The JEV P3 strain was generated and harvested in mice brains according to the protocol in the previous study (Li et al., 2015). The heat-inactivated JEV P3 (heated-JEV P3) was acquired via incubating at 94°C for 15 min (Chang et al., 2015). The ZIKV MR766 was generated in the Vero cell line and collected for experiments. The hBMEC and hBMEC EGFR knockout (EGFR-KO) cell lines were kindly provided by Dr. Xiangru Wang (Huazhong Agricultural University, Wuhan, China) and subcultured in flasks with 10% heat-inactivated fetal bovine serum (FBS), 2 mM L-glutamine, 1 mM sodium pyruvate, vitamins, essential amino acids, nonessential amino acids, and 100 U/ml penicillin-streptomycin in RPMI 1640 medium, and maintained in a humidified incubator (37°C, 5% CO₂) (Wang et al., 2016). The Vero and BHK-21 cells were cultured in Dulbecco's Modified Eagle's Medium (DMEM) (Gibco) plus 10% FBS and 1% penicillin/streptomycin. The cells were starved in a serum-free medium for 12~16 h and subjected to further experimentation when cells were 90~95% confluent.

Human EGF recombinant protein (rhEGF) was purchased from Life Technologies (Gaithersburg, MD USA). Proteinase K, phenylmethylsulfonyl fluoride (PMSF), and Cell Counting Kit (CCK-8) assay were all obtained from Biosharp (Anhui, China). HighGene Transfection reagent (RM09014) was purchased from ABclonal (Wuhan, Hube, China). The EGFR inhibitor AG1478 (HY-13524), Gefitinib inhibitor (HY-50895), ERK inhibitor U0126 (HY-12031), and HSP90/STAT3 inhibitor 17-AAG (HY-10211) were purchased from MedChem Express (Shanghai, China). Antibodies used for Western blotting, anti-phospho-EGFR (Tyr1068), and anti-EGFR antibodies (both rabbit polyclonal antibodies) were obtained from Cell Signaling Technology (Danvers, MA, USA). Anti-phospho-STAT3 (Tyr705) and anti-STAT3 (both rabbit) were purchased from Abcam (Cambridge, MA, USA). Anti-phospho-ERK-T202/Y204 and anti-ERK antibodies (both rabbit) were purchased from ABclonal (Wuhan, Hubei, China). An anti-β-actin antibody was purchased from Proteintech (Chicago, IL, USA). Monoclonal antibodies of JEV envelope (E) and ZIKV non-structural protein 5 (NS5) were courtesy of Dr. Shengbo Cao (Huazhong Agricultural University, Wuhan, China). For immunofluorescence, mouse anti-EGFR antibody was obtained from Abcam and rabbit anti-EEA1 antibody was from Cell Signaling Technology, while Alexa Fluor 488 or Cy3-labeled secondary antibodies and DAPI were obtained from Thermo Fisher Scientific (San José, CA, USA).

When cells reached 100% confluence, hBMECs and Vero cells were serum-starved for 12~16 h before rhEGF was added. After that, cells were washed to prepare for the subsequent experiments. For the inhibition experiment, hBMECs were treated with the inhibitors AG1478, gefitinib, U0126, 17-AAG, or the vehicle control dimethyl sulfoxide (DMSO) at various concentrations for the corresponding times as previously described (András et al., 2005; Maruvada and Kim, 2012). For the virus infection procedure, hBMECs and Vero cells were infected with the JEV at a multiplicity of infection (MOI) of 1 at various times. All virus infection experiments were executed using the above-described procedure unless otherwise stated. The treated cells were then acquired for the following analysis.

Total RNA from treated cells was extracted with TRIzol reagent following the manufacturer's instructions (Invitrogen, Grand Island, NY). The RNA was reverse-transcribed into cDNA using the ReverTra Ace qPCR RT kit (Toyobo, Japan). SYBR Green 2 × mix (Invitrogen) was utilized to perform quantitative real-time PCR using a 7500 Real-Time PCR System (Applied Biosystems). The transcriptional levels of the target mRNA were normalized to β-actin except for the JEV-C gene and ZIKV-NS5 gene. A standard curve was generated to quantify the viral copy numbers, and the pcDNA3.0-HA/JEV-C gene plasmid or the pcDNA3.0-HA/ZIKV-NS5 gene plasmid was used as a template. All amplifications were performed in triplicate, and primers employed for the quantitative real-time PCR are listed in Table 1.

