The cytopathic effect was thereby monitored by microscopic examination, and the infectivity titer was expressed as TCID₅₀/g tissue using the Reed-Muench formula.

After anesthesia with sodium pentobarbital (60 mg kg⁻¹), mice were transcardially perfused with ice-cold PBS followed by 4 % (w/v) paraformaldehyde (PFA) in PBS. The brains were dissected and post-fixed overnight, then trimmed, and routinely paraffin wax embedded. Serial 3–5-μm-thick sections were stained with hematoxylin-eosin (HE) for histological analysis under a light microscopy.

T98G, CCF-STTG1, SK-N-SH, HPDAs, A549, and differentiated THP-1 cells were infected with TBEV WH2012 at a multiplicity of infection (MOI) of 1. To assess the requirement for viral replication in the generation of RANTES, cells were treated with UV-inactivated preparations of TBEV. At the indicated time points, culture supernatants and cell monolayers were harvested for further analysis.

Total RNA was extracted from cell monolayers using Omega HP total RNA Isolation kit (Omega Bio-Tek, Inc., GA). To remove residual genomic DNA, RNA samples were pretreated with RQ1 RNase-free DNase (Promega, WI). Then, RNA was converted to cDNA using M-MLV Reverse Transcriptase (Promega). Real-time PCR was carried out using primers specific for human RANTES (sense 5′-ACCACACCCTGCTGCTTTGC-3′, antisense 5′-CCGAACCCATTTCTTCTCTGG-3′) and TransStart Eco Green qPCR SuperMix kit (TransGen, Beijing, China) on the Bio-Rad CFX96 Real-Time PCR System (Bio-Rad Laboratories, Inc., CA). The data acquisition and analysis were carried out with CFX Manager Software (version 2.1; Bio-Rad). Relative expression of RANTES was calculated using the 2^−ΔΔCt method [22] after normalization with endogenous control β-actin (sense 5′-CGGGAAATCGTGCGTGACAT-3′, antisense 5′-GAACTTTGGGGGATGCTCGC-3′). Results are expressed as the relative fold increase of the stimulated over the mock control group.

The mouse tissue homogenates and cell culture supernatants were assessed for RANTES protein levels using RANTES-specific enzyme-linked immunosorbent assay (ELISA) kit (Boster, Wuhan, China), according to the manufacturer’s instructions. The analytical procedure has been described previously [20].

Cell chemotaxis assay was performed as previously described [23, 24]. Briefly, THP-1 cells (5 × 10⁵ cells/100 μl) were added to the upper chamber of Transwell insert (5-μm polycarbonate filter; Corning, NY). A total of 1-ml culture supernatant from TBEV-infected T98G (MOI = 5) for 72 h was added to the lower chamber of Transwell insert. For the neutralizing experiment, anti-hRANTES Ab (0.5 μg/ml; R&D Systems, Minneapolis, MN) or control normal goat IgG was added to the conditioned media in the lower chamber. Plates were incubated for 4 h at 37 °C and 5 % CO2. Transwell inserts were then removed, and the number of cells migrated to the lower chamber was determined with a TC20 Automated Cell Counter (Bio-Rad). Data are expressed as percentage of the migrated cells in total number of input cells.

Cohorts of mice were treated via ic injection with Met-RANTES (1 μg/mouse; Bachem, Basel, Switzerland), vehicle, anti-RANTES mAb (MAB478), or an IgG2a isotype-matched control mAb (10 μg/mouse; R&D Systems) on days 2 to 8 after ic infection with TBEV WH2012 (10³ TCID₅₀s). Met-RANTES is a recombinant RANTES analog, in which the initiating methionine residue is retained after expression in Escherichia coli cells, resulting in a potent antagonist of the murine RANTES receptors CC chemokine receptor (CCR)5 and CCR1 [25, 26]. The chosen RANTES-neutralizing mAb reacts with murine and human RANTES and no other identified cytokine or chemokine [27, 28]. Mice were monitored daily for signs of neurological disease and survival over a period of 14 days. The survival curves were compared using Kaplan-Meier tests. Both brain virus titers and mice survival rates were estimated at the time points indicated below. The severity of inflammation was determined by staining sections of paraffin-embedded brain tissue with HE by day 8 post infection (p.i.), as after this time, the efficacy of this treatment may decline due to the decay of corresponding molecules within the mouse brain.

