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Communication

Cnidium monnieri Polysaccharides Exhibit Inhibitory Effect on Airborne Transmission of Influenza A Virus

by
Heng Wang
1,2,†,
Yifei Jin
2,†,
Yanrui Li
2,
Yan Wang
2,
Yixin Zhao
2,
Shuang Cheng
1,
Zhenyue Li
1,
Mengxi Yan
1,
Zitong Yang
1,
Xiaolong Chen
1,
Yan Zhang
2,
Zhixin Yang
2,
Zhongyi Wang
2,
Kun Liu
2,* and
Ligong Chen
1,*
1
College of Veterinary Medicine, Hebei Agricultural University, Baoding 071000, China
2
Academy of Military Medical Sciences, Academy of Military Sciences, Beijing 100071, China
*
Authors to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Viruses 2026, 18(1), 86; https://doi.org/10.3390/v18010086
Submission received: 20 December 2025 / Revised: 6 January 2026 / Accepted: 7 January 2026 / Published: 8 January 2026
(This article belongs to the Special Issue Influenza and Other Respiratory Viruses: Prevention, Diagnosis, Treatment: 3rd Edition)
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Abstract

Influenza A virus (IAV) continues to present a threat to public health, highlighting the need for safe and multi-target antivirals. In this study, anti-influenza activity, airborne transmission blocking capacity, and immunomodulatory effects of Cnidium monnieri polysaccharides (CMP) were evaluated. Cytotoxicity in A549 cells was assessed by CCK-8 (CC50 = 8.49 mg/mL), antiviral efficacy against A/California/04/2009 (CA04) by dose–response (EC50 = 1.63 mg/mL), and the stage of action by time-of-addition assays (pre-, co-, post-treatment). A guinea pig model infected with CA04 was used for testing the effect of pre-exposure CMP on transmission, with readouts including nasal-wash titers, seroconversion, lung index, and tissue titers (EID50). RT-qPCR was employed to quantify the mRNA expression levels of proinflammatory cytokines, including TNF-α, IL-1β, and IL-6, in lung tissue, while Western blot analysis was performed to assess the expression and phosphorylation status of key proteins involved in the NF-κB signaling pathway. CMP suppressed viral replication in vitro within non-cytotoxic ranges, and pre-treatment—rather than co- or post-treatment—significantly reduced titers and cytopathic effect, consistent with effects at pre-entry steps and/or host priming. In vivo, pre-exposure CMP lowered nasal shedding, reduced aerosol transmission (3/6 seroconverted vs. 6/6 controls), decreased lung indices, and diminished tissue viral loads; IAV was undetectable in trachea at 7 days post-infection in pre-exposed animals, and nasal-turbinate titers declined relative to infection controls. Moreover, during in vivo treatment in mice, CMP significantly suppressed the levels of inflammatory cytokines (TNF-α, IL-1β, and IL-6) in lung tissue. This effect was mechanistically associated with CMP-mediated regulation of the NF-κB signaling pathway, leading to attenuation of inflammatory responses. These data indicate that CMP combines a favorable in vitro safety and efficacy profile with inhibition of airborne spread in vivo, supporting further mechanistic, pharmacokinetic, and fractionation studies toward translational development.

