# Regulation of detoxifying enzymes expression and restriction of picorna-like virus infection by natural polysaccharide extracts in Drosophila cells

Gabrielle Haas, Mélodie Seiler, Jenny Nguyen, Laurent Troxler, Samuel Pennarun, Elise Lefebvre, Yasmine Benamrouche, Loriane Loizeau, Cody Reinbolt, Ming Liang, et al.

<figure><img src="image_1.png" /></figure>

## ► To cite this version:

Gabrielle Haas, Mélodie Seiler, Jenny Nguyen, Laurent Troxler, Samuel Pennarun, et al. Regulation of detoxifying enzymes expression and restriction of picorna-like virus infection by natural polysaccharide extracts in Drosophila cells. Virology, 2025, 607, pp.110513. 10.1016/j.virol.2025.110513. hal-05016278

HAL Id: hal-05016278

https://hal.science/hal-05016278v1

Submitted on 1 Apr 2025

HAL is a multi-disciplinary open access
archive for the deposit and dissemination of sci-
entific research documents, whether they are pub-
lished or not. The documents may come from
teaching and research institutions in France or
abroad, or from public or private research centers.

L'archive ouverte pluridisciplinaire HAL, est
destinée au dépôt et à la diffusion de documents
scientifiques de niveau recherche, publiés ou non,
émanant des établissements d'enseignement et de
recherche français ou étrangers, des laboratoires
publics ou privés.

<figure><img src="image_2.png" /></figure>
<figure><img src="image_3.png" /></figure>

# Regulation of detoxifying enzymes expression and restriction of picorna-like virus infection by natural polysaccharide extracts in *Drosophila* cells

Gabrielle Haas<sup>a</sup>, Mélodie Seiler<sup>a</sup>, Jenny Nguyen<sup>a</sup>, Laurent Troxler<sup>a</sup>, Samuel Pennarun<sup>a</sup>, Elise Lefebvre<sup>a</sup>, Yasmine Benamrouche<sup>a</sup>, Loriane Loizeau<sup>a</sup>, Cody Reinbolt<sup>a</sup>, Ming Liang<sup>b</sup>, Xiaoliang Lin<sup>b</sup>, Wenzhi Li<sup>b</sup>, Zumeng Xia<sup>b</sup>, Joao T. Marques<sup>c,d</sup>, Jean-Luc Imler<sup>a</sup>,

## ARTICLE INFO

### Keywords:

- Antiviral activity

- Picorna-like virus

- Detoxifying enzymes

- Polysaccharides

- Traditional Chinese medicine

## ABSTRACT

The world is currently witnessing a rise in viral infections, while the availability of antiviral drugs remains limited. Traditional Chinese medicine (TCM) has historically served as a valuable source of novel compounds for disease treatment. In this study, we assessed the antiviral potential of various TCM compounds using *Drosophila melanogaster* as a model organism. Our findings reveal that natural polysaccharide extracts, prepared from 10 commonly used medicinal herbs or fungi, exhibit antiviral activity against two picorna-like viruses. Importantly, the antiviral effect is not directly attributable to the compound itself but is instead mediated by cellular responses induced by treatment with the extract. We observed that the polysaccharide extract triggers a broad transcriptional response, which partially overlaps with NF-κB pathway activation in *Drosophila*. However, the antiviral activity of the extract was independent of classical innate immune pathways, such as RNA interference or NF-κB signaling. Instead, the extract appears to uniquely stimulate detoxification pathways, including upregulation of cytochrome P450 and glutathione S-transferase genes, which correlates with its antiviral effects.

## 1. Introduction

An unprecedented succession of viral epidemics, including H1N1 flu (2009), MERS-Coronavirus (Middle East Respiratory Syndrome Coronavirus or MERS-CoV) (2012), Ebola virus (2014), Zika virus (2015) and SARS-CoV2 (Severe Acute Respiratory Syndrome Coronavirus 2) (2019) have occurred in past 15 years (Lu et al., 2021). It reflects the accelerating globalization of human activities, climate change and the growing human pressure on ecosystems harboring wildlife reservoirs for new viruses, which altogether favor viral transmission (Dobson et al., 2020; Gibb et al., 2020). As a result, we must prepare for new epidemics in the future, to avoid the dramatic human and financial toll of the COVID-19 (Coronavirus Disease, 2019) pandemic. One strategy, which was highly successful against SARS-CoV2, is to rely on vaccines. However, we cannot predict what the next virus of concern will be, and it may be difficult to develop vaccines against some viruses, like HIV (Human

immunodeficiency virus) or HCV (Hepatitis C virus) (Jochmans et al., 2023). Alternatively, intense research efforts have led to the development of drugs used to treat important human viral pathogens. Developing antiviral drugs is challenging because viruses are the smallest pathogens, hijacking cellular molecules for their replication cycle, and offer few targets for intervention. In addition, viruses exhibit high mutation rates, such that they rapidly develop resistance to antiviral drugs. Thus, successful antiviral therapies often rely on the combination of drugs targeting different components of the virus. Altogether, these considerations highlight the necessity to increase the arsenal of compounds that can be used to control viral infections (Dolgin, 2021; Edwards et al., 2022; Lu et al., 2021; Service, 2021).

When it comes to screening for active lead compounds, exploiting the extensive knowledge gathered over centuries on the use of plant extracts in traditional and popular remedies can, arguably, be more efficient than random screening of chemical libraries (Li and Peng, 2013; Mattioli

<footnote><sup>a</sup> Université de Strasbourg, CNRS UPR9022, 67084, Strasbourg, France</footnote>

<footnote><sup>b</sup> Infinitus (China) Company Ltd., Guangzhou, China</footnote>

<footnote><sup>c</sup> Université de Strasbourg, INSERM U1257, CNRS UPR9022, 67084, Strasbourg, France</footnote>

<footnote><sup>d</sup> Department of Biochemistry and Immunology, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, 31270-901, Belo Horizonte, Brazil</footnote>

<footnote>* Corresponding author.
E-mail address: jl.imler@ibmc-cnrs.unistra.fr (J.-L. Imler).</footnote>
et al., 2024). Indeed, drugs derived from natural products represent a significant proportion of modern pharmacopeia (Normile, 2015). Among folk medicines, Traditional Chinese Medicine (TCM) with up to 100,000 recipes documented sticks out for its long standing record, with reports of treatment of infectious diseases using natural based medicine for almost 2000 years (Lin et al., 2016; Mattioli et al., 2024). Several TCM formulations include polysaccharides, which are critical broad mediators of biological functions and have been shown to contribute to the physiopathology of several infectious diseases, e.g. COVID-19 (Buchanan et al., 2022; Griffin and Hsieh-Wilson, 2022; Mattioli et al., 2024; Zhou and Cobb, 2021). For example, polysaccharides have been reported to block viral infection by interfering with the initial step of the infection, namely cell surface attachment and virus entry. Indeed, this critical step of the infection often requires interaction of the viral particles with cell surface glycans (Buchanan et al., 2022; Li and Peng, 2013). Polysaccharides can also bind to and activate cell surface immune receptors, thus acting as immunomodulators. For example, several studies reported stimulation of immunity by the compounds, possibly following engagement of innate immune receptors, and activation of the transcription factor NF-κB (Nuclear Factor kappa-light-chain enhancer of activated B cells) (reviewed in ref (Zhang et al., 2023)).

The fruit fly *Drosophila melanogaster* is a simple model organism with a well characterized immune system that involves signaling pathways that are conserved in humans (Ferrandon et al., 2007; Marques et al., 2024). Here, we evaluated the impact of different commercial formulation of natural polysaccharide extracts on *Drosophila* resistance to viral infection, including the Lingzhihuang Capsule (LZH). LZH is a composite natural product that includes polysaccharide extracts from two species of Chinese medicinal fungi and eight varieties of medicinal herbs. The formulation includes *Ganoderma lucidum*, *Poria cocos*, *Atractylodes macrocephala*, *Polygonatum sibiricum*, *Lycium barbarum*, *Coix lacryma-jobi*, *Ophiopogon japonicus*, *Cuscuta chinensis*, *Schisandra chinensis*, and *Glycyrrhiza uralensis*. Extracts from these components are known to exhibit a diverse array of pharmacological activities, such as anti-inflammatory, anti-tumor, anti-aging, antioxidant, immunomodulatory, neuro-protective, hepatoprotective, and metabolic regulatory effects (Donnapee et al., 2014; Du et al., 2023; Ji et al., 2024; Li et al., 2024; Liu et al., 2024; Luo et al., 2024; Shaikh et al., 2024; Wang et al., 2024, 2023; Xiao et al., 2024; T. Zhang et al., 2024; Y. Zhang et al., 2024). However, there is still poor understanding on the mechanisms of action of these compounds alone or in combination. Liquid chromatography-coupled with mass spectrometry (LC-MS) was recently performed to analyze the detailed chemical constituents of LZH (Huang et al., 2022). This revealed 94 compounds, including triterpenes, flavonoids and organic acids. Interestingly, in silico target prediction and pathway enrichment analysis suggested that the constituents of LZH may connect to signal transduction, oxidation-reduction processes and inflammatory responses. This prompted us to evaluate the potential of natural polysaccharide extracts within LZH in controlling viral infections, using the *Drosophila* model.

Our results suggest that LZH has broad effects on *Drosophila* cells leading to antiviral resistance. In particular, we show that the induction of detoxifying enzymes of the cytochrome P450 and Glutathione S-transferase families correlated with inhibition of viral infection. Overall, our work points to a possible new antiviral mechanism that could be explored to treat virus infections.

# 2. Material and methods

## 2.1. Cell lines

Drosophila S2 and Kc167 cells were cultured in Schneider's *Drosophila* medium (Biowest) supplemented with 10% FBS (fetal bovine serum) (Gibco), 1% GlutaMax-1 (Gibco), 1% Penicillin/Streptomycin (100x; 10 mg/mL/10000 U, Gibco) at 25 °C. For Drosophila S2 *dicer-2* null cells, the culture media is also supplemented with 300 µg/mL

Hygromycin B.