The RANTES promoter reporter construct (pGL2-220) and site-mutated plasmids for cAMP response element (CRE) (CRE Mut), interferon-stimulated response element (ISRE) (ISRE Mut), nuclear factor for interleukin 6 (NF-IL6) (NF-IL6 Mut), nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kB1) (NF-kB1 Mut), and NF-kB2 (NF-kB2 Mut) were kindly given by Dr. Casola (University of Texas Medical Branch, USA) [29]. The dominant-negative mutants of IkB kinase alpha (mIkBα), IRF-3ΔN, and IRF-7ΔN were kind gifts of Prof. S. B. Xiao (Huazhong Agricultural University, China). All plasmids for transfection were prepared with Endo-free Plasmid Midi Kit (Omega Bio-Tek, Inc., GA). Exponentially growing T98G cells in 24-well plates were transfected with 0.1 μg of pRL-TK reporter (Renilla luciferase for internal control) and 0.5 μg of RANTES-pGL2 plasmids (Firefly luciferase, experimental reporter) using the X-tremeGENE HP reagent (Roche, Mannheim, Germany). For co-expression experiments, 0.5 μg of each indicated expression plasmid was added to the reporter vectors. Twenty-four hours after transfection, cells were inoculated with TBEV and harvested at the indicated time interval. Reporter gene activity was measured using a dual-luciferase assay system (Promega, Madison, WI), according to the manufacturer’s instructions.

T98G cells were washed with PBS and lysed with RIPA lysis buffer (Beyotime, Nantong, China) containing protease inhibitor (cOmplete Protease Inhibitor Cocktail Tablets; Roche). Equal amounts of protein were separated on 12 % SDS-PAGE and transferred onto an Immobilon-P PVDF membrane (Millipore, MA). The blots were blocked for 1 h with 2 % bovine serum albumin in Tris-buffered saline (20 mM Tris-HCl, pH 7.4, 0.15 M NaCl) containing 0.1 % Tween 20 (TBST) at room temperature (RT) and reacted overnight at 4 °C with the primary antibodies. The membranes were washed four times with TBST and then incubated with the secondary Ab for 1 h at RT. Horseradish peroxidase-conjugated goat anti-rabbit IgG (#31460) or goat anti-mouse IgG (#31430) secondary antibodies were used (Thermo Scientific Pierce, IL). The membranes were washed and visualized with BeyoECL Plus Chemiluminescent Substrate (Beyotime) for signal detection. Primary antibodies directed against p-IRF-3 (Ser396, 4D4G, #4947), p-TANK-binding kinase 1 (TBK1) (Ser172, D52C2, #5483), retinoic acid-inducible gene 1 (RIG-I) (D14G6, #3743), melanoma differentiation-associated protein 5 (MDA5) (D74E4, #5321), IkBα (#9242), and TBK1 (#3013) were obtained from Cell Signaling Technologies (Beverly, MA). IRF-3 (#11312-1-AP) and β-actin (#60008-1-Ig) antibodies were purchased from Proteintech Group, Inc. (Chicago, IL).

T98G cells were transfected with small interfering RNA (siRNA) duplexes using HiPerFect transfection reagent (Qiagen, Hilden, Germany) according to the manufacturer’s protocols. siRNA oligonucleotides were synthesized (RiboBio, Guangzhou, China) with the following sequences (sense strands): RIG-I, 5′-GGAAGAGGUGCAGUAUAUU-3′; MDA5, 5′-GGUGAAGGAGCAGAUUCAG-3′ [30]. Nonsilencing siRNA with a scrambled sequence were used as a negative control (control siRNA (Ctrl siRNA)). After cultured for 16 h, the medium with transfection reagent was removed. Cells were either mock treated or infected with TBEV at an MOI of 1 for 24 h. Culture supernatants and cell monolayers were harvested for further analysis.

Inhibition of IRF-3 and NF-kB signaling in T98G and CCF-STTG1 cells was performed with BX795 (InvivoGen, CA) and MG132 (Millipore), respectively, as described previously [31, 32]. Briefly, virus inoculation were carried out as mentioned above, followed by treatment with BX795 (2 μM), MG132 (3 μM), or DMSO vehicle in the absence of serum for 36 h, and the levels of RANTES released in culture supernatants were determined by ELISA. IRF-3 and p-IRF-3 were detected by Western blot analysis. Virus infectivity was determined by TCID₅₀ assay as described above.

