U251 cells (human glioma cell line), Vero cells (African green monkey kidney epithelial cell line) and HSF cells (hedgehog fibroblast cells) were maintained in our laboratory. U251 and Vero cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM; Gibco, Waltham, MA, USA) supplemented with 10% (v/v) heat-inactivated fetal bovine serum (FBS; ExCell Bio, Suzhou, China), 100 U/mL penicillin, and 100 μg/mL streptomycin at 37 °C in a humidified incubator with 5% CO₂. The HSF cell dissociation procedure has been described in detail elsewhere (Walmsley et al., 2016). Briefly, hairless ear tissue was collected from adult hedgehogs following anesthesia with 4% isoflurane. The tissue was minced and digested in a solution of collagenase type I (20 mg/mL, Gibco, Waltham, MA, USA) prepared in 50% complete medium (DMEM supplemented with 50% (v/v) FBS, 1% (v/v) MEM non-essential amino acids (MEM NEAA; Gibco, Waltham, MA, USA), 100 U/mL penicillin, and 100 μg/mL streptomycin, and 1‰ (v/v) 2-Mercaptoethanol (2-ME)) at 37 °C in a thermostatic shaker for 2–3 h. Following digestion, cells were mechanically dissociated by pipetting and filtered through a 70 μm cell strainer (Biosharp, Anhui, China) to obtain a single-cell suspension. The isolated cells were initially cultured in 50% complete medium at 37 °C with 5% CO₂. After 24–48 h, the medium was replaced with 10% complete medium (DMEM with 10% (v/v) FBS, 1% (v/v) MEM NEAA, 1‰ (v/v) 2-ME, 100 U/mL penicillin, and 100 μg/mL streptomycin), and cells were cultured until reaching sufficient confluence for experimental use.

The HSV-1 (17⁺ strain) used in this study was preserved in our laboratory. Viral stocks were propagated in Vero cells. At 72 h post-infection, culture supernatants were harvested, clarified by centrifugation at 3,000 × g for 10 min at 4 °C, aliquoted, and stored at −80 °C. To ensure stable virulence and titers, the viral stock was subjected to 3 consecutive low-passage propagations. Viral titers were quantified using a standard plaque assay on Vero cells and expressed as plaque-forming units per milliliter (PFU/mL).

Female C57BL/6 J mice (8 weeks old) were purchased from the Laboratory Animal Center of Shanxi Provincial People’s Hospital (Taiyuan, Shanxi, China). Ifnar^−/− mice were purchased from Cyagen Biosciences Inc. (Guangzhou, Guangdong, China). The mice were group-housed in a controlled environment with a temperature of 22 ± 1 °C and a 12-h light/dark cycle, with free access to autoclaved water and standard rodent chow. All animal care and experiments were approved by the Animal Care Committee of Shanxi Agricultural University (Approval number: SXAU-EAW-2025 M&Rr. BQ.004015379).

Six-month-old female hedgehogs were purchased from Hunan Fulongkang Breeding Co., Ltd. To prepare hedgehog skin extract (HSE), fat-free epidermal tissue was first snap-frozen in liquid nitrogen and ground into a fine powder using a mortar and pestle. The powder was transferred onto ice, homogenized using a Jingxin Auto Sample Grinder-24 L (Shanghai Jingxin Industrial Development Co., Ltd.), and then lysed with 1 mL of ice-cold lysis buffer per portion of powder. The lysis buffer contained 0.5 M Tris–HCl (pH 8.0), 5 mM EDTA (pH 8.0), 150 mM NaCl, 10 mM MgCl₂, 2% (v/v) 2-ME and a protease inhibitor cocktail. The lysate was incubated at 4 °C for 30 min with gentle rotation to ensure complete lysis and subsequently centrifuged at 12,000 rpm for 15 min at 4 °C. The supernatant was carefully harvested, avoiding both the upper lipid layer and bottom insoluble pellet, to obtain a crude protein extract (Yum et al., 2017). To remove endotoxin, the crude extract was dialyzed at 4 °C using an endotoxin-removing dialysis bag with a molecular weight cutoff of 10 kDa, with endotoxin-free PBS as the external dialysis buffer. The external buffer was replaced three times at appropriate intervals to thoroughly eliminate residual detergents, salts, and endotoxins. Following protein concentration determination via the BCA assay, anti-HSV-1 activity was evaluated by seeding U251 cells in 12-well plates at a density of 1.5 × 10⁵ cells/well and culturing them for 12 h. Cells were then infected with HSV-1 at a multiplicity of infection (MOI) of 0.05 and then immediately treated with hedgehog skin extract (25 or 50 μg/mL) or acyclovir (ACV, 5 μg/mL) for 24 h. Finally, total cellular DNA was extracted and analyzed by quantitative PCR (qPCR).