## 2.2. TCM compounds

The TCM compounds provided by the company Infinitus were prepared as follows:

1) Zhen yuan Capsule (ZYC) is formulated from the water extracts of crude polysaccharides derived from *Ganoderma lucidum* (35–42% w/w), *Cuscuta chinensis* (15–20% w/w) and *Codonopsis pilosula* (5–10% w/w) complemented with *Poria cocos*, *Schisandrae chinensis* and *Atractylodes macrocephala*. *G. lucidum* was extracted twice with 100 °C boiling water, 2.5 h with 12 times the volume of water for the first extraction, and 2.0 h with 10 times the volume of water for the second extraction. *C. chinensis*, *C. pilosula*, *P. cocos*, *S. chinensis*, and *A. macrocephala* were extracted twice with 100 °C boiling water: 1.5 h with 12 times the volume of water for the first extraction, and 1.0 h with 10 times the volume of water for the second extraction. The combined extracts were subsequently concentrated using a vacuum concentrator at a temperature of ≤95 °C and a vacuum level ranging from –0.03 to –0.09 MPa. The concentrate was then dried using a spray-drying method with an inlet air temperature controlled between 145 °C and 160 °C until the moisture content was reduced to less than 8%. The final product contains 1300 mg of polysaccharides per 100g of extract.

2) Core ingredients of ZYC (CoreZYC) is formulated from the water extracts of crude polysaccharides derived from *G. lucidum* (50–60% w/w), *C. chinensis* (25–30% w/w), *C. pilosula* (10–15% w/w). *G. lucidum* was extracted twice with 100 °C boiling water, first for 2.5 h with 12 times the volume of water, and second for 2.0 h with 10 times the volume of water. *C. chinensis* and *C. pilosula* were extracted twice with 100 °C boiling water, first for 1.5 h with 12 times the volume of water, and second for 1.0 h with 10 times the volume of water. The combined extracts were subsequently concentrated using a vacuum concentrator at a temperature of ≤95 °C and a vacuum level ranging from –0.03 to –0.09 MPa. The concentrate was then dried using a spray-drying method with an inlet air temperature controlled between 145 °C and 160 °C until the moisture content was reduced to less than 8%. The final product contains 1500 mg of polysaccharides per 100g of extract.

3) LZH Capsule (also known as Royal Lingzhi Plus Capsule) is formulated from the water extracts of crude polysaccharides derived from *G. lucidum* (50–55% w/w), *Coix lacryma-jobi* (10–15% w/w), *P. cocos* (8–15% w/w), complemented with *A. macrocephala*, *Polygonatum sibiricum*, *Lycium barbarum*, *Ophiopogon japonicus*, *C. chinensis*, *S. chinensis* and *Glycyrrhiza uralensis*. The raw materials were subjected to a two-step boiling water extraction process. The first extraction was conducted at 100 °C using 12 times the volume of water for 2.5 h, followed by a second extraction using 10 times the volume of water for 2.0 h. The extract was subsequently concentrated using a vacuum concentrator at a temperature of ≤95 °C and a vacuum level ranging from –0.03 to –0.09 MPa. The concentrated extract was subjected to belt drying under a vacuum pressure of –0.1008 to –0.0978 MPa, with the drying temperature controlled between 80 °C and 100 °C until the moisture content was reduced to less than 9%. The final product contains 800 mg of polysaccharides per 100g of extract.

## 2.3. Preparation of stock solutions for TCM compounds

TCM compounds were dissolved in Schneider's *Drosophila* medium at a concentration of 10 mg/mL. Tyndallization was carried out to sterilize the stock solutions as described (Brown et al., 1979). Briefly, solutions were boiled for 30 min then incubated at room temperature for 24 h, for three consecutive days. After tyndallization, solutions were centrifuged to pellet debris, and the saturated supernatants were filtered with 0.8 µm
pore filters. These 10 mg/mL stock solutions were stored at 4 °C. Control corresponds to Schneider's *Drosophila* medium treated in the same way.

## 2.4. Cell toxicity/viability assay

100,000 cells per well were seeded in 96-well plate and cultured at 25 °C in media supplemented with increasing concentrations of compounds solutions. Cells number was determined at different time point using the CellTiter 96 AQueous One Solution Cell Proliferation Assay (Promega) according to the manufacturer's instructions. Absorbance at 490 nm was recorded using a 96-well plate reader (Varioscan LUX, Thermo Scientific).

## 2.5. Viral infections

S2 cells were pre-cultured for 24-72 h in T25 flasks in media supplemented or not with the TCM compounds, heat-killed *E.coli* (HKEC) (MOI (Multiplicity of Infection) 40), or 100 µM Rifampicin (RFP) depending on the experiments. Then, 100,000 cells were transferred per well in 96-well plate, retreated and infected with Drosophila C Virus (DCV; MOI 1), Cricket Paralysis Virus (CrPV; MOI 0.01), Flock House Virus (FHV; MOI 1), Vesicular Stomatitis Virus (VSV; MOI 1) or Insect Iridescent Virus 6 (IIV6; MOI 1) for 1 h at 4 °C. Viruses were removed and replaced with complete medium ± compounds. Cells were incubated at 25 °C and samples collected at different time post-infection for qPCR analysis. Time points and multiplicity of infection were chosen to avoid saturation of replication based on established kinetics of viral replication previously reported in *Drosophila* cells (de Faria et al., 2022; Khong et al., 2016; Li et al., 2002; Mueller et al., 2010; Reavy et al., 1983). For western blotting and virus titration, cells were seeded in 24-well plate (500,000 cells/well) and processed similarly.

## 2.6. Assays for direct antiviral activity

We based our assays on the methods described in (Tai et al., 2015) to test the following steps. **Virus inactivation assay.** DCV (MOI 1) and CrPV (MOI 0.01) particles were preincubated with LZH-medium at a final concentration of 8 mg/mL for 2 h at 25 °C to observe if the compounds operate directly on viruses. Viruses were added to S2 cells (2 mg/mL final solution) in 96-well plate for 2 h at 25 °C. Then, viruses and compounds were removed and replaced with complete Schneider medium. Cells were incubated at 25 °C and assayed at indicated time after virus removal. **Virus binding assay.** Cells were seeded in LZH-medium at 2 mg/mL and infected with DCV (MOI 1) or CrPV (MOI 0.01) for 1 h at 4 °C to allow virus binding at the cell surface but not the internalization. Then, viruses and compounds were removed and replaced with fresh complete medium, incubated at 25 °C, and assayed at indicated time after virus removal. **Virus internalization assay.** Cells were infected with DCV (MOI 1) or CrPV (MOI 0.01) for 1 h at 4 °C. Viruses were removed and replaced with complete medium containing 2 mg/mL of LZH for 1 h at 25 °C. Then, medium was replaced with complete medium without LZH. Cells were incubated at 25 °C and assayed at indicated time after virus removal. **Virus replication assay.** Cells were infected with DCV (MOI 1) or CrPV (MOI 0.01) for 1 h at 4 °C. Viruses were removed and replaced with complete medium for 1 h at 25 °C. Then, the medium was replaced by LZH-containing medium and cells were incubated at 25 °C until the appropriate time points.

## 2.7. RNA extraction and RT-qPCR

Cells were harvested and cDNA was produced using the Cells-to-CT kit procedure (Invitrogen) as directed by the manufacturer. qPCR was conducted on a CFX384 Touch Real-Time PCR platform (Biorad) or on a QuantStudio 5 Real-Time PCR System (ThermoFisher) using the iTaq Universal SYBRGreen Supermix (Biorad). Normalization was performed with the housekeeping genes *RpL32* (RP49) or *gapdh2*. At least two

independent biological replicates were performed for each experiment. The primer sequences used as listed in Suppl. Table 1.

## 2.8. Virus titration

The 24-well plates containing infected S2 cells were frozen at -80 °C. After 3 cycles of freezing/defreezing, the suspensions were titrated. DCV was titrated on Kc167 cells and CrPV on S2 cells by observation of the cytopathic effect. The Reed-Muench method was used to calculate TCID50 (Tissue Culture Infective Dose 50 %) and converted to PFU (Plaque-Forming Unit) with a conversion factor of 0.7 for both Kc167 and S2 cells.

## 2.9. Western blotting

S2 cells were collected, washed once with ice-cold PBS, then lysed in 2X Protein Sample Buffer (100 mM Tris-HCl pH 6.8, 4 % (w/v) SDS, 20 % glycerol, 0.2 M DTT, 0.5 % bromophenol blue). The protein extracts were run on 10 % SDS-PAGE gel and transferred to a nitrocellulose membrane (GE Healthcare). Membranes were blocked in PBST (PBS-Tween 0.3 %) containing 5 % of fat milk and then incubated with the indicated primary antibodies overnight at 4 °C. After washing, the membranes were incubated with HRP-labeled secondary antibodies for 1 h. Immunoreactive bands were developed using Clarity Western ECL Substrate (Bio-Rad) and Amersham Hyperfilm ECL (GE Healthcare). Guinea pig Anti-DCV RdRp (RNA dependent RNA Polymerase) (1:2000) and guinea pig anti-CrPV RdRp (1:2000) were custom-made by Proteogenix (France). Rabbit anti-DCV VP2 (1:2000) and mouse anti-CrPV Capsid (1:2000) were previously described (Chtarbanova et al., 2014; Dostert et al., 2005). Mouse anti-actin (Gen Tex) was used at 1:1,000, and the secondary antibodies anti-mouse, anti-rabbit or anti-guinea pig IgG-HRP (Amersham) were used at 1:10,000.

## 2.10. Heat-killed *E. coli* preparation

A single colony of *E. coli* was cultured overnight at 37 °C. The bacteria were washed with 1X PBS then heated at 60 °C for 1 h. Additional washes were performed and HKEC were resuspended in 1X PBS. The OD600 (optical density at 600 nm) was measured to determine the bacterial concentration.

## 2.11. Double stranded (ds)RNA preparation and RNA interference (RNAi) in S2 cells

cDNAs were prepared from S2 cells or flies and DNA templates were obtained by RT-PCR using sets of primers containing the T7 polymerase recognition sequence at their 5' end. The list of primers used and their sequence is presented in Suppl. Table 1. dsRNAs were then produced using the T7 MEGAScript kit (Ambion) following the manufacturer protocol and annealed. Product lengths for both DNA and dsRNA were verified by agarose gel electrophoresis. 125,000 S2 cells were bathed in 500 µL of serum-free medium containing 30 µg of dsRNA per well of a 12-well plate for 4 h. The soaked cells were then supplemented with 750 µL double serum medium and powders solution (final concentration of LZH is 2 mg/mL) and incubated for 3 days at 25 °C. Silenced cells were counted, 100,000 cells were seeded in 96-well plate and then were infected with DCV (MOI 1) or CrPV (MOI 0.01). Viruses were removed and replaced with complete medium with or without LZH. Cells were incubated at 25 °C and assayed at indicated time post-infection.

