Skip to main content
NCBI home page
Virologica Sinica logoLink to Virologica Sinica
. 2025 Jul 15;40(4):647–657. doi: 10.1016/j.virs.2025.07.007

Quercetin inhibits herpes simplex virus 1 replication in corneal epithelium and suppresses keratitis progression

Yubin Yu a,b,1, Sihao Liu a,1, Qinghua Liu a, Xiuping Liu a, Kaili Wu a,⁎
PMCID: PMC12414389  PMID: 40675379

Abstract

Quercetin is a natural compound with potent antiviral effects; however, its role in the treatment of herpes simplex keratitis (HSK) remains underexplored. Here, we investigated the antiviral effects of quercetin against herpes simplex virus 1 (HSV-1). By examining different phases of viral infection in human corneal epithelial cells (HCECs), we found that 30 μmol/L quercetin inhibits HSV-1 replication primarily by disrupting viral attachment. RNA-sequencing and subsequent analyses revealed that the nuclear factor E2-related factor 2 (Nrf2) was upregulated by quercetin in a dose-dependent manner. Knocking down Nrf2 partially compromised quercetin's antiviral effect. Importantly, topical application of 100 μmol/L quercetin alleviated HSK severity in mice, reduced viral titers in tears, and inhibited VP16 expression in the cornea and trigeminal ganglia. These findings demonstrate the antiviral effect of quercetin against HSV-1 and provide a foundation for mechanistic studies to elucidate its therapeutic potential in HSK.

Keywords: Quercetin, Herpes simplex virus type 1 (HSV-1), Herpes simplex keratitis (HSK), Nrf2, Antiviral

Highlights

  • •

    Quercetin inhibits HSV-1 replication by disrupting viral attachment.

  • •

    RNA-sequencing analysis was conducted to identify host factors mediating the antiviral effect of quercetin against HSV-1.

  • •

    Nrf2 plays a role in the anti-HSV-1 effects of quercetin.

  • •

    Topical application of quercetin eye drops effectively inhibits the progression of herpes simplex keratitis.

Introduction

Herpes simplex virus type 1 (HSV-1) is known to be a widespread pathogen, with approximately 66.6% of individuals aged 0–49 years having been exposed to the virus (James et al., 2020). The infection process begins with viral attachment to host cells, where viral glycoproteins bind to cellular receptors. This is followed by fusion of the viral envelope with the plasma membrane, which allows the virus to penetrate the cell further (Spear, 2004). In corneal epithelial cells, nectin-1 and herpesvirus entry mediator (HVEM) have been identified as HSV-1 receptors, with nectin-1 demonstrating higher efficiency in promoting viral attachment than HVEM (Shah et al., 2010; Dai et al., 2024). Following primary infection, the virus can establish lifelong latency within the trigeminal ganglion and may be reactivated by factors such as stress, fever, and sunburn (Sibley and Larkin, 2020).

Currently, acyclovir and other nucleoside analogs are the primary topical drugs for managing herpes simplex keratitis (HSK) as they inhibit viral DNA polymerase, thereby impeding viral replication (Sibley and Larkin, 2020). Despite their efficacy, the clinical use of nucleoside analogs is limited by emerging drug resistance issues (Duan et al., 2009). Furthermore, nucleoside analogs can only be incorporated into the replicating viral genome, leading to premature termination of viral DNA synthesis. Consequently, they are ineffective in eradicating latent HSV infections (Whitley and Gnann, 1992). Thus, due to the high infection rate and lack of curative treatment for HSV-1 infection, HSK remains a leading cause of corneal blindness worldwide (Sibley and Larkin, 2020).

Previous studies have reported various medicinal plants and natural compounds effective against HSV-1, including acyclovir-resistant HSV-1, with the flavonoid class of compounds being widely mentioned (Li et al., 2019; Luo et al., 2020; Liu et al., 2021; Xu et al., 2022). Quercetin, a prominent flavonoid, is known for its broad antiviral potential, effective against both DNA and RNA viruses (Di Petrillo et al., 2022). Several mechanisms may be involved, including the inhibition of polymerases, reverse transcriptases, and proteases, suppression of DNA gyrase, and binding to viral capsid proteins. Identified as one of the primary compounds in Houttuynia cordata water extracts, in vitro studies have demonstrated that quercetin can exert antiviral effects in the early stages of HSV-1 infection, and its inhibition was found to be related with NF-κB activation (Hung et al., 2015; Lee et al., 2017). However, the precise mechanism underlying its antiviral activity requires further elucidation. For ocular surface applications, quercetin has been highlighted for its potent antioxidant properties in corneal epithelial cells and as a potential anti-inflammatory drug (Stoddard et al., 2013; Oh et al., 2015; Luan et al., 2023). While the topical use of quercetin is beneficial for conditions such as dry eye and fungal keratitis (Oh et al., 2015; Luan et al., 2023), its potential therapeutic effects on HSK are not yet fully elucidated.

This study investigated the anti-HSV-1 efficacy of quercetin in vitro, explored its underlying mechanisms, and evaluated its therapeutic potential for HSK through the topical application of quercetin eye drops in mouse model.

Results

Quercetin inhibited HSV-1 replication in HCECs at different time regimens and phases of infection

Quercetin is known as 3,3′,4′,5,7-pentahydroxyflavone, and the CCK-8 assay revealed that quercetin showed no or only slight effects on cell viability at concentrations up to 70 μmol/L (Fig. 1A). Thus, concentrations of quercetin below 70 μmol/L can be considered safe for treating HCECs. To explore the potential anti-HSV-1 effect of quercetin on HCECs, 30 μmol/L quercetin was added at different time regimens (Fig. 1B). The results showed that quercetin exhibits strong antiviral effects across different time regimens, including pre-treatment, co-incubation, and post-treatment (Fig. 1C–E). Then, we determined that quercetin has no viral inactivation effect (Fig. 1F, J, K). Further investigation was conducted to determine which phases of virus infection are targeted by quercetin (Fig. 1G). The results revealed that quercetin treatment significantly reduced the attachment of HSV-1, while showing no effect on virus penetration (Fig. 1H and I). Using AutoDock Vina, we identified that quercetin exhibits a potential binding ability to nectin-1, a crucial receptor for the HSV-1 gD protein in HCECs (Fig. 1L). The calculated binding energy between nectin-1 and quercetin was −6.9 kcal/mol, indicating a highly stable binding interaction. Overall, these results suggested that quercetin may inhibit HSV-1 replication in HCECs by blocking the virus's receptor before endocytosis.

