Tetraselmis sp. was mass-cultivated in a semi-open raceway system, as previously described (Kang et al., 2024[13]; Lee et al., 2021[16]). The system has been meticulously engineered in conjunction with a greenhouse, thereby ensuring the sustainable cultivation of marine microalgae irrespective of external conditions (Figure 2A(Fig. 2)). The harvested biomass was lyophilized and stored at −80 °C until use. CA was extracted through a series of steps, including 90 % acetone extraction (Figure 2B(Fig. 2)), filtration using 1 µm filter paper, vacuum evaporation, and purified via a Media Flash C18 column, a Porasil silica column, and a Shimpack C18 column, following established protocols (Kang et al., 2024[13]). The CA structure was characterized by using liquid chromatography-mass spectrometry (LC-MS) and nuclear magnetic resonance (NMR) analyses (Figure 2C(Fig. 2)).

Vero E6 cells were obtained from the American Type Culture Collection (Manassas, VA, USA) and maintained in Dulbecco's Modified Eagle Medium (DMEM; Gibco, Waltham, MA, USA) supplemented with 10 % fetal bovine serum (FBS; Gibco), 1 % 10 mM Hydroxyethyl piperazine ethane sulfonic acid (HEPES; Thermo Fisher Scientific, Waltham, MA, USA), and 1 % antibiotic-antimycotic solution (Thermo Fisher Scientific). Cells were incubated at 37 °C in a humidified atmosphere containing 5 % CO₂. ZIKV was obtained from the Korea Centers for Disease Control and Prevention (KCDC, Cheongju, South Korea). Virus stocks (1 × 10⁵ PFU/mL) were titrated by plaque assay and stored at −80 °C.

Vero E6 cells (2 × 10⁴ cells/well, in 96-well plates) were subjected to an initial incubation period of 16 h, followed by treatment with various concentrations of CA for an additional 72 h. Cell viability was evaluated using the WST-1 assay (DoGen, Seoul, South Korea).

Vero E6 cells (1 × 10⁶ cells/well, in 6-well plates) were incubated in DMEM containing 10 % FBS, 1 % HEPES and 1 % antibiotic-antimycotic solution until confluency was reached. Cells were washed with 1 mL Dulbecco's Phosphate Buffered Saline (DPBS) and applied with CA (2.5, 5, 10, or 20 µM) with 2 mL of DMEM supplemented with 2 % FBS, 1 % HEPES and 1 % antibiotic-antimycotic solution for 2 h prior to infection with ZIKV at 0.01 multiplicity of infection (MOI). Untreated, uninfected cells served as the mock control, while virus-infected but untreated cells served as the positive control (PC). CA-treated, virus-infected cells comprised the experimental groups. After 48 h of incubation, cell culture supernatants were collected for plaque assays, and cells were harvested for RNA-Seq and quantitative PCR (qPCR) analyses to evaluate ZIKV-related gene expression.

Plaque assays were performed as described previously (Kang et al., 2024[13]). Vero E6 cells (1 × 10⁶ cells/well, in 6-well plates) were incubated in DMEM containing 10 % FBS, 1 % HEPES, and 1 % antibiotic-antimycotic solution until confluency was reached. Cell culture supernatants collected for plaque assays as described in the section entitled “Sample treatment and virus infection” were serially diluted 10-fold in DMEM. The prepared cells were washed twice with DPBS, followed by the addition of 0.2 mL DMEM and 0.5 mL of each cell culture supernatant dilution per well. Following a 2 h adsorption period with gentle agitation every 15 min, the inoculum was removed, and 3 mL of DMEM/F12 containing 2 % Oxoid agarose was overlaid onto the cells. The cells were incubated at 37 °C with 5 % CO₂ for 5 d. The cells were fixed with 4 % formaldehyde for 1 h and subsequently stained with 0.1 % crystal violet for 30 min. Viral titers were calculated as follows: PFU/mL = Number of plaques / (dilution factor × volume of inoculum per well).