Hedgehog fibroblast cells were seeded in 12-well plates at a density of 2 × 10⁵ cells per well and cultured for 12 h at 37 °C with 5% CO₂. The cells were then infected with HSV-1 at an MOI of 0.1 for 1 h at 37 °C. After infection, the inoculum was replaced with virus maintenance medium, and incubation continued for 24 h. A blank control group (designated as Vehicle) was included in the experiment. Total RNA was then isolated and subjected to Quantitative RT-PCR (RT-qPCR) analysis.

The cathelicidin sequence of E. europaeus was identified through keyword searches (“Erinaceus europaeus” and “cathelicidin”) in the National Center for Biotechnology Information (NCBI; https://www.ncbi.nlm.nih.gov/). The mature peptide sequence was designated as CathEE. By truncating the template peptide to retain its α-helical functional region, three modified peptides CathEE-1, CathEE-2 and CathEE-3 were obtained. Following the initial screening, the most potent anti-HSV-1 peptide was chosen as the template for rational design. Specific amino acid substitutions and insertions were introduced, resulting in two derivatives designated CathEE-2a and CathEE-2b. Specifically, the peptide was engineered by replacing neutral/polar residues with arginine (Arg) and lysine (Lys) to increase net positive charge, thereby enhancing antimicrobial activity while minimizing toxicity. The proportion of hydrophobic residues was adjusted to approximately 40–60%, thereby enhancing selectivity. Notably, during these modifications, the functional α-helical conformation was preserved, with the polar/nonpolar residue ratio maintained at roughly 1:1. The C-termini of all the peptides were amidated (-NH2). These peptides were chemically synthesized by Gill Biochemical Co., Ltd. (Shanghai, China), with purity exceeding 95% as verified by reversed-phase high-performance liquid chromatography (HPLC) and mass spectrometric (MS) characterization (Supplementary Table 1).

Structural analyses and visualizations of the peptides were performed as follows:

The cytotoxicity of the peptides on U251 cells was assessed using a Cell Counting Kit-8 (CCK-8, Biosharp, Anhui, China). Briefly, U251 cells were seeded into 96-well plates with 1.0 × 10⁴ cells per well for 12 h. Subsequently, cells were exposed to the peptides at concentrations ranging from 0 to 160 μM (0, 1.25, 2.5, 5, 10, 20, 40, 80, and 160 μM). After 24 h treatment, 10 μL CCK-8 reagent was added to each well and incubated for 0.5–4 h at 37 °C. Absorbance was measured at 450 nm using a SpectraMax M2e Multi-Mode Microplate Reader (Molecular Devices, San Jose, CA, USA) with SoftMax Pro 7.0 software.

U251 cells were seeded in 12-well plates at a density of 2 × 10⁵ cells per well (1 mL/well) and cultured for 12 h. Then, they were infected with HSV-1 at 0.05 MOI and treated with the peptides (10 μM) or ACV (10 μM; positive control). After incubation for 24 h, total DNA was extracted, and the relative expression levels of HSV-1 UL30 gene were detected by qPCR.

To assess whether CathEE-2a directly inactivates HSV-1, viral particles were incubated with the indicated concentrations of CathEE-2a at 37 °C for 1, 2, or 4 h. Following incubation, the mixtures were diluted 200-fold to terminate peptide activity. The treated virus was then used to infect U251 cells, and viral replication was evaluated by qPCR analysis of the HSV-1 UL30 gene, with UL30 levels normalized to the 18S rDNA reference gene. A control mixture consisting of HSV-1 and an equivalent amount of CathEE-2a without pre-incubation was included.

To examine whether CathEE-2a interferes with viral attachment, U251 cells were incubated with HSV-1 (MOI = 0.05) in the presence or absence of CathEE-2a at 4 °C for 1 h. After adsorption, unbound virus and peptide were removed by extensive washing, and the amount of cell-associated HSV-1 UL30 levels was quantified by qPCR and normalized to 18S rDNA.