1. Introduction

Influenza A virus (IAV) infects a wide range of hosts and can cross species barriers among birds, mammals, and humans [1,2]. It remains a persistent threat to global public health and causes substantial economic losses to the livestock and poultry industries [3,4,5]. Vaccines and antiviral drugs are the mainstays of prevention and treatment [6,7]. However, vaccine effectiveness depends heavily on accurate strain prediction, while existing antiviral drugs frequently encounter issues of resistance and adverse effects [8,9,10]. Therefore, developing broad-spectrum, low-resistance, and safe antiviral agents remains an urgent necessity [11,12,13]. Natural medicines have attracted increasing attention owing to their multi-target mechanisms and low toxicity [14,15,16]. Among them, Traditional Chinese Medicines (TCM) comprise a large repertoire of bioactive constituents, including polysaccharides, flavonoids, and alkaloids, many of which exhibit antiviral effects against influenza viruses through both direct inhibition and immunomodulatory pathways [17,18].
Excessive cytokine release—cytokine storms and viral pneumonia—often constitute major causes of respiratory injury and mortality in severe influenza patients [19]. Accordingly, modulating cytokine expression is a critical therapeutic strategy for influenza virus infection [20]. The inflammatory response triggered by viral infection relies on the activation and transduction of multiple signaling pathways, with the NF-κB signaling pathway serving as a key mediator in regulating immune and inflammatory responses [21]. As the core transcription factor of the classical NF-κB signaling pathway, p65 serves as a key regulatory protein in cellular responses to pathogen invasion [22]. Under resting conditions, p65 typically binds to the inhibitory protein IκBα and exists in an inactive form within the cytoplasm. When pathogens are recognized by cellular pattern recognition receptors, they activate the upstream IKK complex. IKK subsequently phosphorylates IκBα, leading to its ubiquitination and degradation, thereby releasing p65. p65 activation depends not only on dissociation from IκBα but critically on its own phosphorylation at specific serine residues. Phosphorylated, activated p65 (p-p65) rapidly translocates to the nucleus, binds to κB sites on DNA, and initiates transcription of downstream proinflammatory cytokines (e.g., TNF-α, IL-6), chemokines, and interferons [23]. Accumulating evidence indicates that activation of the NF-κB signaling pathway exacerbates IAV-induced lung injury [14]. Therefore, therapeutic modulation of NF-κB signaling to attenuate inflammatory cytokine production is of particular importance for the development of novel anti-influenza agents.
Aerosol and droplet transmission play a central role in the rapid spread of IAV among humans and animals [24,25]. Fine respiratory particles (<5 µm) exhaled by infected hosts can remain airborne for extended periods, facilitating long-distance dissemination and interspecies transmission [26]. Viral stability and infectivity are strongly influenced by environmental conditions such as temperature and relative humidity, and even short-range aerosol exposure can establish productive infection in mammalian models [27,28]. Airborne IAV has been detected in clinical and agricultural environments, emphasizing the bioaerosol-associated risk of outbreaks in both humans and livestock [29]. Thus, interventions capable of reducing viral shedding and interrupting airborne transmission are of high public-health and veterinary importance [30,31].
Cnidium monnieri (C. monnieri) Cusson (“Shechuangzi”) is a traditional herbal medicine containing abundant coumarins, volatile oils, chromones, flavonoids, and trace elements [32]. Recent pharmacological studies have highlighted its antiviral potential—compound formulations of C. monnieri show synergistic therapeutic effects against viral infections, and its signature coumarins display direct antiviral activities, including inhibition of HSV-1, suppression of HBV secretion, and blockade of HIV-1 Rev nuclear export [33,34,35]. Moreover, C. monnieri demonstrates bronchodilatory and anti-asthmatic properties that may help alleviate influenza-induced respiratory symptoms [36]. Its polysaccharides, the major macromolecular components, have been reported to enhance macrophage activation, humoral immune responses, and T-cell proliferation via upregulating IL-2 and IFN-γ secretion [37,38,39]. However, the anti-influenza potential of C. monnieri polysaccharides (CMP) remains largely unexplored. Here, we evaluated the safety profile, anti-influenza efficacy, capacity to block airborne transmission, and immunomodulatory effects of CMP against influenza A virus.