Fig. 1.

Fig. 1

Open in a new tab

The impact of quercetin on human corneal epithelial cells (HCECs) viability and its ability to inhibit HSV-1 replication at different time regimens and phases of HSV-1 infection. A Cell viability of HCECs under different concentrations (0, 1, 10, 30, 50, 70, 100 μmol/L) of quercetin. B Schematic illustrations of quercetin application against HSV-1 infection in HCECs at different time regimens: pre-treatment, co-incubation, and post-treatment. C–E Quercetin (30 μmol/L) was added into HCECs 2 h before the HSV-1 F strain infection (pre-treatment), together with the HSV-1f infection (co-incubation), or after the HSV-1f infection for 1 h (post-treatment). The relative mRNA expressions of VP16 were evaluated. F In viral inactivation assay, HSV-1 was incubated with 30 μmol/L quercetin at 37 °C for 2 h, the relative mRNA expressions of VP were then evaluated. G–I To determine which phases of virus infection are targeted by quercetin, quercetin (30 μmol/L) was added at the different phases of HSV-1 infection (attachment, penetration), and the relative mRNA expressions of VP16 were evaluated. J, K Quercetin (30 μmol/L) was added at different phases of viral infections, the replication of HSV-1 H129 GFP knock-in strain was assessed using a fluorescence microscope. The extent of viral infection was assessed by measuring GFP fluorescence (n = 3). L Molecular docking verification of quercetin and nectin-1. Three-dimensional structures and hydrogen bonds of the binding pocket were shown. Data are presented as mean ± SD from three independent experiments. ns indicates no statistical significance, ∗∗P < 0.01, and ∗∗∗P < 0.001. P values were calculated with one-way ANOVA (A) or unpaired t-test with Mann-Whitney correction (C–K).

Quercetin inhibited HSV-1 replication in a dose-dependent manner

To further investigate the anti-HSV-1 efficacy of quercetin, we treated HCECs with various concentrations of quercetin in the maintenance medium following infection with the HSV-1. As shown, quercetin at 10 μmol/L induced a significant reduction of the replication of HSV-1 H129 GFP knock-in strain (HSV-1g) (reduced to 45.60%), and concentrations of 20 μmol/L and 30 μmol/L induced more substantial reductions (reduced to 21.46% and 17.09%, respectively). In addition, 10 μmol/L quercetin showed a similar inhibition of HSV-1g with 10 μmol/L acyclovir (reduced to 44.81%) (Fig. 2A, C). Viruses released into the medium were collected, and the viral titer was evaluated by a plaque assay. As shown in Fig. 2B and D, while acyclovir significantly reduced the release of HSV-1, quercetin at 30 μmol/L exhibited comparable antiviral activity to acyclovir. The detection of HSV-1g fluorescence reflected intracellular and extracellular virus levels, while plaque assays quantified the amount of released virus. These results suggested that quercetin inhibited HSV-1 replication in a dose-dependent manner.

Fig. 2.

Fig. 2

Open in a new tab

Quercetin suppressed HSV-1 replication in a dose-dependent manner. A, C HCECs were exposed to HSV-1g (MOI = 0.1) for 1 h, and then treated with DMSO, acyclovir (Acy), or quercetin (Que) at different concentrations for an additional 24 h. The extent of viral infection was assessed by measuring GFP fluorescence (n = 3). B, D HCECs were infected by HSV-1f (MOI = 0.1) for 1 h and then treated with DMSO, acyclovir, or quercetin at different concentrations an additional 24 h. The viral titer was quantified by PFU assay (n = 3). Data are presented as mean ± SD. ∗P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001 compared to the DMSO group. P values were calculated with one-way ANOVA.

Quercetin inhibited the gene expression of HSV-1

The HSV-1 genes have been categorized into immediate early, early, and late genes. Using qRT-PCR, we observed a dose-dependent inhibition on the expression of immediate early (α4), early (UL29), and late (US6, and UL48) HSV-1 genes (Fig. 3A). Quercetin demonstrated a similar inhibitory effect on viral replication at 30 μmol/L compared to acyclovir at 10 μmol/L. Furthermore, we examined the expression of proteins encoded by the viral genes and found that quercetin had dose-dependent inhibitory effects on viral proteins (ICP4 and gD) (Fig. 3B–D). Notably, 30 μmol/L of quercetin had effects comparable to 10 μmol/L of acyclovir. In conclusion, quercetin exhibited dose-dependent inhibition of viral gene expression.

Fig. 3.

Fig. 3

Open in a new tab

Quercetin suppresses gene and protein expressions of HSV-1. A HCECs were infected with HSV-1f (MOI = 0.1) for 1 h before the different agent treatment for an additional 24 h, and the relative expressions of viral genes, including α4, UL29, US6, and UL48, were quantified using qRT-PCR (n = 3). B–D The expressions of viral proteins (ICP4 and gD) were visualized, and their relative expressions were analyzed (n = 3). Data are presented as mean ± SD. ∗∗∗P < 0.001 vs. DMSO group. P values were calculated with one-way ANOVA. Acyclovir, Acy; quercetin, Que.

RNA sequencing analysis identified the Nrf2 as a potential critical factor upregulated by quercetin for inhibiting HSV-1 replication

Since we had already clarified the antiviral attachment effects of quercetin, we conducted further investigations to explore its intracellular actions following endocytosis. The RNA-seq was performed on HSV-1-infected HCECs treated with quercetin to delineate gene expression patterns. In comparison to the HSV-1+DMSO group, a total of 11,660 genes showed altered expression in the HSV-1+Quercetin group, including both upregulated and downregulated genes (Fig. 4A, Supplementary Table S2). Gene ontology (GO) analysis showed significant enrichment of upregulated genes in biological processes such as DNA repair, autophagy, and response to oxidative stress, which may indicate the underlying molecular mechanism (Fig. 4B). As mentioned, the topical application of quercetin has been proven to reduce oxidative stress on the ocular surface efficiently (Stoddard et al., 2013; Oh et al., 2015). Thus, this study further explored the potential relationship between quercetin's abilities for antioxidative stress and anti-HSV-1. To identify critical molecules, we visualized the heatmap displaying differentially expressed genes (DEGs) enriched in response to oxidative stress (Fig. 4C). Among the DEGs, Nrf2 showed a significant difference between cells treated with DMSO or quercetin. As Nrf2 has been reported to repress the replication of HSV-1 (Zhang et al., 2022; Wang et al., 2023), we explored the relative expressions of Nrf2 in cells treated with acyclovir or quercetin at various concentrations. The results consistently showed that Nrf2 expression was upregulated by quercetin in a dose-dependent manner at both the mRNA and protein levels (Fig. 4D–F). These findings suggested that quercetin inhibited viral replication while simultaneously activating the expression of Nrf2.