To assess the effect of CathEE-2a at the post-entry stage, U251 cells were first infected with HSV-1 (MOI = 0.05) at 37 °C for 1 h to allow viral adsorption and entry. The cells were then treated with different concentrations of CathEE-2a for 24 h. Intracellular HSV-1 UL30 gene levels were determined by qPCR using 18S rDNA as the internal reference gene.

BMDMs were generated from the tibias and femurs of mice. Isolated bone marrow cells were cultured in DMEM supplemented with 10% (v/v) FBS, 20 ng/mL macrophage colony-stimulating factor (M-CSF; Merck KGaA, Darmstadt, Germany), 100 U/mL penicillin, and 100 μg/mL streptomycin for 7 days at 37 °C under 5% CO₂ to allow differentiation into macrophages.

Differentiated BMDMs from wild-type (WT) and Ifnar^−/− mice were seeded and allowed to adhere overnight. Cells were then infected with HSV-1 at an MOI of 0.1 and simultaneously treated with CathEE-2a at final concentrations of 5 μM (low dose) or 10 μM (high dose). At 12 h post-infection, cells were harvested for total RNA extraction. The mRNA levels of Ifna, Ifnb, and the interferon-stimulated genes Isg15 and Mx1 were quantified by RT-qPCR, with Hprt as the internal reference gene. At 24 h post-infection, culture supernatants were collected for viral titration by standard plaque assay on Vero cells. In parallel, total cellular DNA was extracted, and HSV-1 UL30 gene levels were quantified by qPCR and normalized to 18S rDNA.

Total RNA was isolated using TRIzol reagent (Invitrogen, Waltham, MA, USA), and cDNA was reverse-transcribed by using the HiScript III RT SuperMix for qPCR (+ gDNA wiper) kit (Vazyme, Nanjing, China). Total DNA was extracted using TIANamp Genomic DNA Kit (TIANGEN, Beijing, China) according to the manufacturer’s instructions. RNA and DNA purity and concentration were determined using a NanoDrop One UV–Vis Spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA).

RT-qPCR and qPCR were performed on a CFX Connect Real-Time System (Bio-Rad, Hercules, CA, USA) with ChamQ Universal SYBR qPCR Master Mix (Vazyme, Nanjing, China), employing the following thermal cycling protocol: initial denaturation at 95 °C for 5 min, followed by 40 cycles of 95 °C for 10 s and 60 °C for 30 s. The reference genes used were as follows: human 18S rDNA or HPRT for U251 cells, hedgehog A-tubulin for hedgehog fibroblasts, and mouse 18S rDNA or Hprt for BMDMs. Primer sequences are listed in Supplementary Table 2.

Female C57BL/6 J mice were acclimated for 7 days and randomly assigned into four groups (n = 5 per group) using a computer-generated randomization scheme. The groups included a vehicle control group (PBS), an HSV-1-infected control group, an HSV-1 + CathEE-2a (10 mg/kg) treatment group, and an HSV-1 + ACV (10 mg/kg) treatment group. The sample size of 5 mice per group was determined based on our previously established and validated HSV-1 infection model, balancing the need for sufficient statistical power with the ethical principle of minimizing animal use in accordance with the 3Rs guidelines (Yisihaer et al., 2025).

After anesthesia by inhalation of 4% isoflurane, all mice except those in the vehicle control group were subplantarly infected with 1.0 × 10⁷ PFU of HSV-1. This inoculation route and strain combination induces a predictable disease course: local inflammation develops at the injection site at 1–3 days post-infection (dpi), followed by viral dissemination to the spinal cord and brain at 4–6 dpi, which subsequently progresses to hindlimb paralysis and mortality at 7–10 dpi. Mice were euthanized at 24 h after the final treatment, which corresponds to 5 dpi. This time point was chosen to capture the peak of viral dissemination to the central nervous system prior to the onset of severe clinical symptoms, allowing analysis of viral load and host responses during the critical early phase of neuroinvasion (Engel et al., 1997; Jia et al., 2025). At 1 h post-infection, CathEE-2a and ACV were administered daily via intraperitoneal injection at a volume of 100 μL per mouse for 5 consecutive days. The vehicle control group and the HSV-1-infected control group both received an equal volume of PBS intraperitoneally. All treatments were coded by an independent researcher not involved in outcome assessment. Investigators responsible for daily body weight monitoring, sample collection and downstream data analysis remained blinded to group assignments throughout the experiment to minimize observer bias (Verhave et al., 2024). At 24 h after the final treatment, all animals were euthanized by cervical dislocation under deep anesthesia induced with 5% isoflurane. Major tissue samples, particularly the brains, were collected for subsequent analysis.