2. Materials and Methods

2.1. Viruses and Cells

Influenza A virus A/California/04/2009 (H1N1) was provided by the Institute of Microbiology, Chinese Academy of Sciences [40], and amplified in 9-day-old specific pathogen-free (SPF) eggs (Beijing Boehringer Ingelheim Viton Biotechnology Co., Ltd. Beijing, China). The harvested whole-capsid fluid was then inoculated into SPF chicken eggs to determine the egg infectious dose 50% (EID50). The titer of the virus stock solution was determined by the chicken embryo titration method to be 107.45 EID50/mL. A549 cells (CCL-185, ATCC) were cultured in Dulbecco’s Modified Eagle Medium (DMEM, Gibco, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (FBS, Gibco, Grand Island, NY, USA) and 100 U/mL penicillin–streptomycin (P/S) under conditions of 37 °C and 5% CO2. H1N1 virus (A/California/04/2009 (CA04)) was serially diluted 10-fold in DMEM medium containing 2% FBS and 2% dual antibiotics. Each dilution was inoculated into a 96-well plate containing A549 cells at 0.1 mL/well. After incubation at 37 °C for 1 h, the incubation supernatant was discarded, and A549 cell maintenance medium (100 μL/well) was added. After 72 h of incubation at 37 °C in a cell culture incubator, the hemagglutination activity of the cell supernatants in each well was measured according to the WHO hemagglutination test guidelines. The number of agglutinations for each concentration gradient was recorded, and the tissue culture infectious dose 50% (TCID50) of the virus was calculated. EID50 and TCID50 values were calculated using the Reed–Muench method.

2.2. Maximum Non-Toxic Concentration in A549 Cells

CMP were purchased from Moqi Biotechnology Co., Ltd. (Shanghai, China). Detailed information on monosaccharide composition, molecular weight distribution, and sample purity is provided in the Supplementary Materials. A solution containing 2% fetal bovine serum (FBS) and 1% (w/v) penicillin–streptomycin in DMEM medium was prepared. A two-fold serial dilution method was used to create a series of solutions at different concentrations (10.125 mg/mL, 6.75 mg/mL, 4.5 mg/mL, 3 mg/mL, 2 mg/mL, 1.33 mg/mL). A549 cells were revived, passaged 3–5 times to stabilize growth, harvested, and seeded uniformly into 96-well plates in DMEM growth medium containing 10% FBS and 2% (w/v) penicillin–streptomycin. After 24–48 h at 37 °C and 5% CO2, when monolayers reached ~80% confluence, medium was removed, cells were washed twice with PBS containing 2% penicillin–streptomycin, and CMP dilutions were added (100 µL/well; five replicates per concentration). Cell-only (vehicle) and blank (medium-only) controls were included [41].
Cell viability was determined using the CCK-8 assay kit (Beyotime Biotechnology, Shanghai, China) [42]. After 72 h infection, wells were washed twice with PBS (2% penicillin–streptomycin), 100 µL fresh medium was added, and 10 µL CCK-8 reagent was added per well per the manufacturer’s instructions. Plates were incubated for 3 h, and absorbance at 450 nm was recorded. Cell viability was calculated as:
Cell Viability% = [(As − Ab)/(Ac − Ab)] × 100%
As = absorbance of the sample well (cells + culture medium + CCK-8 + test compound); Ac = absorbance of the cell control well (cells + culture medium + CCK-8; no test compound); Ab = absorbance of the blank well (culture medium + CCK-8; no cells, no test compound).
The maximum non-toxic concentration was determined from the concentration–viability relationship.

2.3. In Vitro Antiviral Efficacy and EC50 Assay

Starting from the CC50 of CMP in A549 cells, CMP was each two-fold serially diluted in DMEM containing 2% FBS. For infection, A549 cells (prepared as in Section 2.2) were washed twice with PBS (2% penicillin–streptomycin) and inoculated with 100 TCID50 of H1N1 (100 µL/well). After 1 h adsorption at 37 °C, inoculum was removed and serial drug dilutions were added (five replicates per concentration). Following 72 h incubation, cell viability was determined by CCK-8 (Section 2.2), and the EC50 of CMP was calculated from dose–response curves [43].