After ic inoculation of 1-week-old mice with TBEV (10³ TCID₅₀s), most animals developed neurological symptoms, such as hunched posture, loss of balance, hind limb paralysis, and convulsions in the final stage of the disease prior to death (data not shown). Major organs were harvested at various time points to determine virus titers in mice infected with TBEV. Virus was initially detected between 2 and 5 days p.i., with virus titers peaking on day 8 p.i. except for small intestine and heart. With respect to viral distribution, significantly higher virus titers were recovered from the brain, but relatively mediocre levels of viruses were also detected in peripheral tissues (Fig. 1a). Following ic infection of mice with sequentially increasing doses, all tested groups exhibited fatal outcomes, with no significant differences in the mean survival times between the challenge doses (Fig. 1b). However, following ip (Fig. 1c), subcutaneous (sc) [18] or intramuscular (im) (data not shown) infection of groups of mice with sequentially increasing doses, 100 % mortality was not observed even with the highest tested virus challenge dose. These observations indicate that direct CNS infection induces early death in mice even after low-dose challenge.

To gain insight into the neuroinflammatory responses elicited specifically in the CNS compartment, TBEV WH2012 strain was inoculated by the ic route. Time course of acute brain injury was assessed histologically. As depicted in Fig. 2, the HE-stained brain sections of the cerebral cortex obtained from virus-infected group indicate inflammatory cell infiltrates appeared as early as 3 days post challenge. By day 4 p.i., the inflammatory reaction increased. Marked inflammatory cell infiltrates were observed on day 8 p.i. when nearly all infected mice succumbed to infection.

CNS infiltration of peripheral immune cells requires antecedent local expression of cytokines, chemokines, and cell adhesion molecules [33]. To identify the key immune factors induced by TBEV in CNS, brains of virus-infected mice were harvested on days 2, 5, and 8 p.i., and the expression of 40 inflammatory factors was profiled by a mouse-specific protein array. Compared with baseline expression in uninfected controls, the secretion of a panel of immune factors was increased in mouse brains after virus inoculation. Among them, the CC-chemokine RANTES was induced within 5 days after TBEV infection, and the expression levels also remained elevated until the moribund state (Fig. 3a). Below, the expression of RANTES upon TBEV infection is characterized.

To confirm the protein array data, induction of RANTES was examined by ELISA. Mediocre induction of RANTES in response to TBEV infection was found as early as 3 days post challenge (13.9 ± 0.6-fold increase). Then, sustained up-regulation of RANTES in CNS was observed from day 4 to day 8 after infection. Closely mimicking the results from the protein array analysis, a robust increase in RANTES protein expression was observed on day 5 (226.6 ± 11.7-fold increase) and persisted up to day 8 (258.8 ± 10.5-fold increase) p.i. within the brain tissue. Interestingly, the induction pattern of RANTES expression appeared to be coincided with the dynamics of immune cell infiltration in the CNS. It is noteworthy that no concomitant increase of RANTES expression was recorded in tested peripheral tissues and sera (Fig. 3b). Besides, the protein abundance for RANTES (in pg/ml) in the brain was also greater than those observed in tested peripheral tissues and sera following ic TBEV infection (data not shown). To strengthen that the results obtained from 1-week-old mice are also valid in adult mice, RANTES protein levels in the brain and serum were also measured in mice aged 7–8 weeks after ic infection with TBEV. By day 5 p.i., a slight increase in RANTES expression occurred in the brain of adult mice (2.9 ± 0.5-fold increase). TBEV infection caused a dramatic increase in the level of brain RANTES on days 8 (36.4 ± 4.5-fold increase) or 11 (31.3 ± 4.6-fold increase) p.i. compared to mock controls (Fig. 3c).  There was no obvious induction of RANTES production in the serum, and RANTES level in the brain of TBEV-infected adult mice was also significantly higher when compared to that in the serum (data not shown). This higher RANTES concentration in the brain of virus-infected adult mice as compared to that in the serum suggest potential localized inflammatory responses. Taken together, these observations suggest that TBEV induces a significant increase of RANTES production within CNS, which is likely related to the progression of neuroinflammatory responses.

Previous studies have demonstrated that TBEV induced significant alterations in brain physiology that appeared to closely parallel the pattern of RANTES expression. To determine whether antagonizing the activity of this chemokine within CNS would affect disease progression, virus-infected mice were treated with either Met-RANTES or anti-RANTES mAb. As shown in Fig. 4a, b, all infected vehicle, Met-RANTES, isotype control mAb, and anti-RANTES mAb-treated mice eventually succumbed to TBEV infection. However, both Met-RANTES and anti-RANTES mAb-treated mice showed a significant delay in mean time to death after infection, compared with corresponding control groups. The mean survival time for isotype control mAb-treated infected mice was approximately 7.1 days, while anti-RANTES mAb-treated mice succumbed to mortality later after infection (mean days until death, 10.0 days). TBEV-infected Met-RANTES-treated mice lived an average of approximately 4 days longer (mean days until death, 11.3), compared with virus-infected vehicle-treated mice (mean days until death, 7.2). Moreover, TBEV-infected mice treated with Met-RANTES (Fig. 4c) or anti-RANTES mAb (Fig. 4d) exhibited delayed growth retardation, compared with virus-infected vehicle- or isotype Ab-treated animals, respectively. The intensity of symptoms increased gradually up to a moribund state, at a much slower rate for TBEV-infected mice treated with Met-RANTES or anti-RANTES mAb, compared with vehicle- or isotype Ab-injected animals, respectively (data not shown). These results suggest that antagonizing RANTES within CNS affects disease progression following lethal ic challenge with TBEV.