Following fixation in 4% paraformaldehyde for 72 h, mouse brain tissues were processed for paraffin embedding, sectioning, and staining with hematoxylin and eosin (H&E) staining by Servicebio Biotechnology Co., Ltd. (Wuhan, China).

Mouse brain tissues were homogenized using a tissue grinder, and total DNA was extracted from the homogenate supernatants using the TIANamp Genomic DNA Kit (Tiangen Biotech, Beijing, China) according to the manufacturer’s instructions. Absolute quantification of viral copy numbers was performed using a standard curve-method as previously described (Yisihaer et al., 2025). Briefly, the HSV-1 UL30 gene was cloned into the pMD19-T vector, and the resulting recombinant plasmids were transformed into E. coli DH5α competent cells. Following verification by colony PCR and Sanger sequencing, high-purity plasmid DNA was extracted, quantified using a NanoDrop spectrophotometer, and converted to copy numbers (copies/μL). A 10-fold serial dilution series (10¹–10⁸ copies/μL) of the plasmid was then used to generate the standard curve. Viral UL30 gene copy numbers in tissue samples were determined by qPCR using UL30-specific primers, with the standard curve for absolute quantification.

Figures were generated using the GraphPad Prism 9 (Version 9.00; GraphPad Software, Boston, MA, USA). Data are presented as the mean ± SEM. For analyses involving two independent variables, two-way ANOVA was performed, and for comparisons among three or more groups, one-way ANOVA was employed. Both one-way and two-way ANOVA were followed by Tukey’s post-hoc test to control for family-wise error rate. Normality was assessed using Shapiro–Wilk test and Q-Q plots. A p value ≤ 0.05 was considered statistically significant.

To investigate the antiviral potential of hedgehog skin extract (HSE), U251 cells were infected with HSV-1 and subsequently treated with HSE at concentrations of 25 μg/mL and 50 μg/mL, with viral replication quantified via quantitative polymerase chain reaction (qPCR). The data revealed that HSE significantly inhibited HSV-1 replication in a dose-dependent manner, and its inhibitory activity was comparable to that of the positive control ACV (5 μg/mL) (Figure 1A). Previous studies have indicated that cathelicidin (abbreviated as CAMP), an endogenous antimicrobial peptide secreted by epithelial cells of humans and other mammals, exhibits broad-spectrum antimicrobial activity and diverse immunomodulatory functions (Bucki et al., 2010). To identify the antiviral active components in HSE, we further assessed the effect of HSV-1 infection on the expression level of CAMP in hedgehog skin fibroblast (HSF) cells. Compared with the vehicle-treated group, HSV-1 infection significantly upregulated the relative expression level of CAMP. These findings imply that CAMP, whose expression is induced by viral infection, may serve as one of the active antiviral components in hedgehog skin (Figure 1B). To further characterize the candidate antiviral component, we identified a mature cathelicidin peptide (designated as CathEE) derived from E. europaeus in the NCBI database and analyzed its structural features. CathEE exhibits a typical amphipathic α-helical structure (Figure 1C). Subsequent qPCR assays demonstrated that CathEE significantly reduced the UL30/18S ratio relative to the vehicle-treated HSV-1-infected group, confirming its anti-HSV-1 activity (Figure 1D). Additionally, CCK-8 cytotoxicity assays verified that CathEE showed no cytotoxicity toward U251 cells at all tested concentrations (Supplementary Figure 1).

To pinpoint the minimal functional motif responsible for antiviral activity and optimize the efficacy of the native CathEE peptide, we generated a series of truncated CathEE derivatives and further analyzed their molecular structural characteristics as well as anti-HSV-1 activity. As shown in Figure 2A, Schiffer–Edmundson helical wheel projections revealed that CathEE-1, CathEE-2, and CathEE-3 all adopted an amphipathic α-helical conformation, while distinct differences were observed in their amino acid composition and residue spatial distribution, as indicated by the color-coded residues. Consistent with this observation, secondary structure analysis further confirmed that all three peptides folded into a typical α-helical conformation, with only minor differences observed in the flexible regions at the helical terminals (Figure 2B). The surface charge distribution of the tertiary structure demonstrated that prominent positively charged regions (marked in blue) were present on the surfaces of CathEE-1 and CathEE-2, whereas the surface charge distribution of CathEE-3 was relatively uniform (Figure 2C). We next evaluated the antiviral efficacy of these peptides in an HSV-1-infected cell model. Interestingly, all three truncated peptides significantly suppressed HSV-1 replication, among which CathEE-2 showed the strongest inhibitory effect, with activity comparable to that of the positive control ACV, while the inhibitory activities of CathEE-1 and CathEE-3 were slightly weaker but remained statistically significant (Figure 2D). Collectively, these findings indicate that the anti-HSV-1 activity of CathEE peptides is closely related to their structural characteristics, especially the surface charge distribution. Notably, none of the three peptides showed cytotoxicity at the tested concentrations (Supplementary Figure 1).

