Vero cells and PK-15 cells were cultivated in our laboratory using Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin–streptomycin at 37°C in an atmosphere containing 5% CO_2. The variant (PRV-HuN-LD, PRV-YuN-KM) and classical (PRV-HuN-XT) PRV, and HSV-1 strains were isolated and preserved in our laboratory, these strains are utilized to assess the extensive antiviral efficiency of NTZ against infections caused by Alphaherpesvirinae in vitro.

Antibody against PRV gC was kindly provided by Doctor Juan Li in Yunnan Sino-Science Gene Technology CO. ltd (Kunming, China). Antibody against β-actin (ab8226) were purchased from the Abcam (United States). NTZ (Figure 1A) was purchased from MedChemExpress and dissolved in dimethyl sulfoxide (DMSO) to achieve a final concentration of 250 mM.

PK15 or Vero cells were cultured in 96-well plates and treated with DMEM medium supplemented with 10% FBS alongside varying concentrations of NTZ (5.0, 7.5, 10.0, 25.0, 50.0, 100.0, and 200.0 μM) or DMSO for a duration of 48 h. The assessment of cell viability was conducted using the CCK-8 assay, with comprehensive procedural details provided in Tan’s study (16).

The total RNA genome of the cells was extracted using a commercial SteadyPure Universal RNA Extraction Kit (Accurate Biology, Hunan, China). Nearly 1.0 μg of RNA was used to synthesize complementary DNA (cDNA) with the PrimerScript RT Master Mix (Accurate Biology, Hunan, China). RT-qPCR assay was performed to analyze the relative transcript levels of the targeted genes using the 2^-△△CT method as described previously (26), using GAPDH as an internal reference gene. These specific primers were synthesized by Qingke Biotechnology Co., Ltd. (Beijing, China), and their details were presented in Supplementary Table 1.

The relative protein expression levels of PRV-gC were assessed using western blotting assay. In brief, total cellular proteins were extracted using the NP40 lysis buffer containing protease inhibitors. Equal amounts of protein specimens were separated by 10% SDS-PAGE and transferred onto PVDF membranes. The membranes were then blocked with 5% defatted milk powder overnight at 4°C, and incubated with specific primary antibodies (PRV-gC and β-actin) for 4 h at 37°C. Subsequently, the membranes were washed five times with PBST, and incubated with DyLight 800 goat anti-mouse IgG antibodies at 37°C for 1 h. Finally, the target proteins were visualized using an enhanced chemiluminescence substrate (ECL) kit (New Cell & Molecular Biotech, Suzhou, China).

PK15 cells seeded in a 12-well plate were incubated with 4% paraformaldehyde for 15 min at 37°C, followed by a 10-min incubation with 0.1% Triton X-100. After washing three times with PBS, cells were blocked with PBS supplemented with 3% BSA at 37°C for 2 h and and then incubated with mouse anti-PRV gC monoclonal antibody (1:1000 dilution) at 4°C overnight. The cells were washed five times with PBS and subsequently incubated with the FITC-labeled goat anti-mouse secondary antibody (1:3000 dilution; KPL, United States for 45 min in a dark box. Finally, the cells were washed with PBS and subjected to DAPI counterstaining, the green (specific to the PRV-gC protein) and blue (specific to the nucleus) signals were examined by fluorescence microscopy.

Cells cultured in 6-well plates were pre-treated with varying concentrations of NTZ (0, 12.5, 25.0, and 50.0 μM) for 2 h. Subsequently, the cells were infected with PRV strain at a multiplicity of infection (MOI) of 0.1 for 1 h, while maintaining the respective concentrations of NTZ. Cells were washed three times with PBS to remove the supernatants, and then cultured in DMEM medium supplemented with 10% FBS and the corresponding concentrations of NTZ. At 24 h post-infection (hpi), cells were harvested to assess PRV replication via western blotting, IFA, and RT-qPCR analysis. At the same time, the supernatants were collected to assess viral titer using TCID₅₀, as previously described (17).

To assess which phase of the life cycle of PRV has been influenced by NTZ, Vero cells were seeded in 12-well plates and treated with NTZ under different conditions, which included viral inactivation, pre-treatment, co-treatment, and post-treatment (Figure 2A). After these treatments, cells were collected for western blotting analysis at 24 hpi. Meanwhile, the supernatants were obtained to evaluate the viral titers, as previously described (17).

Using the PRV-HuN-LD strain as a model, Vero cells were cultured in 6-well plates and categorized into three distinct groups to examine the antiviral mechanisms of NTZ against PRV infection in vitro, as illustrated in Figure 2D.