RNA-seq was conducted using total RNA extracted from three experimental groups of cells. Total RNA from Vero E6 cells was extracted using the acid guanidinium thiocyanate-phenol-chloroform method (Chomczynski and Sacchi, 1987[6]). For library preparation, the TruSeq Stranded Total RNA with Ribo-Zero H/M/R Gold kit (Illumina, San Diego, CA, USA) was used to deplete ribosomal RNA and generate sequencing-ready libraries. Sequencing was performed on an Illumina platform to obtain paired-end reads (101 bp). Raw reads underwent quality control and adapter trimming to remove low-quality bases and residual adapter sequences, yielding high-quality trimmed reads (Supplementary information, Figure S1-S2). These reads were mapped to the Chlorocebus sabaeus reference genome (NCBI Assembly: GCF_015252025.1) using the HISAT2 program. Alignment files were processed with StringTie to assemble transcripts and quantify gene expression. Transcript abundance was calculated as raw read count, fragments per kilobase of transcript per million mapped reads (FPKM), and transcripts per million (TPM). Differential gene expression analysis was performed using read counts, FPKM, and TPM values in the DESeq2 package. Genes with an absolute fold change ≥ 2 and raw p value < 0.05 (based on the nbinomWaldTest) were considered significantly differentially expressed. Functional classification and gene annotation of differentially expressed genes (DEGs) were carried out using g:Profiler based on gene ontology (GO) and pathway information.

Total RNA from Vero E6 cells was extracted using the acid guanidinium thiocyanate-phenol-chloroform method (Chomczynski and Sacchi, 1987). RNA was reverse-transcribed into complementary DNA (cDNA) using the cDNA synthesis kit (Applied Biosystems, Waltham, MA, USA). qPCR was performed using the SYBR green method on a QuantStudio 3 (Thermo Fisher Scientific) with the cycling conditions as follows: 95 °C for 10 min, followed by 40 cycles of 95 °C for 15 s and 60 °C for 1 min. Gene expression levels were calculated using the crossing point method. Primer sequences are listed in Table 1(Tab. 1).

Vero E6 cells (1 × 10⁵ cells/well, in 8-well cell culture slides) were incubated in DMEM containing 10 % FBS, 1 % HEPES and 1 % antibiotic-antimycotic solution until confluency was reached. Cells were washed with 0.2 mL DPBS and applied with CA (5 µM) with 0.2 mL of DMEM supplemented with 2 % FBS, 1 % HEPES and 1 % antibiotic-antimycotic solution for 2 h prior to infection with ZIKV at 0.01 MOI. Untreated, uninfected cells served as the mock control, while virus-infected but untreated cells served as the positive control (PC). CA-treated, virus-infected cells comprised the experimental groups. After 48 h of incubation, protein expression levels of cells were measured by immunocytochemistry assay. Cells were washed with 0.2 mL DPBS 3 times, and fixed with 4 % paraformaldehyde for 10 min at 37 °C. Cells were washed with 0.2 mL DPBS 3 times again, and permeabilized with 0.2 mL 0.1 % Triton X-100 in 1X DPBS, and incubated for 15 min at 25 °C. Then, cells were washed with 0.2 mL DPBS 3 times, and blocked with 0.25 mL 2 % BSA in 1X DPBS, and incubated for 60 min at 25 °C. After that, cells were immunostained with primary antibody (IFIT2 and IFIT3, Thermo Fisher Scientific), secondary antibody (Alexa Fluor^TM 488, Thermo Fisher Scientific), and hoechst33342 (Invitrogen, Waltham, MA, USA) sequentially. Cell morphology was examined using a Leica THUNDER 3D DMi8 fluorescence microscope (Leica Microsystems, Wetzlar, Germany), and positively stained areas were quantified using ImageJ software.

All in vitro experimental data are presented as the mean ± standard deviation from three independent experiments. Statistical analyses were conducted using GraphPad Prism version 10 (GraphPad Software, San Diego, CA, USA). One-way ANOVA followed by Dunnett's multiple comparison test was used to assess statistical significance. Differences were considered statistically significant at p < 0.05.