## 2.12. Poly-A selection and preparation of RNA libraries

S2 cells were seeded in 12-well plates and treated with HKEC (MOI 40) or with 2 mg/mL of LZH prior infection with DCV (MOI 1) or CrPV (MOI 0.01) in this case. Samples were collected at the indicated time points and total RNA was isolated using TRIzol reagent (Invitrogen)
following the manufacturer's protocol. RNA samples were resuspended in RNAsense-free water (Ambion), their quality verified using a 2100 Bioanalyzer system (Agilent). mRNA libraries were constructed as described in (Olmo et al., 2023). RNA was first enriched for mRNA following the instructions of the kit NEBNext Poly(A) mRNA Magnetic Isolation Module (New England BioLabs inc) and then, the mRNA libraries were prepared with the kit NEBNext Ultra 769 II Directional RNA Library Prep Kit for Illumina (New England BioLabs inc.). Libraries were pooled and sequenced at MGX-Montpellier GenomiX sequencing platform in Montpellier, France.

## 2.13. RNA sequencing analysis

Adapters have been removed from raw reads using the shell written by Brian Bushnell, BBduk from BBMap, version 38.90. Processed reads were aligned to the transcript *Drosophila melanogaster* reference genome (Flybase release 6.32.104) using STAR v2.7.3a (Dobin et al., 2013) and nucleotide alignments were quantified with RSem v1.3.1 (Li and Dewey, 2011). Quantifications were then imported into R v4.2.2 (R Core Team. (2022). R: A language and environment for statistical computing. R Foundation for Statistical Computing, Vienna, Austria. URL https://www.R-project.org/) using tximport v1.26.1 package (Soneson et al., 2016). Data filtering and normalization were performed using the packages DESeq2 v1.38.3 (Love et al., 2014). Tables of the enriched GO ordered from highest rank (Normalized Enrichment score, NES) for each comparison were calculated using the R package clusterProfiler v4.6.2 (Yu et al., 2012). The GO matrix was reduced using the rrvgo v1.10.0 package (Sayols, 2023). Available at: https://sayols.github.io/rrvgo/) and the Keywords Enrichment annotation function (Gu and Hübschmann, 2023). Heatmaps were generated with the ComplexHeatmap v2.14.0 package (Gu et al., 2016). RNA-seq libraries in this study have been deposited on the European Nucleotide Archive (ENA) at EMBL-EBI under accession number PRJEB78990 (https://www.ebi.ac.uk/ena/browser/view/PRJEB78990).

## 2.14. GST (glutathione S-transferase) assay

S2 cells were cultured for 24 h in presence or not of 2 mg/mL of LZH. Then collected pellets were resuspended in GST Assay buffer (100 mM Potassium phosphate buffer, pH7; 2 mM EDTA), sonicated by 6 rounds of 30 s (Bioruptor Plus, Diagenode) and spun at 10,000 g for 15 min at 4 °C. Supernatants were collected and protein concentration was determined with a BCA Protein Assay Kit (Pierce), prior to adjust all the protein samples to 2 µg/µl. The GST activity was measured using the Glutathione S-Transferase Assay kit (Cayman chemical) measuring the conjugation of 1-chloro-2,4-dinitrobenzene (CDNB) with reduced glutathione, following the manufacturer's protocol and calculations. Briefly, after substrate addition, the absorbance at 340 nm is recorded every minute for 10 min using a 96-well plate reader (Varioscan LUX, Thermo Scientific). The absorbance values over time are plotted to determine the reaction rate ($ΔA_{340}/min$) which allowed then to determine GST activity using the formula:

$$ \text{GST activity} = \frac{\Delta A_{340}}{\text{min}} / 0.00503 \, \mu\text{M}^{-1} \times \text{sample dilution} = \text{nmol/min/mL, where } 0.00503 \, \mu\text{M}^{-1} \text{ is the extinction coefficient of CDNB.} $$

Purified GST is assayed in parallel as positive control. Four biological replicates, each containing technical replicate were performed.

## 2.15. Statistics

Statistics were performed with GraphPad Prism software (version 10).

# 3. Results

## 3.1. TCM compounds restrict replication of picorna-like viruses

To assess the potential of TCM to treat viral diseases, we initially tested three different TCM compounds composed of different combinations of natural polysaccharide extracts: Zhen Yuan Capsul (ZYC), only core ingredients of ZYC (CoreZYC) and LZH (See methods for details). We first assessed cell toxicity of these compounds by treating *Drosophila* S2 cells with doses ranging from 0.01 to 2 mg/mL. Cells treated with coreZYC at doses ≥0.5 mg/mL showed reduced numbers, which became apparent as soon as 48h after the beginning of the treatment. By contrast, treatment with LZH did not affect cell number for up to 6 days while a ~40 % decrease in cell numbers was observed for the highest concentration of ZYC after 6 days (Suppl. Fig. 1A). Next, we chose the highest dose without acute toxicity for the 3 compounds (0.5 mg/mL) to treat S2 cells for 72h, before infecting them with a panel of viruses from different families, including Cricket Paralysis Virus (CrPV) (*Dicistroviridae*), Vesicular Stomatitis Virus (VSV, *Rhabdoviridae*), Flock House Virus (FHV, *Nodaviridae*) and Invertebrate Iridescent Virus-6 (IIV6, *Iridoviridae*). In this antiviral assay, we observed a trend for decreased CrPV RNA accumulation in cells treated with the three compounds, which was statistically significant only for LZH at 48h post infection (Suppl. Fig. 1B). A trend for decreased viral RNA accumulation was also observed for LZH at 8h and 24h post-infection upon infection with another virus of the *Dicistroviridae* family, Drosophila C virus (DCV), although the decrease did not reach statistical significance at this dose for this virus (Suppl. Fig. 1C). By contrast, none of the compounds showed antiviral effects against VSV, FHV or IIV6 at 0.5 mg/mL (Suppl. Figs. 1D–F). To confirm our initial observation, we focused on picorna-like viruses (CrPV and DCV) and increased the dose for LZH and ZYC, which did not exhibit cell toxicity at higher concentrations (Suppl. Fig. 1A). At 2 mg/mL, we observed significant reduction in the accumulation of CrPV RNA in cells treated with both LZH or ZYC (Fig. 1A). A similar inhibitory effect was observed in cells infected with DCV (Fig. 1B). Next, we compared viral titers at 24h or 48h after infection of S2 cells treated or not with LZH or ZYC extracts. The decrease in viral RNA was accompanied by a significant reduction in the amount of infectious CrPV and DCV particles when cells were treated with the compounds (Fig. 1C–D). Finally, a reduction in the production of both the RNA-dependent RNA polymerase (RdRp) and the capsid protein (CP) for CrPV and DCV at 24h and 48h after infection was also observed (Fig. 1E–F, Suppl. Fig. 2). Overall, our results reveal that treatment of S2 cells with LZH and ZYC compounds inhibits multiplication of two picorna-like viruses. Based on these results, we decided to further focus on LZH that was the least toxic compound.

To assess whether LZH directly inactivates CrPV, we pre-incubated virions with the compounds for 2h before adding them to the cells. The amount of viral RNA produced after 24h or 48h was similar in non-treated and treated conditions, indicating that LZH does not directly affect the infectivity of virus particles (Fig. 1G). The amount of virus produced was also not significantly affected when LZH was mixed with the virus during adsorption at 4 °C (Fig. 1H), internalization at 25 °C steps (Fig. 1I) or the replication phase (Fig. 1J). Similar results were obtained with DCV (Suppl. Fig. 3). These results suggest that the LZH does not act directly on the virus itself or on a step of its replication cycle but rather induce a refractory state in treated S2 cells. Accordingly, we observed an antiviral effect on CrPV that was stronger the longer cells were pretreated with LZH (Fig. 1K). A similar result was observed for DCV (Fig. 1L).

## 3.2. LZH modulates activation of innate immunity in S2 cells

The above results suggest that LZH may modulate resistance to virus infection in S2 cells. Therefore, we decided to investigate the role of known antiviral pathways in *Drosophila* such as RNA interference
<figure><img src="image_4.png" /><figcaption>Fig. 1. – ZYC and LZH have antiviral properties in *Drosophila* cells</figcaption></figure>

A-F. S2 cells were grown for 72h in presence or not (NT, non treated) of 2 mg/mL of ZYC or LZH (as indicated by the color code) and then infected with CrPV (A, C, E) or DCV (B, D, F). Virus levels were measured at 8, 24 and 48 h post-infection (hpi) by RT-qPCR (A, B). Virus titers were determined at 24 or 48 hpi by TCID50 (C, D). Viral protein levels were analyzed by Western blot for each virus (E, F), using antibodies directed against their respective viral replicase (RdRp) and capsid protein (CP). Actin was used as loading control. Representative blots out of three are shown. NT non treated, Ni non infected.

G-J. To determine at which level LZH is targeting CrPV, we performed inactivation (G), binding (H), internalization (I) and replication assays (J). Cells were infected with CrPV at a MOI (Multiplicity of Infection) of 0.01 and viral RNA levels were determined at different time points by RT-qPCR using primers matching the genome (G-I) or the antigenome (J).

K-L. To evaluate the importance of the pre-incubation of the cells with LZH (2 mg/mL) prior to be challenged with viruses, different pre-incubation times (48, 24 or 0 h) were tested. Then, cells were infected with CrPV (K) or DCV (L) at MOI of 0.01 and 1, respectively. Virus levels were determined at 24 and 48 h post-infection (hpi) by RT-qPCR.