2.4. Time-of-Addition (Drug-Timing) Assay

A549 cells were seeded into 96-well plates and cultured to ~90% confluence. The concentration of CMP solution was 1.63 mg/mL. Dextran T-10 (NDC) was purchased from Solarbio Science & Technology Co., Ltd. (Shanghai, China). It was diluted to a concentration of 200 μg/mL using the same DMEM diluent. Oseltamivir (OS) was purchased from yuanye Bio-Technology Co., Ltd. (Shanghai, China) and prepared at a concentration of 100 μg/mL using the same DMEM diluent [44]. Different experimental groups were established:
(i)
Pre-treatment (Pre Cell): Add CMP solution, 100 µL/well, and incubate at 37 °C, 5% CO2 for 2 h. The supernatant was removed, and cells were inoculated with diluted virus (100 TCID50; 100 µL/well) for 1 h at 37 °C. Inoculum was discarded and replaced with drug-free DMEM containing 2% FBS (DMEM–2% FBS), followed by incubation to 72 h.
(ii)
Co-treatment: CMP and virus inoculum were mixed 1:1 (50 µL + 50 µL) and added to cells for 1 h at 37 °C, 5% CO2. The mixture was then replaced with drug-free DMEM–2% FBS and incubated to 72 h.
(iii)
Post-treatment: Cells were first inoculated with virus (100 TCID50; 100 µL/well) for 1 h at 37 °C Remove the viral supernatant and cover with DMEM–2% FBS. After 2 h, remove the supernatant, DMEM–2% FBS containing CMP was added and cells were incubated to 72 h.
Culture supernatants were collected at 12, 24, 36, 48, 60, and 72 h. Viral titers were determined as TCID50 by the Reed–Muench method (as described in Section 2.1). A negative drug control group administered NDC (NDC) at 2 h post-infection and a positive drug control group administered OS (OS) at 2 h post-infection were established. Lesion development in each group was observed at 36 h post-infection.

2.5. Airborne-Transmission Experiment

To assess the impact of CMP on airborne transmission of influenza virus, guinea pigs were evaluated in CMP-treated and untreated control cohorts. The Hedley strain guinea pigs (body weight 300 g–350 g) used in this study were purchased from Vital River Laboratory Animal Technology Co., Ltd. (Beijing, China). All guinea pigs tested negative for IAV antibodies prior to the start of the experiment.
Thirty-six animals were randomized 1:1 to CA04 infection and CMP administration 2 h before infection groups (n = 18 per group) [45,46]. The treated group received CMP by oral gavage 2 h before inoculation (100 µL, 5 mg/mL); the control group received an equivalent volume of PBS 2 h before inoculation. All animals were anesthetized with isoflurane and intranasally inoculated with 100 µL of CA04 virus (106.0 EID50). Animals were then allocated to three cages per group (6 per cage).
At 24 h post-inoculation, an additional 36 healthy guinea pigs were introduced (those corresponding to the treated cohort received CMP 2 h prior to infection, while those in the control cohort received an equal volume of PBS at the same time point). Three transmission models were established per group (n = 6 each): (i) direct-contact transmission, achieved by co-housing with inoculated animals; (ii) aerosol transmission, by housing in an adjacent cage separated by 5 cm (no physical contact); and (iii) indirect-contact transmission, by placing recipients into cages containing used bedding from inoculated animals. Nasal-wash samples were collected from both inoculated donors and recipients at 1, 3, 5, and 7 days post-infection (dpi) [47].
For sampling, animals were anesthetized with isoflurane. One milliliter of PBS containing penicillin–streptomycin was instilled into one nostril, and the effluent was collected from the contralateral nostril as the nasal wash. Samples were labeled and stored at −80 °C. To minimize cross-contamination, recipient animals were sampled first and inoculated donors last [48].
Viral titers (EID50) in nasal washes were determined in SPF embryonated chicken eggs. Frozen samples stored at −80 °C were thawed at 4 °C and subjected to 10-fold serial dilution in PBS containing antibiotics, with vortex mixing at each step. Diluted samples were inoculated into 9–10-day-old SPF eggs (200 µL/egg; 3 eggs per dilution) and incubated at 37 °C. Embryos dying nonspecifically within 24 h were excluded; remaining eggs were harvested at 72 h, and hemagglutination (HA) was assessed. EID50 values were calculated by the Reed–Muench method.

2.6. Hemagglutination-Inhibition (HI) Assay

At 21 dpi, sera were collected from all animals for HI testing following the OIE Manual of Diagnostic Tests and Vaccines for Terrestrial Animals [49].