CA was extracted from mass-cultivated Tetraselmis sp. using 90 % acetone and subsequently purified, and characterized as a main compound using LC-MS and NMR analyses as described previously (Figure 2(Fig. 2); Kang et al., 2024[13]). The antiviral effect of CA against ZIKV was evaluated by measuring the rate of viral plaque formation (Figure 3(Fig. 3)), with ZIKV-infected cells serving as the PC. CA treatment (2.5, 5, 10, and 20 µM) significantly reduced infectious viral particle levels without inducing cytotoxicity (Figure 3A-C(Fig. 3)). Infected control cells exhibited a plaque-forming unit (PFU) titer of 49,600 ± 14,708 PFU/mL, whereas CA-treated cells showed markedly reduced titers: 2,153 ± 1,608 PFU/mL and 67 ± 115 PFU/mL at 2.5 and 5 µM, respectively. Notably, no infectious viral particles were detected at 10 and 20 µM CA concentrations (Figure 3B-C(Fig. 3)).

To validate the RNA-seq results, the expression levels of IFIT1, IFIT2, IFIT3, and IFIT5 mRNA were assessed by qPCR following CA treatment (Figure 8(Fig. 8)). ZIKV infection markedly upregulated mRNA expression of all four IFITs relative to the levels observed in the untreated, uninfected control group (M). In contrast, CA treatment restored IFIT expression to levels comparable to those in M (Figure 8(Fig. 8)). Also, the protein expression levels of IFIT2 and IFIT3 were assessed by immunocytochemistry following CA treatment (Figure 9-10(Fig. 9)(Fig. 10), and Figure S10). ZIKV infection markedly upregulated IFIT2 and IFIT3 protein expression relative to the levels observed in the untreated, uninfected control group (M). In contrast, CA treatment reduced IFIT2 protein (Figure 9(Fig. 9), and Figure S10a) and IFIT3 protein (Figure 10(Fig. 10), and Figure S10b) expression. These findings indicate that CA exerts antiviral effects against ZIKV infection by modulating the expression of interferon-inducible antiviral effectors, particularly IFIT family members.

The search for antiviral agents from natural sources has intensified in recent years. For instance, oridonin, a diterpenoid compound isolated from Rabdosia rubescens, has been shown to mitigate herpes simplex virus infection (Jiang et al., 2022[12]). Similarly, several flavonoids (baicalein, fisetin, and quercetagetin) exhibit inhibitory activity against chikungunya virus, a mosquito-borne alphavirus (Lani et al., 2016[15]). Lignans and phenylpropanoids derived from the fruits of Illicium verum, a traditional spice, showed antiviral activities against influenza virus A (Li et al., 2022[19]). Extracts and/or compounds from flowering plants such as Lippia alba, Morus spp., Tecoma sp., and Cryptolepis sp. also display antiviral properties against ZIKV, coronaviruses, and tobacco mosaic virus (Reis et al., 2020[24]; Sobrinho et al., 2021[28]; Thabti et al., 2020[31]; Zhou et al., 2025[46]). Despite their potential, the limited biomass availability of terrestrial plants often hampers their industrial applicability. In contrast, Tetraselmis sp., a marine microalga, is amenable to large-scale cultivation, suggesting that CA derived from Tetraselmis sp. represents a viable candidate for commercial antiviral ingredient development.

RNA-seq, a high-throughput next-generation sequencing technique, enables comprehensive profiling of transcriptomes and has transformed transcriptomics by allowing multidimensional analysis of gene expression at scale (Chen et al., 2016[5]; Yang et al., 2018[39]; Zhang et al., 2021[45]). Also, GO analysis is a widely used tool for elucidating the biological functions underlying gene expression patterns (Rosati et al., 2024[26]). In this study, we investigated the underlying mechanisms of CA by analyzing gene expression profiles in ZIKV-infected cells with or without CA treatment via RNA-seq. The results indicate that “response to external stimulus” and “response to biotic stimulus” are key biological processes modulated by CA treatment in virus-infected cells. Interferons (IFNs) are a group of signaling proteins secreted by cells in response to diverse stimuli, particularly viral infections (Deng et al., 2024[7]; Swaraj and Tripathi, 2024[30]; Wu, 2019[34]). For this reason, IFNs are commonly used as drugs to treat viral infection (Zhang et al., 2016[43]). IFN signaling induces the expression of interferon-stimulated genes (ISGs), which act as downstream effectors to restrict viral replication and modulate immune responses. Proteins encoded by ISGs inhibit viral propagation by suppressing viral transcription, translation, and replication. In addition, ISGs contribute to the degradation of viral nucleic acids and reprogram host lipid metabolism to counteract viral infection (Li et al., 2022[17]). The IFIT gene family, classified as ISGs, is a crucial component of the antiviral innate immune response. These proteins inhibit various stages of viral replication, including translation initiation, viral propagation, and protein synthesis (Wu et al., 2023[36]; Xu et al., 2022[38]). Four IFIT family members have been identified in humans, distinguished by the number of tetratricopeptide repeats they contain (Vladimer and Górna, 2014[33]). Our findings, supported by RNA-seq and qPCR analyses, indicate the involvement of the entire IFIT family in the antiviral mechanism of CA.