Values represent the means ± SEM of three independent experiments (n = 3). Statistical significance was determined using two-way Anova test, ns non-significant, \*p < 0.05. \*\*p < 0.01. \*\*\*p < 0.001. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
(RNAi). This response is triggered by the enzyme Dicer-2, which senses
viral double stranded (ds)RNA produced during viral replication to
trigger silencing of the viral RNA via production of small interfering
RNAs (Galiana-Arnoux et al., 2006; Wang et al., 2006). To analyze the

contribution of RNAi, we assessed the antiviral properties of LZH in *Dicer-2* knock-out S2 cells. In the absence of RNAi, we observed that the accumulation of CrPV and DCV RNA was still decreased by LZH, albeit to a lesser extent (Fig. 2A–B). Since LZH had an inhibitory effect even when

<figure><img src="image_5.png" /><figcaption>**Fig. 2.** – LZH drives a Relish-dependent response in *Drosophila* cells.</figcaption></figure>

A-B. Virus levels were analyzed in Dcr2-deficient S2 cells grown for 72 h (h) in non-treated media (NT) or in media containing 2 mg/mL of LZH prior to infection. C-H. S2 cells were grown for 72h in presence of 2 mg/mL LZH and then infected with CrPV (C, E, G) or DCV (D, F, H) and the relative levels of the Relish-target genes *Vago* (C, D), *dSting* (E, F) and *attacin A* (G, H) were analyzed.

I-J. Different components of the STING and IMD pathways including dSting, PGRP-LC, IMD, Ird5 and Relish were silenced in S2 cells using dsRNA and dsRNA against GFP was used as a negative control. Silenced cells were grown in media containing 2 mg/mL of LZH prior to infection with CrPV MOI 0.01 (I) or DCV MOI 1 (J). Virus accumulation was measured at 24 and 48 h post-infection (hpi) by RT-qPCR.

K-L. S2 cells were stimulated with Heat-killed *E. coli* (HKEC; MOI 40) or with 2 mg/mL of LZH (as indicated by the color code) and then infected with CrPV (K) or DCV (L). Virus levels were measured at 8, 24 and 48 hpi by RT-qPCR.

Values represent the means ± SEM of three independent experiments (n = 3). Statistical significance was determined using two-way Anova test, \*p < 0.05, \*\*p < 0.01. \*\*\*p < 0.001. NT, non treated. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
the replication of DCV and CrPV is increased in the absence of Dcr-2 (Compare Fig. 1A–B to Fig. 2A–B), these results suggest that the anti-viral effect of the compound does not fully depend on RNAi and other mechanisms must be involved.

Antiviral innate immunity in insects also involves transcriptional responses induced by the IMD (Immune deficiency), Toll, Jak/STAT (Janus Kinase/Signal Transducer and Activator of Transcription) and STING (Stimulator of Interferon Genes) pathways (Avadhanula et al., 2009; Dostert et al., 2005; Ferreira et al., 2014; Goto et al., 2018; Palmer et al., 2019; Segrist et al., 2021; West et al., 2019; West and Silverman, 2018; Xu et al., 2012). To assess if LZH modulates these pathways, we monitored expression of marker genes in treated cells in the presence of viral infection or not. We did not observe consistent effects of LZH on expression of *drosomycin* and *vir-1* regulated by the Toll and Jak/STAT-pathways, respectively (Suppl. Fig. 4). By contrast, we observed significant up-regulation of the gene *Vago* in cells treated with LZH, independent on the infection with DCV or CrPV (Fig. 2C–D). Interestingly, this gene is up-regulated by viral infection in *Drosophila*, and appears to be regulated by Dicer-2 and the STING pathway

(Deddouche et al., 2008; Hédelin et al., 2024). Of note, the *Sting* gene was also up-regulated by LZH, although the effect was more consistent in the CrPV infection than in the DCV infection experiment (compare panels E and F in Fig. 2). However, the most striking effect was observed for the IMD-regulated gene *Attacin A*, for which we consistently observed significant induction by LZH at all time points tested and independently of the infection with DCV or CrPV (Fig. 2G–H). Induction of *Attacin A* by LZH was as strong as induction by heat-killed *Escherichia coli* (HKEC), a natural activator of the IMD pathway (Suppl. Fig. 5). Altogether, these results indicate that LZH modulates expression of immunity genes controlled by the IMD and STING pathways in S2 cells.

To directly assess whether activation of IMD and STING pathways could account for the antiviral effect of LZH, we used dsRNA mediated gene silencing to target the expression of key components of the IMD or STING pathways, namely *Sting*, *PGRP-LC*, *imd*, *ird5* or *Relish*. We functionally validated gene silencing by monitoring expression of *Attacin A* and *srg3*, two marker genes regulated by the IMD and STING pathways, respectively (Asling et al., 1995; Cai et al., 2020) (Suppl. Fig. 6). Our results show that the antiviral activity of LZH against CrPV was still

<figure><img src="image_6.png" /><figcaption>**Fig. 3.** – A transcriptomic signature of the cellular responses to LZH.</figcaption></figure>

<figure><img src="image_7.png" /></figure>

A. Gene set enrichment analysis was used to analyze the transcriptome of S2 cells treated with LZH for 8, 24 and 48 h compared to the response to heat killed *E. coli* (HKEC). Biological processes significantly up-regulated in at least two time points of LZH treated cells but not HKEC are shown. Sets of biological processes represented by the same genes were highlighted in pink, representing response to toxic substances, yellow for glutathione metabolism and blue for lipid metabolism. NES: normalized enrichment score.

B-D. Heatmaps representing the relative expression of genes belonging to the processes (B) “lipid metabolism” (highlighted in blue in panel A); (C) “response to toxic substances” (pink in panel A); and (D) “glutathione metabolism” (yellow in panel A). Control, LZH or HKEC treated S2 cells are shown at different time points. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
maintained in cells when key components of the IMD and STING pathways were silenced (Fig. 2I). Intriguingly, targeting some of the canonical IMD components (IMD, PGRP-LC, Relish) reduced the antiviral activity of LZH towards DCV even though it had no effect on CrPV (Fig. 2J). Thus, we tested whether activation of the IMD pathway by a HKEC treatment was sufficient to block viral replication. While LZH significantly inhibited accumulation of CrPV or DCV viral RNA, HKEC had no effect on viral replication (Fig. 2K–L). We conclude that LZH modulates expression of a subset of immune genes regulated by the IMD or STING pathways, but the induction of these genes is not sufficient to explain its antiviral activity against CrPV and DCV.

### 3.3. A transcriptional signature for the response to LZH

To further understand the cellular response to LZH, we performed genome-wide transcriptional response of Drosophila S2 cells treated with LZH by high throughput RNA sequencing. S2 cells treated with HKEC served as controls. This analysis confirmed that the compounds up-regulated expression of several biological processes at 8, 24 and 48h post treatment that are also regulated in response to HKEC (Suppl. Table 2). More importantly, LZH also consistently up-regulated ten biological pathways that were not affected by HKEC during the time-course of the treatment (Fig. 3A). The ten pathways contained overlapping genes and could be collapsed into three distinct groups of processes representing detoxification, glutathione and lipid metabolism (Fig. 3A). Analyzing the expression of individual genes related to the three processes affected by LZH treatment, we observed that several were differentially regulated in cells during the time course of treatment with LZH (Fig. 3B–D). Of note, among the most significant genes upregulated by LZH and not HKEC, we observed two large gene families of Glutathione S-Transferase (Gst) and Cytochrome p450 (Cyp) genes

(Fig. 3C–D). To analyze the regulation of these families in more details, we next looked at the expression of all *Gst* and *Cyp* genes expressed in S2 cells. LZH induced only a subset of *Cyp* genes from Clans 3 and 4 as well as the MITO Clan and *Gst* genes belonging to the delta and epsilon classes (Suppl. Fig. 7) (Gonis et al., 2022; Tijet et al., 2001).

To validate our transcriptome analysis, we chose some of the differentially expressed *Cyp* and *Gst* genes upon LZH treatment representing the glutathione metabolism and detoxification pathways to be tested by RT-qPCR. These experiments performed on independent samples confirmed that LZH up-regulated expression of two cytochrome P450 genes involved in detoxification, *Cyp6a8* and *Cyp6a20*, and two glutathione S-transferase genes, *GstD3* and *GstD5* (Fig. 4), in conditions where replication of DCV and CrPV was inhibited (Suppl. Fig. 8). Overall, our data reveal that LZH induces a unique transcriptional response, which is characterized by upregulation of *Gst* and *Cyp* genes that catalyze chemical modifications of a range of organic compounds and are often associated with detoxification pathways.

### 3.4. LZH increases glutathione S-transferase activity in S2 cells

Induction of gene transcription does not necessarily translate into biological activity. To directly monitor glutathione metabolism, we measured GST activity through conjugation of 1-chloro-2,4-dinitrobenzene (CDNB) with reduced glutathione in S2 cell extracts. In this assay, we observed increased transfer of glutathione in extracts from cells treated with LZH, compared to untreated cells (Fig. 5A). Overall, LZH treatment resulted in significantly increased GST activity in the S2 extracts compared to non-treated cells (Fig. 5B). Thus, our unbiased genome-wide analysis of the transcriptome of S2 cells has identified glutathione metabolism as a physiological process directly impacted by treatment with LZH.

<figure><img src="image_8.png" /><figcaption>**Fig. 4.** – LZH induces expression of *Gst* and *Cyp* genes in response to virus infection.</figcaption></figure>

To validate individual genes regulated by LZH identified in the RNAseq, S2 cells were grown for 24h in absence (NT, non treated) or in presence of 2 mg/mL of LZH and then infected with CrPV (A–D) or DCV (E–H). Levels of *Cyp6a8* (A, E) and *Cyp6a20* (B, F), related to the response to toxic substances or *GstD3* (C, G) and *GstD5* (D, H) related to glutathione metabolic process, were analyzed in non-treated (NT) or LZH-treated cells in presence or not of virus. Values represent the means ± SEM of three independent, experiments (n = 3). Statistical significance was determined using two-way Anova test, \*p < 0.05. \*\*p < 0.01. \*\*\*p < 0.001.
<figure><img src="image_9.png" /><figcaption>Fig. 5. – Upregulation of GST activity induced by LZH.</figcaption></figure>

A. GST activity assays were performed on S2 cell extracts cultured for 24h in media containing 2 mg/mL of LZH or in normal media (NT, non treated). The GST assay measures conjugation of 1-chloro-2,4-dinitrobenzene (CDNB) with reduced glutathione. Conjugation is accompanied by an increase in absorbance at 340 nm, which was measured every minute for 10 min. A sample containing purified GST (GST control) served as a positive control, and non-enzymatic samples (NE control) are used to determine background levels. Absorbance levels per minute are shown in graph A.