Immunofluorescence staining of the cells cultured in the 12-well plate was performed to determine virus replication. In brief, the cells were rinsed with 1 × PBS and fixed in 4% paraformaldehyde. The fixed cells were subsequently permeabilized and blocked with 0.2% Triton X-100 and 5% bovine serum albumin (BSA) in 1 × PBS for 2 h at room temperature, followed by incubation with mouse anti-JEV-E or mouse anti-ZIKV-NS5 monoclonal antibody (1:1,000) in a humidified chamber overnight at 4°C. After three washes with 1 × PBS, the cells were incubated with fluorescently labeled anti-mouse IgG for 1 h at room temperature. Then, the cell nuclei were stained with DAPI. Finally, immunostained samples were visualized under fluorescence microscopy or laser confocal microscopy (Nikon STORM).

For Western blotting analyses, briefly, cells were washed with ice-cold 1 × PBS and then whole cell extracts were prepared using 60 μl of RIPA buffer containing protease inhibitor cocktail (Roche) and phosphatase inhibitor cocktail (Roche). Then the lysates were centrifuged and quantified for protein concentration using a BCA protein assay kit (Beyotime, China). Protein samples were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (12%) and then transferred to PVDF membranes using a wet transfer system. The membranes were blocked in 5% BSA in Tris-buffered saline with Tween 20 (TBST) for 1 h at room temperature and were incubated with primary antibodies overnight at 4°C with shaking. After washing thrice with TBST, the membrane was incubated with a horseradish peroxidase (HRP)-labeled secondary antibody and finally visualized.

Plaque assays were performed on BHK-21 cells utilizing the 24-well culture plate. The supernatants were collected from virus-infected cells at indicated time intervals and stored at −80°C after removing cellular debris. The clarified supernatant was subjected to serial dilution and added into confluent BHK-21 cells for 1.5 h, and then the cells were overlaid with DMEM containing 1.5% carboxymethylcellulose (Sigma-Aldrich) for 5 days. Finally, the plaques were counted to calculate viral titers.

The CCK-8 assay was performed to determine cell viability. Briefly, the hBMECs were plated into a 96-well plate at a density of 5,000 cells per well for 24 h and starved for 12~16 h with a serum-free medium. The inhibitor of various concentrations was added into cells for 2 h, and CCK-8 solutions were subsequently added to each well and continuously incubated for approximately 45 min. Finally, the absorbance of the samples was measured at a wavelength of 450 nm in a Spectrophotometer reader (Bio-Rad, CA, USA).

The hBMECs were seeded in the 24-well plate until reaching 100% confluence, and the incubation medium was replaced with serum-free DMEM for 12~16 h. Then, the cells were incubated with JEV of indicated MOI at 4°C for 1 h (attachment assay). For entry assay, the plate was shifted to 37°C containing 5% CO₂ for 2 h, proteinase K (1 mg/ml) was used to remove non-internalized virions, and then 2 mM PMSF in 1 × PBS with 3% bovine serum albumin was applied to inactivated proteinase K as previously described (Dejarnac et al., 2018).

The statistical data are presented as the mean ± SEMs values, with at least three replicates for each treatment. Student's t-test, one-way analysis of variance (ANOVA), or two-way ANOVA were applied to analyze the statistical significance of the differences by using GraphPad Prism (v7.0; GraphPad, La Jolla, CA, USA). A value of p < 0.05 (^*) was considered statistically significant, while values of p < 0.01 (^**), p < 0.001 (^***), and p < 0.0001 (^****) indicated extremely significant differences.