Fig. 4.

Fig. 4

Open in a new tab

RNA sequencing analysis revealed that Nrf2 might be a key factor upregulated by quercetin to inhibit HSV-1 replication. A Volcano plot of DEGs in the HSV-1+DMSO and HSV-1+quercetin groups. Green dots indicated downregulated genes (log2 fold change < −3.32, adjusted P < 0.05), while red dots represented upregulated genes (log2 fold change >3.32, adjusted P < 0.05). B GO enrichment analysis between the groups. The dot plot color denoted the P value, and the dot size denoted gene count. C Heatmap of DEGs related to oxidative stress response. HCECs were treated with indicated agents for 24 h following HSV-1 infection, and the expression levels of Nrf2 were analyzed using qRT-PCR (D) and Western blotting (E, F). Data represent mean ± SD of three independent experiments. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001. P values were calculated with one-way ANOVA. Acyclovir, Acy; quercetin, Que.

Quercetin inhibited the replication of HSV-1 partly via increasing the Nrf2 expression

To further investigate the protective role of Nrf2 against HSV-1 replication, we generated Nrf2-deficient HCECs using siRNA. The deficiency of Nrf2 was confirmed at both the mRNA and protein levels (Fig. 5A–C). Subsequently, to investigate whether quercetin inhibits ICP4 expression and viral replication in an Nrf2-dependent manner, HCECs previously transfected with either Nrf2 siRNA or NC siRNA were infected with HSV-1 and then treated with or without quercetin. Nrf2 silencing significantly augmented ICP4 expression. While quercetin administration markedly reduced ICP4 expression in both groups, ICP4 levels remained significantly higher in the Nrf2 siRNA-transfected cells compared to the NC siRNA-transfected cells (Fig. 5D–G). Notably, quercetin treatment diminished HSV-1 replication in both the Nrf2 knockdown and NC siRNA groups (Fig. 5H and I), suggesting that quercetin's antiviral activity is not exclusively dependent on the Nrf2 pathway. However, the magnitude of this reduction in HSV-1 replication following quercetin treatment was substantially less pronounced in the Nrf2 siRNA group than in the NC siRNA group (Fig. 5H and I). These results suggest that the anti-HSV-1 activity of quercetin is linked to Nrf2 expression.

Fig. 5.

Fig. 5

Open in a new tab

Nrf2 reduces the antiviral activity of quercetin. A Following transfection with either NC siRNA or Nrf2 siRNA, the relative expression of Nrf2 in the cells was measured by qRT-PCR (n = 3). B, C Nrf2 expression in the siRNA-transfected cells was detected by Western blotting, and the relative levels were analyzed (n = 3). D siRNA-transfected cells were treated with or without quercetin after HSV-1 infection, and qRT-PCR evaluated the expressions of ICP4. E–G siRNA-transfected cells were treated with or without quercetin after HSV-1 infection, the expressions of ICP4 and Nrf2 were evaluated by Western blotting. H siRNA-transfected cells were treated with DMSO or quercetin after viral infections, the replication of HSV-1 H129 GFP knock-in strain was assessed using a fluorescence microscope. I The extent of viral infection was assessed by measuring GFP fluorescence (n = 3). Data are presented as mean ± SD. ∗∗P < 0.01, ∗∗∗P < 0.001 vs. NC siRNA group. P values were calculated with unpaired t-test with Mann-Whitney correction. Quercetin, Que.

Topical application of quercetin mitigated the severity of HSK in mouse models

After establishing the antiviral efficacy of quercetin in vitro, we investigated its potential as a potent topical antiviral eye drop against HSV-1 in vivo. In contrast to the concentration used in vitro, a working quercetin concentration of 100 μmol/L was employed in vivo. This adjustment considers the kinetics of eye drops in vivo and the safety of topical quercetin application on the ocular surface of mice, as previously reported in the literature (Yin et al., 2021; McKay et al., 2022). Healthy mice treated with quercetin or 0.1% DMSO showed no visible corneal damage (Supplementary Fig. S1). In the infected mice, eyes treated with DMSO exhibited signs of ocular infection on 3 dpi, with worsening symptoms and higher disease scores on 5, 6, and 7 dpi (Fig. 6A and B). Conversely, the eyes treated with quercetin showed symptoms on 3 dpi that improved significantly in the following days. While mice treated with DMSO exhibited a significant reduction in body weight following HSV-1 infection, mice treated with quercetin showed no significant reduction in body weight (Fig. 6C). The eyes treated with DMSO exhibited a disrupted corneal epithelial structure and typical epithelial thickening, which are indicative of acute infection. In contrast, eyes treated with quercetin exhibited minimal disruption in the corneal structure (Fig. 6D).

Fig. 6.

Fig. 6

Open in a new tab

The topical application of quercetin alleviated the severity of HSK in mouse models. Six-to eight-week-old female SPF BALB/c mice were infected in the right eye with 1 × 106 PFU HSV-1f, followed by topical treatment with 100 μmol/L quercetin or 0.1% DMSO four times daily from 1 dpi. A Images captured by slit lamp of the right eyes of mice treated with different agents on specific dpi. Each image represents findings from 5 mice in a single experiment. B Ocular disease scores (0–5, 5 being severe) in mice (n = 5 in each group). C Body weights of mice (n = 5 in each group). D Corneal histology of eyes at 7 dpi. Each image represents findings from three mice across two independent experiments. E HSV-1 titers in the tears of each mouse at 3 and 5 dpi were analyzed using plaque assays, with n = 6 mice. At 7 dpi, VP16 relative expression levels in corneas F and trigeminal ganglia G were determined by qRT-PCR, respectively (n = 3). Data are presented as mean ± SD. ∗P < 0.05, ∗∗∗P < 0.01, ∗∗∗P < 0.001. n.s. = no significance. P values were calculated with or unpaired t-test with Mann-Whitney correction.