B. Total GST activity in control and treated cells. Data of 4 independent replicates are presented as percentage activity relative to purified GST positive control. Statistical significance was determined using unpaired t-test. \*p < 0.05.

### 3.5. A drug inducing expression of detoxifying genes mimics the antiviral effect of LZH against CrPV and DCV

Our analysis suggests that LZH uniquely impacts glutathione metabolism and detoxification in treated cells and these pathways could account for the antiviral activity of the compound. To directly assess this hypothesis, we tested the effects of the antibiotic rifampicin, that has pleotropic effects and is known to induce expression of enzymes of the cytochrome P450 family involved in detoxification pathways (Fanni et al., 2014). S2 cells treated with increasing doses of rifampicin did not show signs of acute toxicity at concentrations up to 100 µM (Suppl. Fig. 9A). At the same time, we observed induction of *Cyp6a8* and *Cyp6a20* 24h and 48h after rifampicin treatment, which was maximal at 100 µM (Suppl. Figs. 9B–C). A correspondent dose-dependent effect on CrPV replication was observed with a maximum inhibition at 100 µM at both 24h and 48h post infection (Fig. 6A–B). We next treated S2 cells with 100 µM rifampicin or 2 mg/mL of LZH for 24h, before challenging them with DCV and CrPV. We observed that rifampicin treatment resulted in reduced DCV replication 24h post infection (Fig. 6C), an inhibitory effect that correlated with increased expression of *Cyp6a8* and *Cyp6a20* (Fig. 6D–E), thus mimicking the effect of LZH. Similar results establishing a parallel between the effects of LZH and rifampicin on CrPV were observed at 24h and 48h post-infection (Fig. 6F–K). Overall, these results support the hypothesis that induction of cytochrome P450 family genes by LZH or rifampicin mediates an antiviral effect against picorna-like viruses.

## 4. Discussion

### 4.1. An antiviral activity in TCM extracts

Here, we report that treatment of macrophage-like S2 cells from *Drosophila* with a TCM compound, LZH, inhibits the replication of two viruses, DCV and CrPV. These viruses belong to the same family, the *Dicistroviridae*, from the order Picornavirales, which includes several human pathogens such as poliovirus. TCM with antiviral activity have been shown to act either as direct antiviral agents, interacting with components of the virus and interfering with their function, or as host targeted agents (Li and Peng, 2013; Lin et al., 2016; Wang et al., 2015; Zhang et al., 2023). Our results suggest that LZH does not directly target steps of the viral replication cycle but rather induces an antiviral state in treated cells. Indeed, most antivirals in herbal traditional medicine act as immunomodulators, either enhancing expression of antiviral cytokines or repressing inflammatory responses (Lin et al., 2016). For many TCMs, it is still unclear how their components are sensed by the immune

system, although dedicated pattern recognition receptors are known to sense polysaccharides and activate signaling pathways (Reis e Sousa et al., 2024; Zhang et al., 2023). In S2 cells, we observed that LZH induces expression of a subset of NF-κB dependent immune markers regulated by the IMD pathway. Interestingly, this pathway bears similarities with the mammalian TNF receptor pathway (Hoffmann, 2003), which has been shown to be regulated by other TCM compounds, e.g. (Chang et al., 2011; Wang et al., 2011). However, we showed that the sole activation of the IMD pathway is not sufficient to protect S2 cells against DCV or CrPV. Rather than the classical targets of this pathway, our unbiased analysis of the transcriptome revealed a complex response to LZH that impacts many biological processes. LZH-regulated pathways included large gene families, such as Cyp and Gst, known for their involvement in stress responses. This broad response makes sense considering that glycans are critical mediators of many biological functions, in particular in the context of host-pathogen interactions (Buchanan et al., 2022; Griffin and Hsieh-Wilson, 2022; Zhou and Cobb, 2021). In addition, at least 94 different chemicals beyond polysaccharides are present in LZH and could contribute to its biological activities (Huang et al., 2022). Future work will be necessary to define which chemical components within LZH, alone or in combination, are activating specific biological pathways.

### 4.2. LZH and the response to biotic and abiotic stress

Cyp and Gst genes, induced by LZH treatment in *Drosophila* cells, are enzymes found in all kingdoms of life function that catalyze chemical modifications of a range of organic compounds and are involved in detoxification at the interface of environmental responses and metabolism (Dermauw et al., 2020; Nauen et al., 2022). Cyp proteins have a heme containing active site and are predominantly monooxygenases catalyzing oxidative transformation of a large set of endogenous and exogenous substrates (Nebert and Dalton, 2006). Gst enzymes catalyze the conjugation of glutathione to a wide variety of endogenous and exogenous electrophilic compounds (Mazari et al., 2023). Modifications promoted by Cyp and Gst enzymes tend to decrease toxicity or promote excretion of toxic compounds. Of note, previous studies established a connection between the genes encoding Cyp and Gst, as several of them were shown to be induced in response to viral infections -including DCV in *Drosophila* (Dostert et al., 2005; Kemp et al., 2013; Lamiable et al., 2016; Merkling et al., 2015; Palmer et al., 2018). These inductions may reflect the oxidative stress associated with the fly response to viruses. A subset of Cytochrome P450s, including the Cyp6 family, and of Gsts are known to metabolize toxins/xenobiotics and to be induced in response to xenobiotics (Gonis et al., 2022; Le Goff et al., 2006; Nauen et al.,
<figure><img src="image_10.png" /></figure>

<figure><img src="image_11.png" /></figure>

<figure><img src="image_12.png" /></figure>

<figure><img src="image_13.png" /></figure>

<figure><img src="image_14.png" /></figure>

<figure><img src="image_15.png" /></figure>

<figure><img src="image_16.png" /></figure>

<figure><img src="image_17.png" /></figure>

<figure><img src="image_18.png" /></figure>

<figure><img src="image_19.png" /></figure>

<figure><img src="image_20.png" /><figcaption>**Fig. 6.** – Upregulation of detoxification pathways is associated with lower viral replication</figcaption></figure>

A-B. S2 cells were treated with 25, 50 or 100 µM of Rifampicin (RFP, in blue) for 24 h, prior to infection with CrPV. Viral RNA levels were measured at 24 h (A) and 48 h (B) post-infection by RT-qPCR. Values represent the means ± SEM of four independent experiments (n = 4).

C-K. S2 cells were treated with 2 mg/mL of LZH or 100 µM of Rifampicin (RFP) for 24 h prior to infection with DCV (C-E) or CrPV (F-K). Viral RNA levels were measured at 24 h (C, F) or 48 h (I) post infection. The levels of Cyp6a8 (D, G, J) and Cyp6a20 (E, H, K) were also determined. Values represent the means ± SEM of six independent experiments (n = 6). Control cells (CTRL) correspond to non-treated cells.

Statistical significance was determined using one-way Anova (Dunnett's multiple comparison test) (A, B, C, F, I) or two-way Anova (Tukey's multiple comparison test) (D, E, G, H, J, K). ns non-significant, \*p < 0.05. \*\*p < 0.01. \*\*\*p < 0.001. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

2022; Vandenhole et al., 2021). These enzymes may modify lipids to prevent formation of viral factories, or regulate inflammatory-like mediators (e.g., eicosanoids) (Nebert and Dalton, 2006). Interestingly, the mechanisms of induction of these genes remain poorly understood. The best characterized examples for their regulation involve the aryl hydrocarbon receptor (AhR) and the Cap"n"collar (CncC, known as Nrf2 in

mammal) transcription factors, which bind to so-called xenobiotic response elements and antioxidant response elements, respectively (Vandenhole et al., 2021). However, it is likely that other regulatory mechanisms involved in Cyp and Gst responses to stress exist, and it will be interesting to see which of these mediates the induction by LZH. In addition, LZH seems to regulate only specific groups of *Gst* and *Cyp*
genes, which suggests some specificity to the response.

### 4.3. On the role of detoxifying enzymes in the antiviral defense

Our results suggest that detoxifying enzymes are associated with the antiviral activity of LZH. This is supported by the results with Rifampicin, a known inducer of detoxifying enzymes (Hou et al., 2022), that showed similar antiviral activity against DCV and CrPV in *Drosophila* cells. The potential use of Rifampicin as an antiviral drug has been noted since the 1970s (Moss et al., 1969) with activity against many viruses including Vaccinia, influenza and SARS-CoV-2 (Panayiotakopoulos and Papadimitriou, 2022). In the context of infections by DNA viruses, Rifampicin inhibit the virus encoded DNA-dependent RNA polymerases (DdRp), similarly to its effects as an antibacterial drug (Campbell et al., 2001). However, Rifampicin also has inhibitory effects on RNA viruses such as Influenza and SARS-CoV-2 and it is unlikely that it acts at the level of the RNA-dependent RNA polymerases (RdRp) since they are structurally very different from DdRps (Iyer et al., 2003). Interestingly, RdRps from RNA viruses and DdRps from DNA viruses both seem to be resistant to rifampicin inhibition in vitro (Hung et al., 2002; Pilotto et al., 2024), suggesting that RNA polymerases are not the target of the antiviral effect in vivo. Rather, our results connect for the first time the induction of detoxifying enzymes and antiviral activity by Rifampicin. Regarding the mechanism, it will be important to investigate how detoxifying enzymes may affect viral infection and how broad this effect may be.

### 4.4. Concluding remarks and perspectives

In summary, we have identified a cellular response to LZH that correlates with the protection against picorna-like viruses. One caveat of our approach is that it relies on a cell line, which may only incompletely reflect the complexity of a multicellular organism with interconnected independent tissues composed of a multitude of cell types (Edwards and Hartung, 2021). We note however that S2 cells have proven a reliable system to experimentally address innate immunity and host-pathogen interactions ex vivo. Therefore, these cells provide a valuable tool to decipher the mode of action of TCM, which are themselves already complex and contain multiple active components (Huang et al., 2022). Nevertheless, experiments in vivo will be important to validate the role of detoxifying enzymes in the antiviral defense as well as identifying the mechanism by which Cyp and Gst proteins affect the replication of DCV and CrPV in *Drosophila*.