To further optimize the active motif of CathEE-2, we generated two derivatives (CathEE-2a and CathEE-2b) and characterized their structure and antiviral activity. As shown in Figure 3A, both peptides retained the α-helical secondary structure of the parent peptide, but differed in residue composition and surface charge distribution. Notably, CathEE-2a exhibited more concentrated positive charges on the surface of its predicted three-dimensional structure.

We next evaluated their anti-HSV-1 efficacy in HSV-1-infected U251 cells (MOI = 0.05). CathEE-2a treatment led to a more significant reduction in the UL30/18S ratio, while the inhibitory effect of CathEE-2b was relatively weak, indicating that structural modification of CathEE-2a enhanced its antiviral activity (Figure 3B). The CCK-8-based cytotoxicity assay showed that CathEE-2a had a high 50% cytotoxic concentration (CC₅₀ = 77.65 ± 7.64 μM), confirming no obvious cytotoxicity at its effective antiviral concentration (Figure 3C). We further determined the half-maximal inhibitory concentration (IC₅₀) of CathEE-2a. The dose–response curve showed that CathEE-2a inhibited HSV-1 replication in a concentration-dependent manner, with an IC₅₀ of 9.056 ± 1.69 μM (Figure 3D). The selectivity index (SI) of CathEE-2a, calculated as the ratio of CC₅₀ to IC₅₀, was ~8.57, reflecting a favorable therapeutic window for anti-HSV-1 treatment. A high SI value indicates that CathEE-2a exerts potent antiviral effects at concentrations far below those that induce host cell cytotoxicity, highlighting its potential as a safe and effective anti-HSV-1 candidate.

Following the identification of the post-entry stage as the primary mode of CathEE-2a antiviral activity (Supplementary Figure 2), we subsequently assessed whether CathEE-2a modulates host antiviral immune responses. Analysis in U251 cells indicated that CathEE-2a upregulated key components of the type I IFN signaling pathway (Supplementary Figure 3), suggesting a potential immunomodulatory mechanism. To determine whether type I IFN pathway is functionally required for the antiviral activity of CathEE-2a, we performed antiviral assays using BMDMs from WT and Ifnar^−/− mice. In WT BMDMs, CathEE-2a inhibited HSV-1 infection in a dose-dependent manner, with 10 μM exerting a stronger inhibitory effect than 5 μM. In contrast, the antiviral effect was largely abolished in Ifnar^−/− BMDMs at both concentrations (Figures 4A,B), indicating that CathEE-2a requires intact type I IFN signaling for its antiviral activity. To further delineate this mechanism, we assessed the expression of type I IFNs and downstream effector genes. CathEE-2a treatment increased the mRNA levels of Ifna, Ifnb, Isg15, and Mx1 in WT BMDMs in a dose-dependent manner at 12 h post-infection (Figure 4C). Collectively, these findings demonstrate that the anti-HSV-1 activity of CathEE-2a is primarily mediated through canonical type I IFN signaling and exhibits clear dose dependence.

To validate the translational potential of CathEE-2a, we established an HSV-1 infection model via footpad inoculation of WT mice. As shown in Figure 5A, body weight did not differ significantly among the groups, indicating the absence of overt toxicity induced by CathEE-2a treatment. In addition, we measured viral loads in brain tissues, a key target organ of HSV-1, via qPCR. Notably, CathEE-2a significantly reduced brain viral loads at 5 days post infection, with no statistically significant difference observed between the CathEE-2a and ACV groups (Figure 5B). Histopathological analysis of brain tissues using H&E staining revealed that HSV-1 infection triggered marked pathological changes in the hippocampus, including neuronal shrinkage. In contrast, there was less neuronal damage in the hippocampus of the CathEE-2a and ACV treated groups (Figure 5C). Taken together, these results suggest that CathEE-2a can inhibit HSV-1 infection in vivo and alleviate neuronal damage in infected mice, highlighting its promising translational potential.