Significantly lower viral titers in tears were detected following quercetin treatment than those treated with DMSO at 3 and 5 dpi (Fig. 6E). Relative VP16 expression analysis of corneas further demonstrated reduced replication of HSV-1 in eyes treated with quercetin (Fig. 6F). To determine whether quercetin treatment inhibited the transmission of HSV-1 to the trigeminal ganglia (TG), we harvested TGs from infected mice and assayed them to assess the relative expressions of VP16. Mice treated with quercetin exhibited significantly lower expressions of VP16 (P < 0.05) compared to the control group (Fig. 6G).

These results demonstrate that topical quercetin alleviated HSK severity, reduced viral titers in tears, and inhibited viral spread to the trigeminal ganglia.

Discussion

Typically, viral entry involves the binding of viral glycoproteins to cell surface receptors, followed by fusion of the viral envelope with the plasma membrane. Our results demonstrate that quercetin can effectively inhibit the attachment of HSV-1 to HCECs without affecting the penetration ability of HSV-1. Additionally, quercetin does not directly inactivate the virus. Quercetin has been reported to inhibit HSV attachment to the surface of Vero cells, possibly due to the competitive binding of this small molecular compound to cellular receptors such as HVEM or nectin-1 (Hung et al., 2015). While silencing the expression of HVEM in HCECs did not dramatically alter viral entry (Guo et al., 2015), knocking down the expression of nectin-1 using the CRISPR/Cas9 system significantly alleviates HSV-1 infection in vitro (Li et al., 2022b). Here, we analyzed the potential interaction between quercetin and nectin-1 using AutoDock software and identified a strong binding affinity between them. This suggests that quercetin reduces HSV-1 entry by interacting with nectin-1. Further studies are needed to substantiate this hypothesis.

Due to the widespread use of acyclovir, concerns about its side effects and the emergence of acyclovir-resistant strains have highlighted the need for new antiviral drugs (Rousseau et al., 2022). We demonstrated that quercetin blocked the replication of HSV-1 in a dose-dependent manner, consistent with previous studies (Hung et al., 2015; Lee et al., 2017). Furthermore, 30 μmol/L quercetin exhibited an equivalent capability against HSV-1 F strain as 10 μmol/L acyclovir in HCECs, indicating that quercetin could be a potential drug for the therapy of HSK. Notably, 10 μmol/L quercetin showed an equivalent anti-HSV-1 effect to acyclovir at 10 μmol/L when HCECs were infected by HSV-1 H129 GFP knock-in strain (HSV-1g). This phenomenon, where different drug concentrations are required to resist two different HSV-1 strains, is consistent with our previous research findings (Li et al., 2022a). Previous studies have reported that infection of rabbit corneas with ten different HSV-1 strains resulted in observing typical HSK symptoms in all rabbits at the endpoint. However, the progression of infection varied among the different strains (Hill et al., 1987). Thus, while various HSV-1 strains can be used as experimental models for studying viral characteristics, employing different HSV-1 strains within a single experiment may improve the reliability of the results. We were unable to study the effects of quercetin on acyclovir-resistant HSV-1 strains here due to the lack of these virus strains. However, a previous study demonstrated that water extracts of Houttuynia cordata could inhibit the replication of acyclovir-resistant HSV-1, with quercetin being identified a major contributing compound (Hung et al., 2015).

Viral infection relies on the genomic activity of host cells. After entering the cell, the viral genome is transported into the cell nucleus, where it undergoes transcription and translation before the DNA recombination with viral proteins and further infection with neighboring cells (Zhu and Viejo-Borbolla, 2021). Therefore, inhibiting the transcription and translation of viral genes within cells is also a viable antiviral strategy. Our study demonstrated that quercetin treatment post-HSV-1 infection significantly reduced the expression of viral genes at both mRNA and protein levels, indicating a potential intracellular effect of quercetin against HSV-1. Based on RNA-sequencing analysis and siRNA treatment, we found that the inhibition effect of quercetin on HSV-1 is partly due to the upregulation of the expression of Nrf2. In most instances, Nrf2 activation shields host cells from viral infections (Zhang et al., 2022; Muzammil et al., 2024). Cellular assays have shown that activating Nrf2 not only suppresses SARS-CoV-2, Zika virus, Ebola virus, and influenza A virus replication but also lowers mortality rates and extends lifespan post-infection in vivo (Olagnier et al., 2020; Muzammil et al., 2024). As for HSV-1, Zhang et al. reported that upregulating the expression of Nrf2 by transfecting cells with plasmid could significantly inhibit HSV-1 replication (Zhang et al., 2022). Furthermore, using the agonist of Nrf2, such as tBHQ, could also protect the cells from HSV-1 infection. These all indicated the importance of Nrf2 in HSV-1 replication. Our in vitro results have demonstrated that quercetin's anti-HSV-1 replication activity is partly attributable to its regulatory effect on Nrf2. Further investigation is required using Nrf2-targeting siRNA or Nrf2-specific knockout mice within an HSK model to fully elucidate the role of quercetin-mediated Nrf2 regulation in the progression of HSK. Furthermore, we have verified that quercetin inhibits HSV-1 replication via a dual pathway—by impeding HSV-1 from attaching to host cells and by modulating Nrf2 expression. Future research will integrate these two factors to ascertain whether they are the primary determinants of quercetin's anti-HSV-1 activity. Additionally, RNA-sequencing data will be further analyzed to explore other potential underlying mechanisms.

Ocular topical application of quercetin has been reported to be effective against fungal keratitis and dry eye (Oh et al., 2015; Luan et al., 2023). We found that quercetin at a concentration of 100 μmol/L is safe and effective for treating HSK in vivo. A higher drug concentration is usually required in vivo compared to in vitro, as eye drops typically have poor bioavailability due to rapid nasolacrimal drainage and limited corneal permeability (Sharma and Taniguchi, 2017; Han et al., 2023). Our results found that quercetin eye drops were effective in ameliorating corneal ulceration and reducing viral replication in the corneas of mice infected with HSV-1, consistent with in vitro studies where quercetin inhibited HSV-1 replication in HCECs. Furthermore, in addition to improving corneal symptoms in mice, our findings suggested that topical treatment with quercetin also reduced viral transmission from the cornea to the trigeminal ganglia. Since HSV-1 establishes latent infections in the trigeminal nerve, the result of the present study suggested that quercetin may have some inhibitory effect on latent infections. Subsequent research should aim to determine whether quercetin can inhibit the neuronal transport of HSV-1 to the trigeminal ganglia. Furthermore, a systematic evaluation of varied quercetin concentrations and administration schedules at distinct stages of HSK progression is imperative.