According to the IFN-related genes identified to be markedly altered in succession at three time points in our unpublished hBMECs RNA-seq data and similar results in Li's report on RNA-seq of JEV-infected mouse brain (Supplementary Figure S1A) (Li et al., 2017), a protein to protein interaction (PPI) network was constructed by STRING, from which the emergence of EGFR caught our attention (Supplementary Figure S1B). Nevertheless, in the monolayer of hBMECs, there was no significant difference both in transcription and translation levels of EGFR at 12, 36, and 72 h post-infection (hpi) (Supplementary Figure S1C). Many viruses activate EGFR through phosphorylation at the early stage of infection, including ZIKV, PEDV, and IAV (Yang et al., 2018; Sabino et al., 2021; Wang et al., 2021). To figure out whether EGFR could be activated by JEV through phosphorylation, phosphorylation on tyrosine sites of EGFR in hBMECs at the early phase of JEV infection was examined. hBMECs were treated with JEV, heat-inactivated JEV (heated-JEV), and DMEM, respectively, and phosphorylated EGFR was determined by Western blotting at 0, 10, 20, 30, 60, and 120 min post-treatment. Simultaneously, the total EGFR expression levels were also measured. The accumulation of phosphorylated EGFR appeared at 10 min and was sustained for at least 2 h in JEV-infected hBMECs but not in heated-JEV or mock-infected hBMECs (Figure 1A). The inactivation of heated-JEV was verified by plaque assay, in which no live virus particles were detected (data not shown). Next, Vero cells were utilized to determine whether the activation of EGFR occurred in other cell types besides hBMECs. It was observed that the phosphorylation level of EGFR was also boosted in Vero cells, which is similar to the result in hBMECs (Figure 1B). These results demonstrated that JEV but not heated-JEV could induce the phosphorylation of EGFR with no remarkable effect on the expression of total EGFR. EGF, one of the ligands of EGFR, was utilized as a positive stimulator in the activation of EGFR (Yang et al., 2018; Kim et al., 2020). To determine if cells are responsive to EGF, cells were treated with rhEGF, which could prompt the receptor dimerization, autophosphorylation, and activation of EGFR (Herbst, 2004; Xiong et al., 2020). The phosphorylation of EGFR was induced at 10 min and reached the peak at 30 min in hBMECs, but the highest level was around 10 min in Vero cells, and the total EGFR showed no noticeable change over time (Figures 1C,D). The divergence of the highest levels of phosphorylated EGFR in hBMECs and Vero cells is probably owing to the different biological characteristics of the two cell types, while both cells are consistent with the previous report that prolonged stimulation with EGF leads to the degradation of ligand-induced phosphorylated EGFR (Schlessinger, 1986; Sorkin, 2001). Taking all these observations together, these results suggested that JEV infection activates EGFR through phosphorylation at the early stage of infection.

To explore whether the intracellular localization of EGFR could be affected by JEV, hBMECs were infected with JEV at an MOI of 1. Simultaneously, hBMECs were treated with rhEGF as a positive control. The specific antibodies of EGFR (red) and EEA1 (endosome marker, green) were utilized to determine the localization of EGFR under JEV infection, which was observed by confocal laser scanning microscopy. As shown in Figure 2A, compared to hBMECs in the mock group, both in JEV- and rhEGF-treated hBMECs, cell membrane EGFR substantially decreased and EGFR in cell cytoplasm and nucleus increased and presented as intracellular dot-like structures, which is concomitant with the increasing co-localization of EGFR and endosomal marker EEA1 (Figure 2A). Thereafter, the infection of JEV in hBMECs was measured with JEV E-specific antibody (green) by confocal laser scanning microscopy (Figure 2B). These results suggested that EGFR is internalized after JEV infection. Previous data demonstrated that EGFR localization might be changed at the early stage of JEV infection. To further confirm the result, hBMECs were infected with JEV at an MOI of 10, and then images were captured by confocal microscopy. The majority of the EGFR is located at the plasma membrane in uninfected hBMECs, while contrastingly, the dot-like structures near the cytoplasm and nucleus appeared at 30 min and persisted up to 120 min after JEV infection (Figure 2C). The altered subcellular localization of EGFR in hBMECs indicates that EGFR is internalized at the early phase of JEV infection.

For further study, two EGFR tyrosine kinase inhibitors, AG1478 and gefitinib, were applied, which were functionally approved in the previous research (Dhar et al., 2018; Li et al., 2021). To determine the toxic effects of the inhibitors, the cell viability of hBMECs was assessed upon inhibitor treatment. The hBMECs were seeded in 96-well plates and treated with AG1478 or gefitinib with concentrations from 0.5 to 100 μM for 48 h. In gefitinib-treated hBMECs, there were still more than 70% of cells that remained viable at the dose of 50 μM; for AG1478 treatment, in the concentration of 50 μM, 90% of cells remained viable. Hence, based on the CC₅₀ (cell cytotoxicity at 50%), 25 μM was chosen as the highest dose of treatment in the following study for both inhibitors (Figure 3A). Afterward, hBMECs and Vero cells were pretreated with AG1478 or gefitinib in different concentrations. It was observed that the tyrosine phosphorylation of EGFR abrogated markedly after pretreatment, which confirmed that AG1478 and gefitinib could be used as inhibitors of EGFR phosphorylation in both hBMECs and Vero cells (Figure 3B).