2.7. Virus Tissue Distribution and Lung Index

Guinea pigs were randomized into two groups (n = 12/group): CA04 infection and CMP administration 2 h before infection groups. The procedure is the same as in 2.5. Both groups inoculated with CA04 virus (106.0 EID50)., At 1, 3, 5, and 7 dpi, three animals per group were euthanized. Lung, nasal turbinates, and trachea were collected, homogenized in 1 mL PBS (tissue homogenizer; QIAGEN, Hilden, Germany), and clarified at 12,000× g, 4 °C, 10 min [50]. Supernatants were stored at −80 °C and titrated for EID50 in eggs as above. An additional 24 guinea pigs were used to establish a blank control group receiving only CMP and a blank control group receiving only PBS, with 12 animals in each group. Lung index (lung weight/body weight) was determined on the same days to assess disease severity.

2.8. Effects of CMP on Cytokine-Related mRNAs in Virus-Infected Mouse Lungs

The experiment utilized 140 female Balb/c mice weighing 18–20 g, purchased from Vital River Laboratory Animal Technology Co., Ltd. (Beijing, China). During the study period, mice were housed in an IVC system and provided with standard chow and water. The 140 mice were randomly divided into 7 groups of 20 mice each: negative control group (NC), virus-infected group (PC), negative drug treatment group (NDC), oseltamivir treatment group (OS), low-dose Cnidium monnieri polysaccharide treatment group (CMP-L), medium-dose Cnidium monnieri polysaccharide treatment group (CMP-M), and high-dose Cnidium monnieri polysaccharide treatment group (CMP-H). Mice in all groups except the NC group were lightly anesthetized with isoflurane and intranasally inoculated with 50 μL of H1N1 subtype influenza A virus (CA04, 106.0 EID50) per mouse. Mice in the NC group received intranasal inoculation with PBS at the same dose. All groups of mice received treatment starting on the day of infection, 2 h after establishing the influenza virus-infected mouse model, with continuous treatment for 5 days. Treatment regimens were as follows: CMP-L, CMP-M, and CMP-H groups received CMP at concentrations of 2 mg/mL, 5 mg/mL, and 10 mg/mL, respectively, at 50 μL per mouse; OS group received OS treatment at a dose of 20 mg/kg, diluted in phosphate-buffered saline (PBS) at a concentration of 5 mg/mL [51]; NDC group received NDC administration. NDC was diluted to a concentration of 10 mg/mL in phosphate-buffered saline (PBS) and administered at a dose of 50 μL per animal.
NC group and PC group mice were administered PBS at 100 μL per animal. All treatments were administered via oral gavage once daily at the same time each day. Five mice were randomly selected from each group at 1, 3, 5, and 7 dpi, euthanized by decapitation, the collected mouse lung tissue was divided into two portions. The first portion was homogenized and subjected to viral load determination (EID50), calculated using the Reed–Muench method. The second portion was used for RNA extraction and subsequent measurement of cytokine-related mRNA expression levels. Total RNA was extracted from mouse lung tissue using the RNeasy Plus Universal Kit (73404, Qiagen, Germany). Reverse transcription was performed using the Prime Script™ RT Kit (RR047A, Takara, Dalian, China). Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) was performed using Power SYBR™ Green PCR Master Mix. RT-qPCR was conducted on an ABI 7500 Fast Real-Time PCR System (Thermo Fisher Scientific, Waltham, MA, USA). The RT-qPCR program consisted of 50 °C for 2 min, 95 °C for 10 min, 40 cycles: 95 °C for 15 s, 60 °C for 1 min. Data analysis employed the 2−ΔΔCt method. PCR primers synthesized by Sangon Biotech Co., Ltd. (Shanghai, China). Sequences as follows: TNF-α forward, 5′-CCCTCACACTCAGATCATCTTCT-3′; TNF-α reverse, 5′-GCTACGACGTGGGCTACAG-3′; IL-1β forward, 5′-GCAACTGTTCCTGAACTCAACT-3′; IL-1β reverse, 5′-ATCTTTTGGGGTCCGTCAACT-3′; IL-6 forward, 5′-TAGTCCTTCCTACCCCAATTTCC-3′; IL-6 reverse, 5′-TTGGTCCTTAGCCACTCCTTC-3′; GAPDH forward, 5′-AGGTCGGTGTGAACGGATTTG-3′; GAPDH reverse, 5′-TGTAGACCATGTAGTTGAGGTCA-3′.