Conclusions

In summary, our findings indicate that quercetin effectively suppresses HSV-1 replication by impending viral attachment and upregulating Nrf2 expression. In post-treatment regimen, it inhibits viral transcription and protein expression. Moreover, topical application of quercetin significantly alleviates the symptoms of HSK and blocks the transmission of HSV-1 to the TG. Future studies are needed to further substantiate the contribution of quercetin-induced Nrf2 upregulation to HSK pathogenesis.

Materials and methods

Cells and cell culture

Vero cells (African green monkey kidney cells, ATCC® CCL-81™) were maintained in Dulbecco's modified Eagle's medium (DMEM; Invitrogen, Carlsbad, USA) supplemented with 10% fetal bovine serum (FBS; Zetalife, San Francisco, USA), along with 100 U/mL penicillin and 100 μg/mL streptomycin (Invitrogen), following the method described in prior studies (Duan et al., 2012). The DMEM/F12 culture (Invitrogen) medium containing 10% FBS, 0.01 μg/mL human epidermal growth factor (Sigma-Aldrich, Saint Louis, USA), 5 μg/mL insulin (Sigma-Aldrich), 100 U/mL of penicillin, and 100 μg/mL of streptomycin was used to culture human corneal epithelial cells (HCECs; RRID: CVCL_1272), as described in our previous publication (Yu et al., 2023). The cultured cells were maintained at 37 °C in a 5% CO2 atmosphere within dishes. All cell lines underwent testing and were confirmed negative for mycoplasma contamination.

Cytotoxicity assay

Incubation of HCECs in a 96-well plate overnight was followed by 24 h treatment with quercetin at various concentrations. The plate was rinsed with PBS and incubated for an additional 2 h in DMEM/F12 supplemented with cell counting kit-8 (CCK-8, Dojindo Labs, Kumamoto, Japan) after removing the quercetin-containing medium. Cell viability was subsequently assessed using the Synergy H1mfd system (BIOTEK, Winooski, USA).

Virus

The origin of the HSV-1 F strain (HSV-1f) and the HSV-1 H129 GFP knock-in strain (HSV-1g) used in this study was consistent with our previous reports (Zeng et al., 2017; Duan et al., 2022). According to our previous report, the virus was cultured and titrated on Vero cells, with viral titers analyzed by the plaque assay (Duan et al., 2022). Briefly, an overnight incubation of Vero cells was performed in a 12-well plate. Following a 1 h infection with varying dilutions of HSV-1, the cells were washed three times with PBS and then incubated in DMEM supplemented with 1% agarose for an additional 72 h. Cells were fixed overnight using a formalin solution and subsequently stained with crystal violet (Beyotime, Shanghai, China). The mean plaque count was determined using ImageJ software (NIH, Bethesda, USA) based on data from three separate experiments.

Virus infection in vitro

HSV-1 infection of the cells was carried out according to a previously described method with slight modifications (Duan et al., 2022; Li et al., 2022a). HSV-1f and HSV-1g, dissolved in infection medium (serum-free DMEM or DMEM/F12) at a multiplicity of infection (MOI) of 0.1, were applied to cells for 1 h, with gentle shaking every 15 min. Cells were washed three time with PBS after discarding the infection medium, and cells were then incubated in maintenance medium (2% FBS in DMEM or DMEM/F12) for 24 or 48 h. We explored different time-based treatment regimens of quercetin using a modified version of a previously described protocol (Li et al., 2022a). In brief, dimethyl sulfoxide (DMSO; MedChemExpress, Middlesex County, USA), quercetin (Selleck, Houston, USA), or acyclovir (Selleck) were employed in pre-treatment, co-incubation, or post-treatment conditions (Fig. 1B). In the pre-treatment, HCECs were exposed to quercetin for 2 h prior to HSV-1 infection. Co-incubation refers to the simultaneous culture of quercetin and HSV-1 during the 1 h infection. Post-treatment involved adding quercetin to the maintenance medium following HSV-1 infection. The GFP fluorescence of HSV-1g in cells was recorded at 24 h post-infection, and the fluorescence intensity was measured using ImageJ.

Anti-attachment, anti-penetration, and viral inactivation assay

As seen in Fig. 1G, following our previously published protocol with modifications, HCECs were treated with quercetin (30 μmol/L) at different phases of HSV-1 infection (Duan et al., 2022).

For the anti-attachment assay, HCECs were first chilled at 4 °C for 1 h. Next, cells were exposed to HSV-1 at an MOI of 0.1 for 1 h at 4 °C, with or without the addition of quercetin. Cells were rinsed with ice-cold PBS to eliminate quercetin and any unattached virus before they were cultured in maintenance medium for either 24 or 48 h.

In the anti-penetration assay, HCECs were first chilled and then infected with HSV-1 at 4 °C for 1 h. Cells were then incubated at 37 °C for 30 min, with or without quercetin. Subsequently, cells were washed and treated with citrate buffer (pH 3.0) for 1 min to inactivate any non-penetrated virus. Finally, cells were transferred to maintenance medium and incubated for 24 or 48 h.

For the viral inactivation assay, HSV-1 was incubated with 30 μmol/L quercetin at 37 °C for 2 h. The mixture was then diluted 100-fold and used to infect HCECs, which were subsequently incubated for 24 or 48 h.

Quantitative real-time polymerase chain reaction (qRT-PCR)

A previously described qRT-PCR protocol was modified for the present study (Yu et al., 2023). A Total RNA Extraction Kit (Bioer, Hangzhou, China) was employed to isolate total RNA and genomic DNA from tissue and cell samples. Following cDNA synthesis, qPCR was conducted using Taq Pro Universal SYBR qPCR Master Mix (Vazyme, Nanjing, China) in accordance with the manufacturer's instructions. The primer sequences utilized are listed in Supplementary Table S1.

Western blotting

HCECs were lysed and then treated with SDS-PAGE loading buffer (Beyotime) for 10 min. As previously reported (Yu et al., 2023), we separated the samples on SDS-PAGE and transferred them to a PVDF membrane. After blocking, the membrane was incubated overnight with the primary antibody at 4 °C. Following incubation with the secondary antibody, proteins were detected using Ncm ECL High (New Cell & Molecular, Suzhou, China). We used GAPDH to normalize signal across samples. The primary antibodies included HSV VP16 monoclonal antibody (1:1000, Santa Cruz, Dallas, USA), HSV gD monoclonal antibody (1:3000, Santa Cruz), Nrf2 monoclonal antibody (1:1000, Zen-Biosciences, Chengdu, China) and GAPDH monoclonal antibody (1:5000, Affinity Biosciences, Changzhou, China).