Our observations raise several important questions that will have to be addressed in future studies. One of them pertains to the active compound(s) in the LZH capsule that mediate the antiviral activity and the effect on the expression of a subset of Cyp and Gst genes. Identification of these compounds will pave the way to the identification of the cellular receptor(s) that detects them and to the characterization of the signaling pathway(s) they regulate. In parallel, these genes provide markers that will be useful to design reporter assays for rapid screening of new formulae of TCM or other compounds that may have antiviral properties.

#### CRediT authorship contribution statement

**Gabrielle Haas:** Writing – review & editing, Writing – original draft, Validation, Supervision, Project administration, Methodology, Investigation, Formal analysis, Data curation, Conceptualization.
**Mélodie Seiler:** Methodology, Investigation.
**Jenny Nguyen:** Methodology, Investigation.
**Laurent Troxler:** Formal analysis, Data curation.
**Samuel Pennarun:** Methodology, Investigation.
**Elise Lefebvre:** Methodology, Investigation.
**Yasmine Benamrouche:** Methodology, Investigation.
**Loriane Loizeau:** Methodology, Investigation.
**Cody Reinbolt:** Investigation.
**Ming Liang:** Supervision, Project administration.
**Xiaoliang Lin:** Supervision, Project administration.
**Wenzhi Li:** Supervision,

**Project administration.** Zumeng Xia: Resources. **Joao T. Marques:** Writing – review & editing, Writing – original draft, Validation, Supervision, Project administration, Investigation, Formal analysis, Conceptualization.
**Jean-Luc Imler:** Writing – review & editing, Writing – original draft, Validation, Supervision, Investigation, Funding acquisition, Conceptualization.

#### Funding

This project was funded by the Hoffmann Infinitus Program and National Key R&D Program of China (2024YFE0104900). The funders had no role in study design, data collection and analysis.

#### Declaration of competing interest

The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Jean-Luc Imler reports financial support was provided by Infinitus (China) Company Ltd. Joao Trindade Marques reports financial support was provided by Infinitus (China) Company Ltd. Ming Liang, Xiaoliang Lin, Wenzhi Li, Zumeng Xia reports financial support was provided by Infinitus (China) Company Ltd. Jean-Luc Imler reports a relationship with Infinitus (China) Company Ltd that includes: consulting or advisory and funding grants. Joao Trindade Marques reports a relationship with Infinitus (China) Company Ltd that includes: consulting or advisory. Ming Liang, Xiaoliang Lin, Wenzhi Li, Zumeng Xia reports a relationship with Infinitus (China) Company Ltd that includes: employment. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

#### Acknowledgements

We would like to thank Jules Hoffmann for initiating this program and for discussions. We also acknowledge the help of Carine Meignin and other members of our department for advice and suggestions throughout this project. We thank the technical staff for their essential support, particularly Anaïs Hils, whose assistance in preparing specialized media for the flies during the initial phase was instrumental. Additionally, we would like to thank the MGX-Montpellier Genomix platform for the RNA sequencing.

#### Appendix A. Supplementary data

Supplementary data to this article can be found online at https://doi.org/10.1016/j.virol.2025.110513.

#### Data availability

Data will be made available on request.

#### References

Asling, B., Dushay, M.S., Hultmark, D., 1995. Identification of early genes in the Drosophila immune response by PCR-based differential display: the Attacin A gene and the evolution of attacin-like proteins. Insect Biochem. Mol. Biol. 25, 511–518. https://doi.org/10.1016/0965-1748(94)00091-c.

Avadhanula, V., Weasner, B.P., Hardy, G.G., Kumar, J.P., Hardy, R.W., 2009. A novel system for the launch of alphavirus RNA synthesis reveals a role for the Imd pathway in arthropod antiviral response. PLoS Pathog. 5, e1000582. https://doi.org/10.1371/journal.ppat.1000582.

Brown, J.V., Wiles, R., Prentice, G.A., 1979. The effect of a modified Tyndallization process upon the sporeforming bacteria of milk and cream. Int. J. Dairy Technol. 32, 109–112. https://doi.org/10.1111/j.1471-0307.1979.tb01907.x.

Buchanan, C.J., Gaunt, B., Harrison, P.J., Yang, Y., Liu, J., Khan, A., Giltrap, A.M., Le Bas, A., Ward, P.N., Gupta, K., Dumoux, M., Tan, T.K., Schimaski, L., Daga, S., Picchiotti, N., Baldassarri, M., Benetti, E., Fallerini, C., Fava, F., Giliberti, A., Koukos, P.I., Davy, M.J., Lakshminarayanan, A., Xue, X., Papadakis, G., Deimel, L.P., Casablancas-Antrás, V., Claridge, T.D.W., Bonvin, A.M.J.J., Sattentau, Q.J., Furini, S., Gori, M., Huo, J., Owens, R.J., Schaffitzel, C., Berger, I., Renieri, A., Gen-
Covid Multicenter, Study, Naismith J.H., Baldwin, A.J., Davis, B.G., 2022. Pathogen-sugar interactions revealed by universal saturation transfer analysis. Science 377, eabm3125. https://doi.org/10.1126/science.abm3125.

Cai, H., Holleufer, A., Simonsen, B., Schneider, J., Lemoine, A., Gad, H.H., Huang, Jingxian, Huang, Jieqing, Chen, D., Peng, T., Marques, J.T., Hartmann, R., Martins, N.E., Imler, J.-L., 2020. 2'3'-cGAMP triggers a STING- and NF-κB-dependent broad antiviral response in Drosophila. Sci. Signal. 13, eabc4537. https://doi.org/10.1126/scisignal.abc4537.

Campbell, E.A., Korzheva, N., Mustaev, A., Murakami, K., Nair, S., Goldfarb, A., Darst, S. A., 2001. Structural mechanism for rifampicin inhibition of bacterial RNA polymerase. Cell 104, 901–912. https://doi.org/10.1016/S0092-8674(01)00286-0.

Chang, J.S., Wang, K.C., Shieh, D.E., Chiang, L.C., 2011. Liu-He-Tang inhibited plaque formation by human respiratory syncytial virus infection in cell lines of the human respiratory tract. J. Ethnopharmacol. 137, 1149–1155. https://doi.org/10.1016/j.jep.2011.07.043.

Chtarbanova, S., Lamiable, O., Lee, K.-Z., Galiana, D., Troxler, L., Meignin, C., Hetru, C., Hoffmann, J.A., Daeffler, L., Imler, J.-L., 2014. Drosophila C virus systemic infection leads to intestinal obstruction. J. Virol. 88, 14057–14069. https://doi.org/10.1128/JVI.02320-14.

de Faria, I.J.S., Aguiar, E.R.G.R., Olmo, R.P., Alves da Silva, J., Daeffler, L., Carthew, R. W., Imler, J.-L., Marques, J.T., 2022. Invading viral DNA triggers dsRNA synthesis by RNA polymerase II to activate antiviral RNA interference in Drosophila. Cell Rep. 39, 110976. https://doi.org/10.1016/j.celrep.2022.110976.

Deddouche, S., Matt, N., Budd, A., Mueller, S., Kemp, C., Galiana-Arnoux, D., Dostert, C., Antoniewski, C., Hoffmann, J.A., Imler, J.-L., 2008. The DEXD/H-box helicase Dicer-2 mediates the induction of antiviral activity in drosophila. Nat. Immunol. 9, 1425–1432. https://doi.org/10.1016/j.ni.1664.

Dermauw, W., Van Leeuwen, T., Feyereisen, R., 2020. Diversity and evolution of the P450 family in arthropods. Insect Biochem. Mol. Biol. 127, 103490. https://doi.org/10.1016/j.ibmb.2020.103490.

Dobin, A., Davis, C.A., Schlesinger, F., Drenkow, J., Zaleski, C., Jha, S., Batut, P., Chaisson, M., Gingeras, T.R., 2013. STAR: ultrafast universal RNA-seq aligner. Bioinformatics 29, 15–21. https://doi.org/10.1093/bioinformatics/bts635.

Dobson, A.P., Pimm, S.L., Hannah, L., Kaufman, L., Ahumada, J.A., Ando, A.W., Bernstein, A., Busch, J., Daszak, P., Engelmann, J., Kinnaird, M.F., Li, B.V., Loch-Temzelides, T., Lovejoy, T., Nowak, K., Roehrdanz, P.R., Vale, M.M., 2020. Ecology and economics for pandemic prevention. Science 369, 379–381. https://doi.org/10.1126/science.abc3189.

Dolgin, E., 2021. The race for antiviral drugs to beat COVID – and the next pandemic. Nature 592, 340–343. https://doi.org/10.1038/d41586-021-00958-4.

Donnapee, S., Li, J., Yang, X., Ge, A., Donkor, P.O., Gao, X., Chang, Y., 2014. Cuscuta chinensis Lam.: a systematic review on ethnopharmacology, phytochemistry and pharmacology of an important traditional herbal medicine. J. Ethnopharmacol. 157, 292–308. https://doi.org/10.1016/j.jep.2014.09.032.

Dostert, C., Jouanguy, E., Irving, P., Troxler, L., Galiana-Arnoux, D., Hetru, C., Hoffmann, J.A., Imler, J.-L., 2005. The Jak-STAT signaling pathway is required but not sufficient for the antiviral response of drosophila. Nat. Immunol. 6, 946–953. https://doi.org/10.1038/nli237.

Du, X., Lou, N., Hu, S., Xiao, R., Chu, C., Huang, Q., Lu, L., Li, S., Yang, J., 2023. Anti-aging of the nervous system and related neurodegenerative diseases with Chinese herbal medicine. Am J Alzheimers Dis Other Denem 38, 15333175231205445. https://doi.org/10.1177/15333175231205445.

Edwards, A., Hartung, I.V., 2021. No shortcuts to SARS-CoV-2 antivirals. Science 373, 488–489. https://doi.org/10.1126/science.abj4988.

Edwards, A.M., Baric, R.S., Saphire, E.O., Ulmer, J.B., 2022. Stopping pandemics before they start: lessons learned from SARS-CoV-2. Science 375, 1133–1139. https://doi.org/10.1126/science.abn1900.