Viral Attachment Assay: Vero cells were pre-treated with either DMEM or DMEM supplemented with NTZ (50.0 μM) for 2 h. Subsequently, 10⁶ TCID50 of PRV-HuN-LD was added to the cell plates and incubated for 2 h at 4°C to facilitate viral attachment. After removing the supernatants, the cells were washed three times with cold PBS. Total DNA was extracted from the cells using a commercial DNA/RNA extraction kit (Takara, Dalian, China). The number of PRV DNA copies was determined using qPCR, as previously described (27).

Viral Entry Assay: Vero cells were incubated with 10⁶ TCID₅₀ of PRV-HuN-LD for 2 h at 4°C to facilitate virus attachment. After removing the supernatants, the cells were treated with DMEM medium containing 10% FBS, with or without NTZ (50.0 μM) for 2 h at 37°C to allow for virus entry. Total DNA was extracted from the cells using a commercial DNA/RNA extraction kit (Takara, Dalian, China). The number of PRV DNA copies was determined using qPCR, as previously described (27).

Viral release assay: Vero cells were infected with 10³ TCID₅₀ of PRV-HuN-LD for 2 h at 37°C to permit viral entry. After removing the supernatants, the cells were washed three times with PBS. DMEM supplemented with 10% FBS, with or without NTZ (50.0 μM), was then added, and the cells were incubated for an additional 4 h at 37°C. The supernatants were then harvested at 26 hpi, to evaluate the viral titers as previously described (28).

PK15 cell monolayers, achieving approximately 80% confluence in a 6-well plate, were infected with PRV-HuN-LD at a MOI of 0.1 for 2 h. Following the infection, cells were rinsed with PBS and incubated with NTZ (50 μM) or DMSO for 24 h. The cells in each group were washed with precooled PBS and total RNA was extracted utilizing Trizol reagents (Hunan Aikerui Biotechnology Co., Ltd., Changsha, China). The concentration and purity of the RNA samples were assessed using a NanoDrop spectrophotometer (Thermo Fisher, United States).

Eukaryotic mRNA was enriched using Oligo (dT) magnetic beads, and then fragmented into short segments with a fragmentation buffer. The obtained mRNA served as a template for the synthesis of first-strand cDNA utilizing random primers. Following this, a second-strand labeling buffer and a second-strand end repair enzyme mix were incorporated into the reaction system to generate second-strand cDNA. The double stranded cDNA fragments were purified with AMPure XP beads and underwent end repair. A sequencing linker was ligated to the fragments, and size selection was performed using AMPure XP beads. The cDNA library was enriched through PCR amplification. After the completion of the library construction, the library was diluted to a concentration of 1 ng/μL. The inserted size was assessed using the Agilent 2,100 Bioanalyzer (Agilent Technologies, CA, United States). RT-qPCR was performed to improve or ensure the quality of the constructed library, and the constructed library was dispatched to a commercial company (Wuhan Jinkairui Biotechnology Co., Ltd., Wuhan, China) for sequencing on the Illumina HiSeq 2,500 platform.

Raw reads that included adapter and poly-N sequences, as well as those of low-quality reads, were removed. The remaining raw reads were aligned to the genomes of the PRV variant strain (GenBank accession numbers: KP098534) and the Sus scrofa reference genome using the Hisat2 software. For each sequenced sample, the mapped reads were assembled using StringTie software (29).

A total of 10 core genes linked to antioxidative stress pathway, the MAPK signaling pathway, and the Wnt/β-catenin signaling pathway-related genes, were selected based on the results of transcription analysis to verify the differential expression of DEGs through RT-qPCR. The primers targeting these genes were shown in Supplementary Table 1.

All experiments conducted in this study were executed with more than three independent replicates. The data obtained from different groups were subjected to statistical analysis using a t-test, facilitated by GraphPad Prism Version 8.0 software (GraphPad Software, La Jolla, CA, United States). A p value of less than 0.05 was deemed statistically significant, *p < 0.05, **p < 0.01, ***p < 0.001, while ns indicates no statistical significance.

According to the findings of prior studies, we assessed the cytotoxic effects of NTZ at various concentrations on Vero and PK15 cells for 48 h utilizing the CCK-8 assay. As shown in Figures 1B,C, the highest non-toxic concentration of NTZ for PK15 cell and Vero cells was approximately 75.0 μM and 50.0 μM, respectively. The 50% cytotoxicity concentrations were determined to be 203.0 ± 3.65 μM for PK15 cells, and 217.2 ± 4.28 μM for PK15 cells.

The antiviral efficacy of NTZ against PRV-HuN-LD infection was assessed using Vero and PK15 cell lines. Each cell line was pre-treated with varying concentrations of NTZ (12.5, 25.0, and 50.0 μM), and then infected with PRV at a MOI of 0.1. Following a 24-hpi period, both the cells and supernatants were harvested for western blotting, IFA, RT-qPCR, and TCID₅₀ to evaluate viral replication. The findings indicated that NTZ treatment resulted in a does-dependent reduction in the relative expression of PRV gC in Vero cells. Meanwhile, the relative mRNA expression levels of the PRV gB gene, as well as the viral titers in PRV-infected cells, were significantly higher compared to those in NTZ-treated cells (Figures 3A–C,G). Additionally, similar antiviral properties of NTZ against PRV infection were also monitored in PK15 cells (Figures 3D–F,G).