RNA-seq and pathway enrichment analysis

HCECs were infected with HSV-1 F strain and subsequently treated with either DMSO or 30 μmol/L quercetin. Total RNA was extracted, and its concentration, integrity, and purity were subsequently assessed. The input materials included RNA samples with total amounts exceeding 400 ng and RNA integrity numbers above 7, with three biological replicates per group. Sequencing libraries were prepared using the NEBNext Ultra™ RNA Library Prep Kit for Illumina (New England Biolabs, Ipswich, USA). After cluster generation and sequencing of the libraries, the reference genome index was constructed, and paired-end clean reads were aligned to the reference genome using HISAT2 (version 2.0.5). Additionally, reads for each gene were quantified, and differential gene expression was analyzed using the DESeq2 R package (version 1.20.1). Gene ontology (GO) enrichment for differentially expressed genes (DEGs; P value < 0.05) was analyzed using the clusterProfiler R package.

RNA silencing

In this experiment, a negative control siRNA (NC-siRNA; purchased from RiboBio, Guangzhou, China) and a siRNA targeting Nrf2 (Nrf2-siRNA; 5′-GAGAAAGAATTGCCTGTAA-3′) were used. Transfection was performed using Lipofectamine 3000 Transfection Reagent (Invitrogen), according to the methodology described in our previous study (Yu et al., 2023). Cells were distributed into 6-well plates and left to incubate overnight. Before transfection, the medium was exchanged for an antibiotic-free culture medium. Transfection was performed by mixing 30 nmol/L of NC-siRNA or Nrf2-siRNA with diluted Lipofectamine 3000 and incubating for 15 min. The effectiveness of Nrf2 silencing was assessed through qRT-PCR and Western blotting analysis.

Molecular docking

Molecular docking was employed to verify potentially significant active components and their key targets, as outlined below: the three-dimensional structure file of nectin-1 was first retrieved from the RCSB Protein Data Bank (http://www.rcsb.org/). Nectin-1 was crystallized after solvent molecules and ligands were removed, polar hydrogens were added, and the energy minimization process was applied. We optimized the chemical compound's three-dimensional structure using the MM2 force field and adjusted the number of rotatable bonds. PubChem Compounds database (https://pubchem.ncbi.nlm.nih.gov/) was used to obtain the molecular structures of quercetin. Molecular docking software AutoDock Vina was used for scoring, with a threshold of −5 kcal/mol.

Murine HSV-1 infection

In this study, 6–8 week old female specific-pathogen-free (SPF) BALB/c mice were utilized. After being anesthetized with 1% pentobarbital, the mice underwent corneal sacrifice using a 3 × 3 crosshatch pattern, followed by inoculation with 1 × 106 PFU HSV-1f in the right eye. Starting from 1 day post-infection (dpi), the right eye of each mouse was treated four times daily with either 100 μmol/L quercetin or 0.1% DMSO. Body weight and disease scores were recorded at specified time points following infection. Scoring was conducted using the following scoring system (Jaishankar et al., 2018): 0 for no symptoms, 1 for mild swelling of the eyelids, 2 for moderate swelling with some crusting, 3 for moderate swelling with more than 50% crusting, 4 for severe crusting, and 5 for the eye being completely swollen shut. As per the previous report, cotton swabs harvested tear samples (n = 6) on 3, 5, and 7 dpi (Tan and Xia, 2020). Before being frozen at −80 °C, all swabs were immersed in 1 mL of DMEM, and titers of HSV-1 in tear samples were determined using the plaque assay. We serially diluted the tear sample 10-fold and titrated it on Vero cell monolayers, and the viral titer was expressed as PFU/mL. At 7 dpi, the mice were sacrificed, and corneal and trigeminal ganglia (TG) samples were collected, and tissue homogenates were prepared. The VP16 levels in the samples were measured by PCR assays. A color digital camera (DS-Ri2, Nikon) attached to the slit-lamp microscope was used to collect corneal graphs.

Statistical analysis

All experiments in this study were conducted with a minimum of three independent replicates. The data was analyzed using one-way ANOVA and an unpaired t-test with Mann-Whitney correction in Prism version 9.2.0. A significance level of P < 0.05 was considered statistically significant, with the results expressed as means with standard deviations (±SD).

Data availability

All data relevant to the study are included in the article.

Ethics statement

Mice studies have been approved by the Animal Ethics Committee of the Zhongshan Ophthalmic Center (O2022018) according to the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research.

Author contributions

Yubin Yu: conceptualization, investigation, data curation, methodology, formal analysis, writing - original draft. Sihao Liu: investigation and data curation. Qinghua Liu: methodology and formal analysis. Xiuping Liu: investigation and formal analysis. Kaili Wu: conceptualization, supervision, formal analysis, writing-review & editing and funding acquisition.

Conflict of interest

The authors declare that they have no conflict of interest.

Acknowledgments

We thank Professor Minhua Luo from Wuhan Institute of Virology for providing the HSV-1f and HSV-1g strain. This work was supported by the National Natural Science Foundation of China (No. 81970848).

Footnotes

Appendix A

Supplementary data to this article can be found online at https://doi.org/10.1016/j.virs.2025.07.007.

Appendix A. Supplementary data

The following are the Supplementary data to this article:

Supplementary Material
mmc1.docx (132KB, docx)
Supplementary Table S2
mmc2.xls (5.5MB, xls)

Supplementary Figure S1.

Supplementary Figure S1

Open in a new tab

The topical application of quercetin at 100 μmol/L showed no significant toxicity to the mouse cornea. Six to eight-week-old SPF BALB/c female mice received right ocular infection with 1 × 106 PFU HSV-1f, and were subsequently treated topically with 100 μmol/L quercetin or 0.1 % DMSO four times daily, commencing 1 dpi. A Images captured by slit lamp of the right eyes of mice treated with different agents on day 0 and day 7. Each image represents findings from 5 mice in a single experiment. B Corneal histology of eyes on 7 dpi. Each image is representative of 3 mice in a single experiment.