In the RNA-seq analysis on the antiviral activity of CA, a limitation of this study is the absence of a control group that exclusively received CA, which hinders the ability to ascertain whether the observed effects were solely attributable to the antiviral action of CA or were also influenced by its direct cellular toxicity or transcriptional modulation. Another limitation is that the experiments were conducted using a single cell line, and it is possible that the results may not fully reflect physiological diversity. To overcome these limitations and more clearly validate the antiviral activity of CA, further studies should include well-structured experimental designs involving multiple cell lines, time-course experiments, and appropriate control groups such as non-treated, non-infected cells (Mock), CA-treated cells (Control), ZIKV-infected cells (PC), CA-treated, ZIKV-infected cells (CA), and vehicle controls.

Antiviral mechanisms are closely associated with the viral replication cycle. Since certain viruses share similarities in their replication processes, some antiviral agents can inhibit multiple viral infections-these are known as broad-spectrum antivirals. Such agents function either by enhancing host antiviral responses or by suppressing host factors essential for viral replication. Thus, these agents are not directly influenced by viral genetic control, and the likelihood of resistance mutations is relatively low. Therefore, the discovery of broad-spectrum antivirals has gained increasing attention in recent years. Notably, several chlorophyll derivatives have been reported to exhibit antiviral activity against dengue virus (Azizullah et al., 2014[1]) and hepatitis C virus (Ratnoglik et al., 2014[23]). In addition, chlorophyll derivatives are known to possess a wide range of biological activities, including anticancer, antimutagenic, antioxidant, radioprotective, antimicrobial, and anti-inflammatory effects. Also, CA is consumable as a dietary component, and it holds potential as a functional food ingredient or nutraceutical supplement for health improvement. In future studies, it will be important to evaluate the antiviral efficacy of CA not only against flaviviruses but also across a broader spectrum of viral species, and to further assess its potential as a food or pharmaceutical material through comprehensive ADMET profiling and long-term safety studies.

Throughput comparison between raw data and trimmed data; Q30 value comparison between raw data and trimmed data; Heat map for differentially expressed genes (DEGs) identified using criteria of absolute fold change ≥ 2 and raw p < 0.05 in at least one comparison; Heatmap of 130 DEGs from the intersecting sets; Bar plots for enrichment analysis of Gene Ontology (GO) terms related to Biological Process among the 130 DEGs; Bar plots for enrichment analysis of GO terms related to Cellular Component among the 130 DEGs; Dot plot of significantly enriched GO Cellular Component terms among the 130 DEGs; Bar plots for enrichment analysis of GO terms related to Molecular Function among the 130 DEGs; Dot plot of significantly enriched GO Molecular Function terms among the 130 DEGs; Corrected integrated density of the stained cells with IFIT2 (A) and IFIT3 (B) by immunocytochemistry(PDF); List for all identified genes; Heatmap category for 130 differentially expressed genes; List for DEGs upregulated by CA treatment following ZIKV infection; List for DEGs downregulated by CA treatment following ZIKV infection (Excel).

This research was supported by Korea Institute of Marine Science and Technology Promotion (KIMST) funded by the Ministry of Oceans and Fisheries, Korea (20210466).

The manuscript was written by all authors. All authors have given approval of the final version of the manuscript. Conceptualization, writing-original draft preparation, NK; Methodology, investigation, EAK; Data curation, Formal analysis, YJL; Data curation, Formal analysis, SYH; Software, JHH; Validation, Resources, WKL; Visualization, Resources, YKR; Resources, TK; Writing-review and editing, project administration, supervision, SJH.

All data in this study can be downloaded from the links in this article. The data supporting the findings of this study are available from the corresponding author upon request.

The authors declare that they have no conflict of interest.

No artificial intelligence tools were used in the preparation, writing, editing, or analysis of this manuscript.