Previous studies gave evidence that the EGFR pathway plays a role in producing various viruses, such as DENV, ZIKV, etc (Ueki et al., 2013; Lin et al., 2019; Chuang et al., 2020). To explore whether EGFR is involved in the JEV infection process, cells were pretreated with AG1478 or gefitinib in different concentrations. It was observed that the level of viral RNA (JEV-C gene) was dramatically decreased in hBMECs treated with 0.5 μM of AG1478, 1 μM of gefitinib, or higher doses (Figure 3C). Further study demonstrated that both 10 μM AG1478 and 25 μM gefitinib memorably inhibited viral replication at 24 h and 48 h after JEV infection (Figure 3D). Consistent with the transcription levels, viral protein (JEV-E) expression was downregulated in cells treated with 10 μM AG1478 or 25 μM gefitinib at 24 hpi (Figure 3E). As shown in the immunofluorescence assay, the number of JEV-positive cells was significantly less in AG1478- or gefitinib-treated hBMECs than in a mock-treated control group (Figure 3F). Furthermore, the plaque assay illustrated that the production of viral particles was markedly reduced with inhibitor treatment in hBMECs (Figure 3G). Then, we investigated whether activated EGFR could also affect the infection of other CNS-invading flaviviruses in hBMECs. It was reported that ZIKV persistently infects hBMECs, and another report revealed that EGFR is related to the ZIKV life cycle (Mladinich et al., 2017; Sabino et al., 2021). Thus, the replication of ZIKV was determined in AG1478- and gefitinib-treated hBMECs. The result showed a significant reduction in the expression of ZIKV protein (NS5) in the inhibitor-treated groups compared to that in the mock group, which is consistent with the result of JEV infection (Supplementary Figures S2A,B). Taken together, the results demonstrated that activated EGFR is associated with the infection of JEV and ZIKV in hBMECs.

The above-mentioned results showed a positive correlation between the activated EGFR and viral infection in hBMECs. To further evaluate the function of EGFR in JEV infection, EGFR knockout (KO) hBMEC cell lines were utilized, which were previously generated by using clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9-mediated gene editing method (Fu et al., 2018). First, the expression of EGFR was confirmed in EGFR-KO cells by Western blotting (Figure 4A). The viral infection was drastically reduced in EGFR-KO cells both in RNA and protein levels in a time-dependent manner compared to that observed in wild-type (WT) hBMECs (Figures 4B,C). As shown in the immunofluorescence assay, the number of JEV-positive cells was significantly decreased in EGFR-KO hBMECs than in WT hBMECs (Figure 4D). Additionally, the plaque assay showed the viral titer was much lower in the supernatant of EGFR-KO hBMECs than that of WT hBMECs at 24, 48, and 72 hpi, which is consistent with the result of RNA and protein levels (Figure 4E). Furthermore, similar to JEV infection, the depression of ZIKV infection was also found in EGFR-KO hBMECs (Supplementary Figures S3A–C). These findings support the proposition that EGFR is crucial in mediating Flaviviridae infection in hBMECs.