References

  1. Dai Y., Idorn M., Serrero M.C., Pan X., Thomsen E.A., Narita R., Maimaitili M., Qian X., Iversen M.B., Reinert L.S., Flygaard R.K., Chen M., Ding X., Zhang B.-c., Carter-Timofte M.E., Lu Q., Jiang Z., Zhong Y., Zhang S., Da L., Zhu J., Denham M., Nissen P., Mogensen T.H., Mikkelsen J.G., Zhang S.-Y., Casanova J.-L., Cai Y., Paludan S.R. TMEFF1 is a neuron-specific restriction factor for herpes simplex virus. Nature. 2024;632:383–389. doi: 10.1038/s41586-024-07670-z. [DOI] [PubMed] [Google Scholar]
  2. Di Petrillo A., Orrù G., Fais A., Fantini M.C. Quercetin and its derivates as antiviral potentials: a comprehensive review. Phytother Res. 2022;36:266–278. doi: 10.1002/ptr.7309. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Duan F., Ni S., Nie Y., Huang Q., Wu K. Small interfering RNA targeting for infected-cell polypeptide 4 inhibits herpes simplex virus type 1 replication in retinal pigment epithelial cells. Clin. Exp. Ophthalmol. 2012;40:195–204. doi: 10.1111/j.1442-9071.2011.02668.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Duan F., Zeng W., Zhang Y., Li D., Wu K. Lipopolysaccharide enhances HSV-1 replication and inflammatory factor release in the ARPE-19 cells. Heliyon. 2022;8 doi: 10.1016/j.heliyon.2022.e11787. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Duan R., de Vries R.D., van Dun J.M., van Loenen F.B., Osterhaus A.D., Remeijer L., Verjans G.M. Acyclovir susceptibility and genetic characteristics of sequential herpes simplex virus type 1 corneal isolates from patients with recurrent herpetic keratitis. J. Infect. Dis. 2009;200:1402–1414. doi: 10.1086/606028. [DOI] [PubMed] [Google Scholar]
  6. Guo H., Pang K., Wei Y., Yi C., Wu X. Herpes virus entry mediator in human corneal epithelial cells modulates the production of inflammatory cytokines in response to HSV type 1 challenge. Ophthalmic Res. 2015;54:128–134. doi: 10.1159/000437209. [DOI] [PubMed] [Google Scholar]
  7. Han Y., Guo S., Li Y., Li J., Zhu L., Liu Y., Lv Y., Yu D., Zheng L., Huang C., Li C., Hu J., Liu Z. Berberine ameliorate inflammation and apoptosis via modulating PI3K/AKT/NFκB and MAPK pathway on dry eye. Phytomedicine. 2023;121 doi: 10.1016/j.phymed.2023.155081. [DOI] [PubMed] [Google Scholar]
  8. Hill J.M., Rayfield M.A., Haruta Y. Strain specificity of spontaneous and adrenergically induced HSV-1 ocular reactivation in latently infected rabbits. Curr. Eye Res. 1987;6:91–97. doi: 10.3109/02713688709020074. [DOI] [PubMed] [Google Scholar]
  9. Hung P.Y., Ho B.C., Lee S.Y., Chang S.Y., Kao C.L., Lee S.S., Lee C.N. Houttuynia cordata targets the beginning stage of herpes simplex virus infection. PLoS One. 2015;10 doi: 10.1371/journal.pone.0115475. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Jaishankar D., Yakoub A.M., Yadavalli T., Agelidis A., Thakkar N., Hadigal S., Ames J., Shukla D. An off-target effect of BX795 blocks herpes simplex virus type 1 infection of the eye. Sci. Transl. Med. 2018;10 doi: 10.1126/scitranslmed.aan5861. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. James C., Harfouche M., Welton N.J., Turner K.M., Abu-Raddad L.J., Gottlieb S.L., Looker K.J. Herpes simplex virus: global infection prevalence and incidence estimates, 2016. Bull. World Health Organ. 2020;98:315–329. doi: 10.2471/BLT.19.237149. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Lee S., Lee H.H., Shin Y.S., Kang H., Cho H. The anti-HSV-1 effect of quercetin is dependent on the suppression of TLR-3 in Raw 264.7 cells. Arch Pharm. Res. 2017;40:623–630. doi: 10.1007/s12272-017-0898-x. [DOI] [PubMed] [Google Scholar]
  13. Li F., Song X., Su G., Wang Y., Wang Z., Jia J., Qing S., Huang L., Wang Y., Zheng K., Wang Y. Amentoflavone inhibits HSV-1 and ACV-resistant strain infection by suppressing viral early infection. Viruses. 2019;11:466. doi: 10.3390/v11050466. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Li J., Cheng C., Lin T., Xue R., Liu X., Wu K. Efficacy of sodium polyanethol sulfonate on herpes simplex virus-1 infection in vitro. Mol. Vis. 2022;28:516–525. [PMC free article] [PubMed] [Google Scholar]
  15. Li Y., Wei Y., Li G., Huang S., Xu J., Ding Q., Hong J. Targeting NECTIN-1 based on CRISPR/Cas9 system attenuated the herpes simplex virus infection in human corneal epithelial cells in vitro. Transl. Vis. Sci. Technol. 2022;11:8. doi: 10.1167/tvst.11.2.8. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Liu Y., You Q., Zhang F., Chen D., Huang Z., Wu Z. Harringtonine inhibits herpes simplex virus type 1 infection by reducing herpes virus entry mediator expression. Front. Microbiol. 2021;12 doi: 10.3389/fmicb.2021.722748. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Luan J., Zhu Y., Lin J., Zhang Y., Xu Q., Zhan L., Tian X., Zhao G., Peng X. Quercetin protects against Aspergillus fumigatus keratitis by reducing fungal load and inhibiting TLR-4 induced inflammatory response. Cytokine. 2023;171 doi: 10.1016/j.cyto.2023.156356. [DOI] [PubMed] [Google Scholar]
  18. Luo Z., Kuang X.P., Zhou Q.Q., Yan C.Y., Li W., Gong H.B., Kurihara H., Li W.X., Li Y.F., He R.R. Inhibitory effects of baicalein against herpes simplex virus type 1. Acta Pharm. Sin. B. 2020;10:2323–2338. doi: 10.1016/j.apsb.2020.06.008. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. McKay T.B., Kivanany P.B., Nicholas S.E., Nag O.K., Elliott M.H., Petroll W.M., Karamichos D. Quercetin decreases corneal haze in vivo and influences gene expression of TGF-beta mediators in vitro. Metabolites. 2022;12:626. doi: 10.3390/metabo12070626. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Muzammil K., Sabah Ghnim Z., Saeed Gataa I., Fawzi Al-Hussainy A., Ali Soud N., Adil M., Ali Shallan M., Yasamineh S. NRF2-mediated regulation of lipid pathways in viral infection. Mol. Aspect. Med. 2024;97 doi: 10.1016/j.mam.2024.101279. [DOI] [PubMed] [Google Scholar]