Fanni, D., Ambu, R., Gerosa, C., Nemolato, S., Castagnola, M., Van Eyken, P., Faa, G., Fanos, V., 2014. Cytochrome P450 genetic polymorphism in neonatal drug metabolism: role and practical consequences towards a new drug culture in neonatology. Int. J. Immunopathol. Pharmacol. 27, 5–13. https://doi.org/10.1177/039463201402700102.

Ferrandon, D., Imler, J.-L., Hetru, C., Hoffmann, J.A., 2007. The Drosophila systemic immune response: sensing and signalling during bacterial and fungal infections. Nat. Rev. Immunol. 7, 862–874. https://doi.org/10.1038/nli2194.

Ferreira, A.G., Naylor, H., Esteves, S.S., Pais, I.S., Martins, N.E., Teixeira, L., 2014. The Toll-dorsal pathway is required for resistance to viral oral infection in Drosophila. PLoS Pathog. 10, e1004507. https://doi.org/10.1371/journal.ppat.1004507.

Galiana-Arnoux, D., Dostert, C., Schneemann, A., Hoffmann, J.A., Imler, J.-L., 2006. Essential function in vivo for Dicer-2 in host defense against RNA viruses in drosophila. Nat. Immunol. 7, 590–597. https://doi.org/10.1038/nli1335.

Gibb, R., Redding, D.W., Chin, K.Q., Donnelly, C.A., Blackburn, T.M., Newbold, T., Jones, K.E., 2020. Zoonotic host diversity increases in human-dominated ecosystems. Nature 584, 398–402. https://doi.org/10.1038/s41586-020-2562-8.

Gonis, E., Fraichard, S., Chertemps, T., Hecker, A., Schwartz, M., Canon, F., Neiers, F., 2022. Expression patterns of Drosophila melanogaster glutathione transferases. Insects 13, 612. https://doi.org/10.3390/insects13070612.

Goto, A., Okado, K., Martins, N., Cai, H., Barbier, V., Lamiable, O., Troxler, L., Santiago, E., Kuhn, L., Paik, D., Silverman, R., Hartmann, R., Liu, J., Peng, T., Hoffmann, J.A., Meignin, C., Daeffler, L., Imler, J.-L., 2018. The Kinase IKKβ regulates a STING- and NF-κB-Dependent antiviral response pathway in Drosophila. Immunity 49, 225–234.e4. https://doi.org/10.1016/j.immuni.2018.07.013.

Griffin, M.E., Hsieh-Wilson, L.C., 2022. Tools for mammalian glycoscience research. Cell 185, 2657–2677. https://doi.org/10.1016/j.cell.2022.06.016.

Gu, Z., Eils, R., Schlesner, M., 2016. Complex heatmaps reveal patterns and correlations in multidimensional genomic data. Bioinformatics 32, 2847–2849. https://doi.org/10.1093/bioinformatics/btw313.

Gu, Z., Hübschmann, D., 2023. simplifyEnrichment: a bioconductor package for clustering and visualizing functional enrichment results. Genom. Proteom. Bioinform. 21, 190–202. https://doi.org/10.1016/j.gpb.2022.04.008.

Hédelin, L., Thiébaut, A., Huang, J., Li, X., Lemoine, A., Haas, G., Meignin, C., Cai, H., Waterhouse, R.M., Martins, N., Imler, J.-L., 2024. Investigating the evolution of Drosophila STING-dependent antiviral innate immunity by multispecies comparison of 2'3'-cGAMP responses. Mol. Biol. Evol. 41, msae032. https://doi.org/10.1093/molbev/msae032.

Hoffmann, J.A., 2003. The immune response of Drosophila. Nature 426, 33–38. https://doi.org/10.1038/nature02021.

Hou, W., Nsengimana, B., Yan, C., Nashan, B., Han, S., 2022. Involvement of endoplasmic reticulum stress in rifampicin-induced liver injury. Front. Pharmacol. 13, 1022809. https://doi.org/10.3389/fphar.2022.1022809.

Huang, M., Yu, S., Shao, Q., Liu, H., Wang, Yingchao, Chen, H., Huang, Y., Wang, Yi, 2022. Comprehensive profiling of Lingzhihuang capsule by liquid chromatography coupled with mass spectrometry-based molecular networking and target prediction. Acupuncture Herbal Med. 2, 58. https://doi.org/10.1097/HM9.00000000000000012.

Hung, M., Gibbs, C.S., Tsiang, M., 2002. Biochemical characterization of rhinovirus RNA-dependent RNA polymerase. Antivir. Res. 56, 99–114. https://doi.org/10.1016/S0166-3542(02)00101-8.

Iyer, L.M., Koonin, E.V., Aravind, L., 2003. Evolutionary connection between the catalytic subunits of DNA-dependent RNA polymerases and eukaryotic RNA-dependent RNA polymerases and the origin of RNA polymerases. BMC Struct. Biol. 3, 1. https://doi.org/10.1186/1472-6807-3-1.

Ji, R., Wang, Z., Kuang, H., 2024. Extraction, purification, structural characterization, and biological activity of polysaccharides from Schisandra chinensis: a review. Int. J. Biol. Macromol. 271, 132590. https://doi.org/10.1016/j.ijbiomac.2024.132590.

Jochmans, D., Laporte, M., Neyts, J., 2023. Antiviral strategies for epidemic and pandemic preparedness. Cell Host Microbe 31, 856–860. https://doi.org/10.1016/j. chom.2023.05.012.

Kemp, C., Mueller, S., Goto, A., Barbier, V., Paro, S., Bonnay, F., Dostert, C., Troxler, L., Hetru, C., Meignin, C., Pfeffer, S., Hoffmann, J.A., Imler, J.-L., 2013. Broad RNA interference-mediated antiviral immunity and virus-specific inducible responses in Drosophila. J. Immunol. 190, 650–658. https://doi.org/10.4049/jimmunol.1102486.

Khong, A., Bonderoff, J., Spriggs, R., Tammpere, E., Kerr, C., Jackson, T., Willis, A., Jan, E., 2016. Temporal regulation of distinct internal ribosome entry sites of the Dicistroviridae cricket paralysis virus. Viruses 8, 25. https://doi.org/10.3390/v8010025.

Lamiable, O., Kellenberger, C., Kemp, C., Troxler, L., Pelte, N., Boutros, M., Marques, J. T., Daeffler, L., Hoffmann, J.A., Roussel, A., Imler, J.-L., 2016. Cytokine Diederl and a viral homologue suppress the IMD pathway in Drosophila. Proc. Natl. Acad. Sci. U. S. A 113, 698–703. https://doi.org/10.1073/pnas.1516122113.

Le Goff, F., Hilliou, F., Siefried, B.D., Boundy, S., Wajnberg, E., Sofer, L., Audant, P., ffrench-Constant, R.H., Feyereisen, R., 2006. Xenobiotic response in Drosophila melanogaster: sex dependence of P450 and GST gene induction. Insect Biochem. Mol. Biol. 36, 674–682. https://doi.org/10.1016/j.ibmb.2006.05.009.

Li, B., Dewey, C.N., 2011. RSEM: accurate transcript quantification from RNA-Seq data with or without a reference genome. BMC Bioinf. 12, 1–16. https://doi.org/10.1186/1471-2105-12-323.

Li, H., Li, W.X., Ding, S.W., 2002. Induction and suppression of RNA silencing by an animal virus. Science 296, 1319–1321. https://doi.org/10.1126/science.1070948.

Li, H., Ye, Z., Zheng, S., Su, Z., 2024. Polysaccharides targeting autophagy to alleviate metabolic syndrome. Int. J. Biol. Macromol. 283, 137393. https://doi.org/10.1016/j.jibiomac.2024.137393.

Li, T., Peng, T., 2013. Traditional Chinese herbal medicine as a source of molecules with antiviral activity. Antivir. Res. 97, 1–9. https://doi.org/10.1016/j. antiviral.2012.10.006.

Lin, L.L., Shan, J.J., Xie, T., Xu, J.Y., Shen, C.S., Di, L.Q., Chen, J.B., Wang, S.C., 2016. Application of traditional Chinese medical herbs in prevention and treatment of respiratory syncytial virus. Evid. base Compl. Alternative Med. 2016, e6082729. https://doi.org/10.1155/2016/6082729.

Liu, J., Zhang, B., Wang, L., Li, S., Long, Q., Xiao, X., 2024. Bioactive components, pharmacological properties and underlying mechanism of Ganoderma lucidum spore oil: a review. Chin Herb Med 16, 375–391. https://doi.org/10.1016/j. chmed.2023.09.007.

Love, M.I., Huber, W., Anders, S., 2014. Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol. 15, 1–21. https://doi.org/10.1186/s13059-014-0550-8.

Lu, L., Su, S., Yang, H., Jiang, S., 2021. Antivirals with common targets against highly pathogenic viruses. Cell 184, 1604–1620. https://doi.org/10.1016/j. cell.2021.02.013.

Luo, W., Zhang, K., Wang, Y., Ye, M., Zhang, Y., Xu, W., Chen, L., Li, H., 2024. The rhizome of Atractylodis macrocephala koidz.: a comprehensive review on the traditional uses, phytochemistry and pharmacology. Chem. Biodivers., e202401879. https://doi.org/10.1002/cbdv.202401879.

Marques, J.T., Meignin, C., Imler, J.-L., 2024. An evolutionary perspective to innate antiviral immunity in animals. Cell Rep. 43, 114678. https://doi.org/10.1016/j. celrep.2024.114678.

Mattioli, L.B., Frosini, M., Corazza, I., Fiorino, S., Zippi, M., Micucci, M., Budriesi, R., 2024. Long COVID-19 gastrointestinal related disorders and traditional Chinese
medicine: a network target-based approach. Phytother Res. 38, 2323-2346. https://doi.org/10.1002/ptr.8163.

Mazari, A.M.A., Zhang, L., Ye, Z.-W., Zhang, J., Tew, K.D., Townsend, D.M., 2023. The multifaceted role of glutathione S-transferases in health and disease. Biomolecules 13, 688. https://doi.org/10.3390/biom13040688.