2.9. Regulatory Effects of CMP on NF-κB p65 and p-NF-κB p65 Protein Expression in Mouse Lung Tissue

A total of 35 female Balb/c mice were randomly divided into 7 groups of 5 mice each. Following the grouping method described in Section 2.8, they were randomly divided into 7 groups of 5 mice each, following the grouping method described in Section 2.8. An influenza virus-infected mouse model was established via intranasal inoculation, using the same inoculation method as in Section 2.8. Two hours after establishing the influenza virus-infected mouse model, mice in each group began treatment on the day of infection, continuing for 5 consecutive days. Treatment and administration methods were identical to those in Section 2.8. At 5 dpi, five mice from each group were sacrificed. Lung tissue was collected from these mice and lysed using RIPA buffer (Epizyme, Shanghai, China) to extract total protein from mouse lung tissue. Protein concentration was measured using the BCA Protein Quantification Kit (Beyotime Biotechnology, Shanghai, China). Equal amounts of protein were subjected to SDS-PAGE electrophoresis and transferred to PVDF membranes (Merck Millipore, Burlington, MA, USA). Subsequently, membranes were incubated overnight with mouse monoclonal antibodies: NF-κB p65 (1:3000, Proteintech, Manchester, IL, USA), phosphorylated NF-κB p65 (1:5000, Proteintech, Manchester, USA), and GAPDH (1:8000, Proteintech, Manchester, USA), followed by detection with HRP-conjugated goat anti-rabbit or anti-mouse IgG. Bands were visualized by Clarity Western ECL Substrate (Bio-Rad, Hercules, CA, USA) in a luminescent image analyzer. The protein expression levels were quantified using ImageJ software (version 1.54g; National Institutes of Health, Bethesda, MD, USA) and normalized with GAPDH. The level of protein phosphorylation was determined as the ratio of gray values for phosphorylated protein to total protein.

2.10. Statistical Analysis

Data are presented as mean ± SD. Analyses were performed using GraphPad Prism 10.5.0 (GraphPad Prism software, Inc San Diego, CA, USA). ANOVA with appropriate post hoc multiple-comparisons tests was used for continuous data, and viral titers were log10-transformed when applicable. Proportions (e.g., transmission/seroconversion rates) were compared using Fisher’s exact test. All tests were two-tailed; p < 0.05 was considered significant.

3. Results

3.1. Cytotoxicity and Antiviral Efficacy of CMP

CMP was evaluated for cytotoxicity in A549 cells using the CCK-8 assay across graded concentrations to define a safe working range and an effective anti-influenza dose. CMP exhibited a CC50 of 8.49 mg/mL (95% CI: 8.25–8.73 mg/mL) in A549 cells (Figure 1A). Within the non-cytotoxic range, treatment of H1N1 (CA04)–infected A549 cells with CMP suppressed viral replication in a concentration-dependent manner, yielding an EC50 of 1.63 mg/mL (95% CI: 1.43–1.82 mg/mL) (Figure 1B).

3.2. Time-of-Addition Assay

Experimental results demonstrated that pre-treatment (CMP administration 2 h before CA04 infection) significantly reduced viral titers at all examined time points (Figure 1C). At 36 h post-infection, co-treatment (CMP administration along with CA04 infection) and post-treatment (CMP administration 2 h after CA04 infection) also significantly decreased viral titers, with pre-treatment showing the most pronounced inhibitory effect. These findings suggest that CMP may exert antiviral activity during the early phase of infection; however, further mechanistic studies are needed to determine the specific stage of the viral life cycle targeted by CMP.