Next, Vero cells were pre-treated with NTZ (50.0 μM) for 1 h, and then infected with PRV at MOIs of 0.5, 0.1 or 0.02. Supernatants and cells were collected at 24 hpi to quantify viral titers, The results demonstrated that viral titers in the supernatants and cells from PRV-infected groups were significantly higher than those from NTZ-treated groups, which was in a manner dependent on the MOI (Figure 3H).

The aforementioned results indicated NTZ effectively inhibited PRV-HuN-LD infection across Vero and PKL15 cell lines. Subsequently, additional strains, including PRV-YuN-KM (a different variant of the PRV strain), PRV-HuN-XT (a classical PRV strain), and HSV-1 strains, were employed to further investigate the antiviral efficacy of NTZ against a broad spectrum of herpesvirus infections in Vero cells. Cells were pre-treated with NTZ at concentrations of 25 or 50 μM for 2 h, followed by infection with PRV-YuN-KM, PRV-HuN-XT, or HSV-1 with an MOI of 0.1. At 24 hpi, supernatants were collected for viral titers analysis, the cells were harvested to assess the mRNA expression levels of gB gene for both PRV and HSV-1 strains. As illustrated in Supplementary Figure 1, the transcription levels of the gB genes for PRV and HSV-1 in Vero cells exhibited a significant reduction when compared to the DMSO-treated control group (Supplementary Figure 1A). Similarly, the viral titers of both PRV and HSV-1 strains in the DMSO-treated cells were markedly higher than those observed in the NTZ-treated group (Supplementary Figure 1B).

In the following study, the impact of drug addition at various time points on PRV infection was examined through western blotting and viral titer analysis (Figure 2A). The results indicated that NTZ demonstrated a significant antiviral effect against PRV infection in the post-treatment model, while no such effect was observed in the pre-treatment or co-treatment models (Figures 2B,C). The viral titer analysis revealed inhibition rates approaching 100% in the post-treatment scenario (Figure 2C). Next, we aimed to determine the specific phase of the viral life cycle-namely viral binding, entry, and release phases, at which NTZ treatment exhibits its antiviral effects against PRV infection (Figure 2D). However, no significant variations in viral titers or viral copies were monitored in the experimental models assessing viral absorption, entry, and release (Figure 2E). In conclusion, NTZ effectively suppressed PRV infection in vitro by targeting viral replication phase, while exhibiting no effects on other stages of the viral life cycle.

To explore the potential cellular mechanisms underlying the antiviral effects of NTZ against PRV infection, an analysis of the impact of NTZ on the host transcription in PRV-infected PK15 cells was conducted using RNA sequencing (RNA-Seq; Figure 4A). The findings revealed that a total of 1729 differentially expressed genes (DEGs) were detected in PRV-infected PK15 cells when compared to mock cells, comprising 1,444 up-regulated DEGs and 285 down-regulated DEGs (Figure 4B). In contrast, a total of 1891 DEGs were identified in PRV-infected PK15 cells relative to NTZ-treated PRV-infected cells, which included 949 up-regulated DEGs and 942 down-regulated DEGs (Figure 4C).

In terms of the DEGs between PRV-infected cells and NTZ-treated PRV-infected cells, the Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis demonstrated that a significant proportion of DEGs were associated with human diseases, encompassing pathways in cancer, lipid and atherosclerosis, and chemical carciingenesis-reactive oxygen species (Figures 4D,E). The findings from the heat map analysis focused on the oxidative stress pathway revealed an up-regulation of several antioxidant genes, specifically HMOX1, NFE2L1, MAPK11, MAP3K5, MAPK12, and AKR1C8 (Figure 4F). Conversely, there was a down-regulation of certain oxidant genes, including CYTB, PIK3R3, FOS, and DUOX2, in PK15 cells infected with PRV and treated with NTZ, in comparison to the PRV-infected cells (Figure 4F).

In order to evaluate the accuracy and reliability of the RNA-Seq analysis results, the relative mRNA expression levels of genes associated with the antioxidative stress pathway (COX3, ND2, ND3, SLC37A2, and PBK), the MAPK signaling pathway (GADD45A, DSUP4), and the Wnt/β-catenin (USP4 and COL17A1) signaling pathway-related genes were analyzed using the RT-qPCR method. The results obtained from the RNA-Seq and RT-qPCR analyses across different groups demonstrated a high degree of consistency (Figure 4G).