In an effort to identify the potential contributing factors to the enhanced survival of anti-RANTES-treated mice, we assess virus replication in the brain of infected mice. As shown in Fig. 4e, no significant difference was observed in TBEV titer recovered from the brain of Met-RANTES-treated mice compared with that in vehicle-injected mice. On day 2, 5, and 8 p.i., virus titers in brain tissue were also similar between the infected anti-RANTES mAb-treated and isotype Ab-treated groups. Morphologically, treatment of TBEV-infected mice with either RANTES receptor antagonist Met-RANTES or anti-RANTES mAb resulted in a significant decrease in local infiltration of inflammatory cells. In addition, less severe perivascular cuffing and hemorrhage within brains after Met-RANTES or anti-RANTES mAb administration, compared to the pathological manifestations present within the cerebral cortex of vehicle or isotype Ab-treated mice was found (Fig. 4f). Therefore, it is likely that blockade of RANTES within CNS reduced accumulation of infiltrating cells and extended the survival of mice after TBEV infection. Overall, our data highlight a potential role for RANTES within CNS in affecting neuroinflammation during TBEV infection.

The primary target of TBEV is the brain. To investigate the mechanism by which TBEV induces RANTES in the CNS, the ability of TBEV infection to trigger RANTES expression in human brain-derived cell lines was assessed. TBEV significantly induced RANTES production both at the messenger RNA (mRNA) and protein levels in human glioblastoma cells T98G at 24 h p.i. and reached a high level at 72 h p.i. compared with mock-infected controls (Fig. 5a, b). Human astrocytoma cells CCF-STTG1 and neuroblastoma cells SK-N-SH were also capable of significantly expressing RANTES in response to TBEV infection, but the level of RANTES expression was generally much lower than that in T98G cells (Fig. 5c–f). To examine whether TBEV infection induces RANTES expression in primary CNS cells, further experiments were conducted using HPDAs as a model. RANTES transcription was induced by TBEV infection, with the increase started at 24 h p.i. and peaked at 72 h p.i. (Fig. 5g). The protein levels of RANTES reached the peak at 72 h p.i., and the pattern was similar to that of the three cell lines described above (Fig. 5h). In tested virus-infected brain cells, the up-regulation of RANTES gene expression by TBEV was likely replication dependent, since UV inactivation dramatically impaired the ability of TBEV to induce RANTES mRNA expression and protein secretion. To reveal the kinetics of virus replication, virus titers were measured for the indicated time points by TCID₅₀ assay. As shown in Fig. 5i, TBEV replicated efficiently in all cell lines tested. Increasing titers of infectious virus were observed after 24 h p.i., and the maximum titer of virus was attained after 72 h p.i. Given the above-described RANTES expression profile, its production appeared to be significantly induced at time points that coincided with significant virus replication in human brain-derived cells. It was also found that viral replication was coupled to RANTES up-regulation in alveolar epithelial A549 cells (Additional file 1: Figure S1A–C). However, though TBEV could replicate in human monocytic cell line THP-1, RANTES production was not significantly induced after infection of this cell line, demonstrating a cell type-specific pattern of RANTES production (Additional file 1: Figure S1D–F). To conclude, the human brain cells are capable of significantly producing RANTES upon TBEV infection, although the level of induced RANTES may vary depending on the cell type.

We further determined the chemotactic activity of the secreted RANTES from TBEV-infected T98G cells using a transwell migration assay. Supernatants from these cultures were more potent in chemoattracting THP-1 cells, compared with those from uninfected control cells. Notably, the chemoattractant activities of these conditioned medium were largely reduced when incubated with anti-hRANTES Ab, suggesting that RANTES is a functionally active chemotactic component secreted by TBEV-infected T98G cells (Fig. 5j).