3.3. CMP Reduces Aerosol Transmissibility of Influenza Virus

In the CA04 infection group exposed to aerosol transmission, CA04 virus was detectable in nasal lavage fluid from all guinea pigs at 1, 3, 5, and 7 dpi (Figure 2A). In contrast, no viral titers were detected in nasal lavage fluid from guinea pigs in the CMP-administered group exposed to aerosol transmission at 1 dpi, and viral titers were detected only in nasal lavage fluid from 3 guinea pigs (Figure 2B). CMP reduced the aerosol transmission rate of CA04 from 100% (6/6) to 50% (3/6). This indicates that CMP can decrease the aerosol transmission efficiency of influenza viruses.

3.4. CMP Lowers Seroconversion in the Aerosol-Exposed Group

In the aerosol-exposed cohort, antibodies were detected in the serum of all guinea pigs in the CA04 infection group, whereas antibodies were detected in the serum of only 3 guinea pigs in the CMP-administered group, CMP reduced the aerosol transmission rate of CA04 from 100% (6/6) to 50% (3/6) (Figure 2C), consistent with the reduced nasal-wash titers noted above.

3.5. CMP Decreases the Lung Index in Infected Guinea Pigs

Lung index was measured at 1, 3, 5, and 7 dpi. The CA04 infection group showed significantly elevated lung index at 3, 5, and 7 dpi versus negative controls (Figure 2D). Pre-exposure administration of CMP significantly lowered the lung index relative to the infection group, indicating in vivo protection.

3.6. CMP Decreases Viral Replication in the Nasal Turbinates

In the respiratory tracts of infected guinea pigs, viral titers in the nasal turbinates of the CA04 infection group showed an increasing trend from 1 to 3 dpi, followed by a decreasing trend from 5 to 7 dpi (Figure 2E). In contrast, viral titers in the CMP-administered group exhibited a consistent downward trend and remained consistently lower than those in the CA04 infection group (Figure 2F). This suggests that CMP may suppress viral replication in the nasal turbinates.

3.7. Effects of CMP on Cytokines in the Lungs of Infected Mice

As shown in Figure 3A, at 3 dpi, the viral titer in lung tissue of the PC group was significantly higher than that in the CMP-H group (p < 0.001). Compared with the PC group, viral titers were reduced to varying degrees in all three treatment groups (CMP-L, CMP-M, CMP-H). At 1, 3, 5, and 7 dpi, expression levels of TNF-α (B), IL-1β (C), and IL-6 (D) in lung tissue were significantly higher in PC group mice than in the NC group (p < 0.001). No significant differences were observed between the NDC group and PC group. Compared with the PC group, all four treatment groups (OS, CMP-L, CMP-M, and CMP-H) exhibited varying degrees of suppression in TNF-α, IL-1β, and IL-6 expression. Cytokine expression levels significantly increased with infection duration, with oseltamivir and CMP-M exerting the most pronounced effects on cytokine levels.

3.8. The Regulatory Role of CMP in the NF-κB Signaling Pathway

As shown in Figure 3E,F, compared with the NC group, the PC group exhibited significantly elevated NF-κB p65 protein phosphorylation levels (p < 0.001). Compared with the PC group, the NF-κB p65 protein phosphorylation levels were significantly reduced in both the OS group and the treatment groups (CMP-L and CMP-M groups) (p < 0.05, p < 0.001), with the most pronounced reduction observed in the OS group and CMP-M group. These findings suggest that the NF-κB signaling pathway may play a crucial role in the immune modulation of CMP hosts.