As EGFR is positively correlated with viral infection in hBMECs, the underlying specific mechanism needs to be further elucidated. The previous report had identified EGFR as a critical regulator in JEV entry into human neuronal cells (Xu et al., 2016). Here, RT-qPCR was performed to validate whether EGFR is related to JEV entry into hBMECs. However, pretreatment with EGFR inhibitor AG1478 or gefitinib did not change either the attachment or entry of JEV to hBMECs compared to the control group treated with DMSO (Figure 5A). Additionally, consistent with inhibitor treatment, no significant difference was observed in viral attachment and entry between EGFR-KO hBMECs and WT hBMECs (Figure 5B). Since EGFR does not affect JEV attachment and entry in hBMECs, how does EGFR facilitate JEV infection in hBMECs? It has been widely reported that EGFR participates in the regulation of host innate immunity during viral infection (Lupberger et al., 2013; Qiu et al., 2020; Wang et al., 2021). For example, respiratory virus (RSV) induced EGFR phosphorylation, which inhibited interferon regulatory factor 1 (IRF1)-regulate interferon-lambda (IFN-λ) production and breakdown of airway epithelium antiviral response (Kalinowski et al., 2018). Besides, in COVID-19 therapy, EGFR signaling inhibitors potentiated the IFN-I response, thereby considered to be an attractive therapeutic strategy (Matsuyama et al., 2020). On the other hand, regarding the tight correlation between interferon response and JEV infection, it was also reported that interferon signaling affects the inoculation dose-independent mortality in JEV attacked mice (Aoki et al., 2014). Besides, restricted viral propagation was observed when IFN-β production was enabled by neutralizing miR-301a in mouse neurons (Hazra and Kumawat, 2017), In vitro, porcine IFN-α inhibited JEV replication, and the overexpression of ISG15 showed antiviral activity against JEV infection (Hsiao et al., 2010; Liu et al., 2013). Based on the studies and previous PPI analysis (Supplementary Figure S1B), we speculated that EGFR might serve as a regulator in interferon signaling of host innate immune response in JEV-infected hBMECs. Hence, the expression of several interferons and interferon-stimulated genes (ISGs) was measured. The result showed that the gene expression of type I IFNs (IFN-α and IFN-β), type III IFNs (IFN-λ1, and IFN-λ2, 3), and ISG15 were significantly increased in EGFR-KO hBMECs compared to the WT hBMECs under JEV infection (Figure 5C). Collectively, these data demonstrated the negative correlation between EGFR and interferon signaling in JEV-infected hBMECs.

Next, the downstream signaling of EGFR stimulated by JEV was investigated. As previously reported, the phosphorylation of ERK or signal transducers and activators of transcription 3 (STAT3) signaling was downstream of EGFR cascade to virus infection (Kung et al., 2011; Xu et al., 2013; Ding et al., 2017). Therefore, the phosphorylation of STAT3 and ERK was measured by Western blotting in hBMECs treated with JEV, rhEGF, or DMEM. Phosphorylation levels of STAT3 and ERK were increased in the hBMECs treated with JEV and rhEGF but not DMEM (Figure 6A). The decreased phosphorylation of STAT3 and ERK was observed at 30 min post-infection with the treatment of the inhibitor AG1478 and gefitinib, but not at 10 min (Figure 6B). It was assumed that the effects of EGFR on STAT3 and ERK may be induced later. Thus, the phosphorylation of STAT3 and ERK in hBMECs was determined at 60 and 120 min with the treatment of AG1478 or gefitinib. Decreased phosphorylation of STAT3 and ERK appeared in EGFR inhibitor-treated hBMECs and EGFR-KO hBMECs compared to the WT hBMECs (Figures 6C,D).

The use of 17-AAG, an inhibitor of heat shock protein 90 (HSP90), or U0126, an ERK inhibitor, has been shown to disrupt the phosphorylation of STAT3 or ERK, respectively, in hBMECs (Maruvada and Kim, 2012; Zhao et al., 2017). To further verify that STAT3 and ERK are related to virus replication during JEV infection, these two inhibitors were applied to the cells to inhibit phosphorylation of STAT3 or ERK (Figure 6E). When compared to mock-treated cells, a markedly impaired production of JEV viral RNA and E protein, as well as viral particles in U0126, was observed, but this change was not noticed in 17-AAG-treated hBMECs (Figures 6F–H). Previous reports have demonstrated that ERK negatively regulates the interferon signaling-mediated antiviral response (Yang and Ding, 2019; Freed et al., 2021). Therefore, the expression of interferons and ISGs was also measured with U0126 treatment in JEV-infected hBMECs. The expression levels of genes ISG15, IFN-α, IFN-β, IFN-λ1 and IFN-λ2, 3 were significantly increased in U0126-treated hBMECs compared to control during JEV infection, which probably is responsible for the dropped replication of JEV in hBMECs (Figure 6I). These findings support that the involvement of the EGFR-ERK pathway represses the host interferon signaling, leading to an acceleration of viral replication in hBMECs.