To determine whether TBEV infection transactivates the RANTES promoter, a reporter plasmid pGL-220 which contains the sequence from nucleotides −220 to −55 (relative to the mRNA start site) was co-transfected with pRL-TK into T98G cells, followed by TBEV infection. T98G was chosen as a cell model due to its relatively high transfection efficiency and the ability of the cells to produce high levels of RANTES transcripts upon TBEV infection. As shown in Fig. 6a, TBEV infection of transfected T98G cells induced a dose-dependent increase of luciferase activity, with the maximal stimulation detected at an MOI of 5. UV-inactivated virus was not capable of inducing RANTES transcription, confirming our previous observation that RANTES induction requires productive TBEV infection. The TBEV-induced promoter activation was also time-dependent, with the increase of luciferase activity started at 24 h and reached the highest levels at 48 h p.i. (Fig. 6b).

The RANTES promoter comprises five pivotal binding sites for the transcription factors CRE, ISRE, NF–IL-6, NF-kB1, and NF-kB2 (Fig. 6c) [29]. To determine the contributions of individual cis regulatory elements in conferring responsiveness to TBEV infection, we tested the impact of mutations at these sites on TBEV-induced RANTES production. As shown in Fig. 6d, mutation of the ISRE site not only affected the promoter basal activity but also nearly completely abolished TBEV-induced RANTES promoter activation. Mutation of the NF-kB1 or NF-kB2 site also reduced the basal activity, with the latter decreasing the TBEV-induced luciferase activity, although to a much lesser extent. Taken together, these data indicate that ISRE element of RANTES promoter is possibly mainly responsible for TBEV-induced RANTES expression.

To clarify the essential role of ISRE, the effects of ISRE-binding IRF-3 and IRF-7 transcription factors lacking their DNA-binding domains (mutants IRF-3ΔN and IRF-7ΔN, respectively) on TBEV-induced RANTES expression were determined. In reporter gene assays, compared with corresponding controls, over-expression of IRF-3ΔN dramatically inhibited the ability of TBEV in activating RANTES gene transcription, whereas co-transfection with IRF-7ΔN in TBEV-infected T98G cells did not have such impact (Fig. 6a). Moreover, over-expression of a nondegradable IkBα mutant (mIkBα) had no effect on activation of the RANTES promoter (Fig. 6b).

To determine whether TBEV trigger RANTES expression by directly inducing activation of known IRF-3 pathway component, we performed Western blot analysis to detect the levels of endogenous RIG-I, MDA5, TLR3, p-TBK-1, p-inhibitor-kB kinase epsilon (IKKε), and p-IRF-3 following infection of T98G cells with TBEV. As depicted in Fig. 6c, TBEV induced up-regulation of RIG-I and MDA5 as early as by 24 h p.i., whereas no detectable TLR3 and p-IKKε were seen during the process of infection in T98G cells (data not shown). Concomitantly, virus replication also activates specific virus-induced kinase TBK-1, which regulates the phosphorylation and thus the activation of IRF-3 [34]. Indeed, phosphorylated forms of IRF-3 (p-IRF-3) were clearly detectable by 24 h p.i. However, we failed to detect a significant decrease in the level of endogenous IkBα, which is a hallmark of IkBα degradation and NF-kB activation.

The abovementioned results indicate that the expression of both RIG-I and MDA5 is greatly enhanced in response to TBEV infection. To explore the role of these proteins in modulation of virus-induced RANTES production, we performed knockdown of RIG-I and MDA5 using siRNA. As shown in Fig. 7d, Ctrl siRNA did not affect the virus-induced activation. Knockdown of either RIG-I or MDA5 led to a significant decrease in the amount of RANTES chemokine production under the experimental conditions. These results indicate the involvement of endogenous RIG-I and MDA5 in the up-regulation of RANTES upon TBEV infection in T98G cells.

To confirm the actual contribution of TBK-1/IRF-3 module to the TBEV-induced RANTES expression, T98G cells were treated with BX795, an inhibitor blocking TBK1- and IKKε-mediated activation of IRF-3, followed by TBEV infection. BX795 significantly blocked the TBEV-induced phosphorylation of IRF-3 at Ser396. Furthermore, secretion of RANTES from T98G and CCF-STTG1 cells was significantly blocked in the presence of BX795. In contrast, treatment with MG132, a potent inhibitor of NF-kB activation, had no effect on RANTES release in virus-infected cells. Therefore, it appears that TBEV-induced production of RANTES is unlikely dependent on the NF-kB signaling pathway (Fig. 7e). It should be noted that both BX795 and MG132 did not significantly block virus replication under the conditions of the experiment (Additional file 2: Figure S2). These results suggest that activation of IRF-3 signaling pathway is most likely to be involved in the TBEV-induced RANTES expression.