Merkling, S.H., Overheul, G.J., Van Mierlo, J.T., Arends, D., Gilissen, C., Van Rij, R.P., 2015. The heat shock response restricts virus infection in Drosophila. Sci. Rep. 5, 12758. https://doi.org/10.1038/srep12758.

Moss, B., Rosenblum, E.N., Katz, E., Grimley, P.M., 1969. Rifampicin : a specific inhibitor of Vaccinia virus assembly. Nature 224, 1280-1284. https://doi.org/10.1038/2241280a0.

Mueller, S., Gausson, V., Vodovar, N., Deddouche, S., Troxler, L., Perot, J., Pfeffer, S., Hoffmann, J.A., Saleh, M.-C., Imler, J.-L., 2010. RNAi-mediated immunity provides strong protection against the negative-strand RNA vesicular stomatitis virus in *Drosophila*. Proc. Natl. Acad. Sci. USA. 107, 19390-19395. https://doi.org/10.1073/pnas.1014378107.

Nauen, R., Bass, C., Feyereisen, R., Vontas, J., 2022. The role of cytochrome P450s in insect toxicology and resistance. Annu. Rev. Entomol. 67, 105-124. https://doi.org/10.1146/annurev-ento-070621-061328.

Nebert, D.W., Dalton, T.P., 2006. The role of cytochrome P450 enzymes in endogenous signalling pathways and environmental carcinogenesis. Nat. Rev. Cancer 6, 947-960. https://doi.org/10.1038/nrc2015.

Normile, D., 2015. Nobel for antimalarial drug highlights East-West divide. Science. https://doi.org/10.1126/science.350.6258.265.

Olmo, R.P., Todjro, Y.M.H., Aguiar, E.R.G.R., de Almeida, J.P.P., Ferreira, F.V., Armache, J.N., de Faria, I.J.S., Ferreira, A.G.A., Amadou, S.C.G., Silva, A.T.S., de Souza, K.P.R., Vilela, A.P.P., Babarit, A., Tan, C.H., Diallo, M., Gaye, A., Paupy, C., Obame-Nkoghe, J., Visser, T.M., Koenraadt, C.J.M., Wongsokarijo, M.A., Cruz, A.L. C., Prieto, M.T., Parra, M.C.P., Nogueira, M.L., Avelino-Silva, V., Mota, R.N., Borges, M.A.Z., Drumond, B.P., Kroon, E.G., Recker, M., Sedda, L., Marois, E., Imler, J.-L., Marques, J.T., 2023. Mosquito vector competence for dengue is modulated by insect-specific viruses. Nat Microbiol 8, 135-149. https://doi.org/10.1038/s41564-022-01289-4.

Palmer, W.H., Joosten, J., Overheul, G.J., Jansen, P.W., Vermeulen, M., Obbard, D.J., Van Rij, R.P., 2019. Induction and suppression of NF-κB signalling by a DNA virus of Drosophila. J. Virol. 93, e0144318. https://doi.org/10.1128/JVI.01443-18.

Palmer, W.H., Medd, N.C., Beard, P.M., Obbard, D.J., 2018. Isolation of a natural DNA virus of Drosophila melanogaster, and characterisation of host resistance and immune responses. PLoS Pathog. 14, e1007050. https://doi.org/10.1371/journal.ppat.1007050.

Panayiotakopoulos, G.D., Papadimitriou, D.T., 2022. Rifampicin for COVID-19. WJV 11, 90-97. https://doi.org/10.5501/wjv.v11.i20.

Pilotto, S., Sýkora, M., Cackett, G., Dulson, C., Werner, F., 2024. Structure of the recombinant RNA polymerase from african swine fever virus. Nat. Commun. 15, 1606. https://doi.org/10.1038/s41467-024-45842-7.

Reavy, B., Crump, W.A.L., Moore, N.F., 1983. Characterization of cricket paralysis virus- and *Drosophila* C virus-induced RNA species synthesized in infected *Drosophila melanogaster* cells. J. Invertebr. Pathol. 41, 397-400. https://doi.org/10.1016/0022-2011(83)90206-6.

Reis e Sousa, C., Yamasaki, S., Brown, G.D., 2024. Myeloid C-type lectin receptors in innate immune recognition. Immunity 57, 700-717. https://doi.org/10.1016/j.immuni.2024.03.005.

Sayols, S., 2023. rrvgo: a Bioconductor package for interpreting lists of Gene Ontology terms. MicroPubl. Biol. https://doi.org/10.17912/micropub.biology.000811.

Segrist, E., Dittmar, M., Gold, B., Cherry, S., 2021. Orally acquired cyclic dinucleotides drive dSTING-dependent antiviral immunity in enterocytes. Cell Rep. 37, 110150. https://doi.org/10.1016/j.celrep.2021.110150.

Service, R.F., 2021. A call to arms. Science 371, 1092-1095. https://doi.org/10.1126/science.371.6534.1092.

Shaikh, S., Ahmad, K., Lim, J.H., Ahmad, S.S., Lee, E.J., Choi, I., 2024. Biological insights and therapeutic potential of Glycyrrhiza uralensis and its bioactive compounds: an updated review. Arch Pharm. Res. (Seoul). https://doi.org/10.1007/s12272-024-01522-0.

Soneson, C., Love, M.I., Robinson, M.D., 2016. Differential Analyses for RNA-Seq: Transcript-Level Estimates Improve Gene-Level Inferences. https://doi.org/10.1268/f1000research.7563.2.

Tai, Chen-Jei, Li, C.-L., Tai, Cheng-Jeng, Wang, C.-K., Lin, L.-T., 2015. Early viral entry assays for the identification and evaluation of antiviral compounds. Jove J. 53124. https://doi.org/10.3791/53124-v.

Tijet, N., Helvig, C., Feyereisen, R., 2001. The cytochrome P450 gene superfamily in *Drosophila melanogaster*: annotation, intron-exon organization and phylogeny. Gene 262, 189-198. https://doi.org/10.1016/S0378-1119(00)00533-3.

Vandenhoele, M., Dermauw, W., Van Leeuwen, T., 2021. Short term transcriptional responses of P450s to phytochemicals in insects and mites. Current Opinion in Insect Science, Insect genomics * Development and regulation 43, 117-127. https://doi.org/10.1016/j.cois.2020.12.002.

Wang, J., Li, S., Zhang, H., Zhang, X., 2024. A review of Lycium barbarum polysaccharides: extraction, purification, structural-property relationships, and bioactive molecular mechanisms. Carbohydr. Res. 544, 109230. https://doi.org/10.1016/j.carres.2024.109230.

Wang, K.-C., Chang, J.-S., Chiang, L.-C., Lin, C.-C., 2011. Sheng-Ma-Ge-Gen-Tang (Shoma-kakkon-to) inhibited cytopathic effect of human respiratory syncytial virus in cell lines of human respiratory tract. J. Ethnopharmacol. 135, 538-544. https://doi.org/10.1016/j.jep.2011.03.058.

Wang, L., Yang, R., Yuan, B., Liu, Y., Liu, C., 2015. The antiviral and antimicrobial activities of licorice, a widely-used Chinese herb. Acta Pharm. Sin. B 5, 310-315. https://doi.org/10.1016/j.apsb.2015.05.005.

Wang, S., He, F., Wu, H., Xiang, F., Zheng, H., Wu, W., Li, S., 2023. Health-promoting activities and associated mechanisms of polygonati rhizoma polysaccharides. Molecules 28, 1350. https://doi.org/10.3390/molecules28031350.

Wang, X.-H., Aliyari, R., Li, W.-X., Li, H.-W., Kim, K., Carthew, R., Atkinson, P., Ding, S.-W., 2006. RNA interference directs innate immunity against viruses in adult Drosophila. Science 312, 452-454. https://doi.org/10.1126/science.1125694.

West, C., Rus, F., Chen, Y., Kleino, A., Gangloff, M., Gammon, D.B., Silverman, N., 2019. IIV-6 inhibits NF-κB responses in Drosophila. Viruses 11, 409. https://doi.org/10.3390/v11050409.

West, C., Silverman, N., 2018. p38b and JAK-STAT signaling protect against Invertebrate iridescent virus 6 infection in Drosophila. PLoS Pathog. 14, e1007020. https://doi.org/10.1371/journal.ppat.1007020.

Xiao, Y., Hu, Z., Liu, H., Jiang, X., Zhou, T., Wang, H., Long, H., Li, M., 2024. A review on antitumor effect of pachymic acid. Medicine (Baltim.) 103, e39752. https://doi.org/10.1097/MD.00000000000039752.

Xu, J., Grant, G., Sabin, L.R., Gordesky-Gold, B., Yasunaga, A., Tudor, M., Cherry, S., 2012. Transcriptional pausing controls a rapid antiviral innate immune response in Drosophila. Cell Host Microbe 12, 531-543. https://doi.org/10.1016/j.chom.2012.08.011.

Yu, G., Wang, L.-G., Han, Y., He, Q.-Y., 2012. clusterProfiler: an R Package for comparing biological themes among gene clusters. OMICS A J. Integr. Biol. 16, 284-287. https://doi.org/10.1089/omi.2011.0118.

Zhang, L., Ye, X., Liu, Y., Zhang, Z., Xia, X., Dong, S., 2023. Research progress on the effect of traditional Chinese medicine on the activation of PRRs-mediated NF-κB signaling pathway to inhibit influenza pneumonia. Front. Pharmacol. 14. https://doi.org/10.3389/fphar.2023.1132388.

Zhang, T., Chen, M., Li, D., Sun, Y., Liu, R., Sun, T., Wang, L., 2024. Extraction, purification, structural characteristics, bioactivity and potential applications of polysaccharides from Semen Coicis: a review. Int. J. Biol. Macromol. 272, 132861. https://doi.org/10.1016/j.ijbiomac.2024.132861.

Zhang, Y., Chen, B., Zhang, H., Zhang, J., Xue, J., 2024. Extraction, purification, structural characterization, bioactivities, modifications and structure-activity relationship of polysaccharides from Ophiopogon japonicus: a review. Front. Nutr. 11, 1484865. https://doi.org/10.3389/fnut.2024.1484865.

Zhou, J.Y., Cobb, B.A., 2021. Glycans in immunologic health and disease. Annu. Rev. Immunol. 39, 511-536. https://doi.org/10.1146/annurev-immunol-101819-074237.