  21. Oh H.N., Kim C.E., Lee J.H., Yang J.W. Effects of quercetin in a mouse model of experimental dry eye. Cornea. 2015;34:1130–1136. doi: 10.1097/ICO.0000000000000543. [DOI] [PubMed] [Google Scholar]
  22. Olagnier D., Farahani E., Thyrsted J., Blay-Cadanet J., Herengt A., Idorn M., Hait A., Hernaez B., Knudsen A., Iversen M.B., Schilling M., Jørgensen S.E., Thomsen M., Reinert L.S., Lappe M., Hoang H.D., Gilchrist V.H., Hansen A.L., Ottosen R., Nielsen C.G., Møller C., van der Horst D., Peri S., Balachandran S., Huang J., Jakobsen M., Svenningsen E.B., Poulsen T.B., Bartsch L., Thielke A.L., Luo Y., Alain T., Rehwinkel J., Alcamí A., Hiscott J., Mogensen T.H., Paludan S.R., Holm C.K. SARS-CoV2-mediated suppression of NRF2-signaling reveals potent antiviral and anti-inflammatory activity of 4-octyl-itaconate and dimethyl fumarate. Nat. Commun. 2020;11:4938. doi: 10.1038/s41467-020-18764-3. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Rousseau A., Pharm S.B., Gueudry J., Deback C., Haigh O., Schweitzer C., Boutolleau D., Labetoulle M. Acyclovir-resistant herpes simplex virus 1 keratitis: a concerning and emerging clinical challenge. Am. J. Ophthalmol. 2022;238:110–119. doi: 10.1016/j.ajo.2022.01.010. [DOI] [PubMed] [Google Scholar]
  24. Shah A., Farooq A.V., Tiwari V., Kim M.J., Shukla D. Hsv-1 infection of human corneal epithelial cells: receptor-mediated entry and trends of re-infection. Mol. Vis. 2010;16:2476–2486. [PMC free article] [PubMed] [Google Scholar]
  25. Sharma A., Taniguchi J. Review: emerging strategies for antimicrobial drug delivery to the ocular surface: implications for infectious keratitis. Ocul. Surf. 2017;15:670–679. doi: 10.1016/j.jtos.2017.06.001. [DOI] [PubMed] [Google Scholar]
  26. Sibley D., Larkin D.F.P. Update on herpes simplex keratitis management. Eye (Lond.) 2020;34:2219–2226. doi: 10.1038/s41433-020-01153-x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Spear P.G. Herpes simplex virus: receptors and ligands for cell entry. Cell. Microbiol. 2004;6:401–410. doi: 10.1111/j.1462-5822.2004.00389.x. [DOI] [PubMed] [Google Scholar]
  28. Stoddard A.R., Koetje L.R., Mitchell A.K., Schotanus M.P., Ubels J.L. Bioavailability of antioxidants applied to stratified human corneal epithelial cells. J. Ocul. Pharmacol. Therapeut. 2013;29:681–687. doi: 10.1089/jop.2013.0014. [DOI] [PubMed] [Google Scholar]
  29. Tan T., Xia L. TRIM21 aggravates herpes simplex virus epithelial keratitis by attenuating STING-IRF3-mediated type I interferon signaling. Front. Microbiol. 2020;11:703. doi: 10.3389/fmicb.2020.00703. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Wang H., Jia X., Zhang M., Cheng C., Liang X., Wang X., Xie F., Wang J., Yu Y., He Y., Dong Q., Wang Y., Xu A. Isoliquiritigenin inhibits virus replication and virus-mediated inflammation via NRF2 signaling. Phytomedicine. 2023;114 doi: 10.1016/j.phymed.2023.154786. [DOI] [PubMed] [Google Scholar]
  31. Whitley R.J., Gnann J.W., Jr. Acyclovir: a decade later. N. Engl. J. Med. 1992;327:782–789. doi: 10.1056/NEJM199209103271108. [DOI] [PubMed] [Google Scholar]
  32. Xu L., Zhong X.L., Xi Z.C., Li Y., Xu H.X. Medicinal plants and natural compounds against acyclovir-resistant hsv infections. Front. Microbiol. 2022;13 doi: 10.3389/fmicb.2022.1025605. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Yin J., Peng X., Lin J., Zhang Y., Zhang J., Gao H., Tian X., Zhang R., Zhao G. Quercetin ameliorates Aspergillus fumigatus keratitis by inhibiting fungal growth, toll-like receptors and inflammatory cytokines. Int. Immunopharmacol. 2021;93 doi: 10.1016/j.intimp.2021.107435. [DOI] [PubMed] [Google Scholar]
  34. Yu Y., Li K., Xue R., Liu S., Liu X., Wu K. A20 functions as a negative regulator of the lipopolysaccharide-induced inflammation in corneal epithelial cells. Exp. Eye Res. 2023;228 doi: 10.1016/j.exer.2023.109392. [DOI] [PubMed] [Google Scholar]
  35. Zeng W.B., Jiang H.F., Gang Y.D., Song Y.G., Shen Z.Z., Yang H., Dong X., Tian Y.L., Ni R.J., Liu Y., Tang N., Li X., Jiang X., Gao D., Androulakis M., He X.B., Xia H.M., Ming Y.Z., Lu Y., Zhou J.N., Zhang C., Xia X.S., Shu Y., Zeng S.Q., Xu F., Zhao F., Luo M.H. Anterograde monosynaptic transneuronal tracers derived from herpes simplex virus 1 strain H129. Mol. Neurodegener. 2017;12:38. doi: 10.1186/s13024-017-0179-7. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Zhang L., Wang J., Wang Z., Li Y., Wang H., Liu H. Upregulation of nuclear factor E2-related factor 2 (Nrf2) represses the replication of herpes simplex virus type 1. Virol. J. 2022;19:23. doi: 10.1186/s12985-021-01733-7. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Zhu S., Viejo-Borbolla A. Pathogenesis and virulence of herpes simplex virus. Virulence. 2021;12:2670–2702. doi: 10.1080/21505594.2021.1982373. [DOI] [PMC free article] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplementary Material
mmc1.docx (132KB, docx)
Supplementary Table S2
mmc2.xls (5.5MB, xls)

Data Availability Statement

All data relevant to the study are included in the article.

Articles from Virologica Sinica are provided here courtesy of Wuhan Institute of Virology, Chinese Academy of Sciences

RESOURCES