4. Discussion

The present study demonstrated that CMP significantly inhibited replication of the CA04 influenza A virus in vitro and in vivo and partially blocked airborne transmission in a guinea pig model. These findings position CMP as a natural, low-toxicity antiviral candidate for prevention and control of influenza infection and spread, complementing current vaccine and drug-based strategies [17,52]. The transmission-blocking signal in our model aligns with the central role of airborne spread in influenza epidemiology and pathogenesis [1,53].
Aerosol interruption is particularly consequential because airborne spread is a major driver of IAV dissemination across hosts and settings [54]. Fine respiratory particles (<5 µm) can remain suspended for prolonged periods, traverse short to intermediate distances, and deposit deeply in the lower respiratory tract, thereby increasing infection probability and disease severity [55]. Environmental conditions—especially temperature and relative humidity—modulate virion stability and transmissibility, further amplifying outbreak risk in seasonally favorable climates and enclosed environments [56]. Airborne IAV and other respiratory viruses have been directly detected in exhaled breath and high-risk venues such as live-poultry markets, linking bioaerosol burden to human and animal exposure [27]. Ferret and guinea-pig models demonstrate that strictly airborne exposure suffices to initiate productive infection, underscoring aerosols as a self-sustaining transmission route independent of contact or fomites [57,58]. Treatment with Lentinan (LNT) significantly reduced viral shedding in the nasal cavity, decreased the transmission efficiency of SARS-CoV-2 aerosols, and potentially lowered the infectiousness of affected individuals [45]. Interventions that lower emitted or inhaled viral dose—whether by reducing upper-airway shedding or enhancing mucosal defenses—are therefore expected to curb onward spread [59]; our finding that CMP reduces nasal shedding and seroconversion in the aerosol-exposed cohort is consistent with this paradigm and suggests potential utility as a complementary, low-toxicity countermeasure at the human–animal interface.
Polysaccharides, as a class of important biomacromolecules, exhibit remarkable “multi-target” activity in the field of biomedicine. Extensive scientific research confirms that polysaccharides not only directly inhibit various viruses but also broadly modulate the immune system, exerting clear protective effects in infection or injury models. Houttuynia cordata polysaccharides enhance survival rates in H1N1-infected mice, mitigate lung and intestinal damage, reduce viral replication, suppress inflammatory responses, protect the intestinal barrier, and regulate mucosal immunity [60]. The antiviral effects of Isatis root polysaccharides are closely linked to their inhibition of TLR-3 signaling pathway activation, thereby mitigating the upregulation of pro-inflammatory factors induced by influenza viruses [61]. Astragalus polysaccharides primarily function by blocking viral adsorption. They also activate macrophages, enhancing phagocytosis, and induce the production of multiple cytokines including interleukin-1 (IL-1) and tumor necrosis factor-alpha (TNF-α). This significantly improves clinical symptoms and lung damage in mice infected with H1N1 virus [62]. Schisandrin polysaccharides have been shown to interfere with the nuclear export process of viral nucleoproteins, thereby inhibiting viral replication [63]. This study revealed that beyond direct antiviral effects, CMP significantly suppressed H1N1 virus-induced p65 phosphorylation. This suggests that CMP may mitigate virus-induced inflammatory damage not only by directly inhibiting viral replication but also by intervening in the excessive activation of the NF-κB signaling pathway. (Figure 4). This provides potential molecular evidence for its “multi-target” antiviral and immunomodulatory effects, addressing the limitations of single-target antiviral drugs [46,47,48]. This characteristic not only broadens the spectrum of action for Chinese herbal polysaccharides but also lays the foundation for developing novel natural antiviral drugs. Such adjunctive strategies are particularly relevant given the historical emergence of resistance to neuraminidase inhibitors and the variable clinical performance of existing agents under field conditions [9,64]. By lowering viral load and reducing emission or susceptibility at the host level [65], CMP may contribute to reduced transmission efficiency, echoing evidence that interventions diminishing exhaled virus can curb spread [66,67,68].
This study suggests that CMP modulates the NF-κB pathway in an influenza virus infection model. However, whether this regulatory effect is specific to virus-induced inflammation or inherent to CMP’s broad-spectrum anti-inflammatory properties remains to be elucidated [69]. Future research should compare CMP’s impact on NF-κB and related inflammatory pathways under various classical pro-inflammatory stimuli (e.g., LPS, TNF-α) to clarify its spectrum of action and potential targets [70,71]. This work is crucial for precisely defining CMP’s clinical application scenarios, such as its specialized use for viral pneumonia or as a broad-spectrum anti-inflammatory agent. The study found that CMP’s in vitro cytotoxic concentration (CC50) is significantly higher than its effective in vivo dose. This discrepancy stems from the fundamental differences between static high exposure in vitro and dynamic absorption, metabolism, and immune regulation in vivo [72]. In vivo experiments revealed that CMP’s inhibition of NF-κB p65 phosphorylation did not exhibit strict linear dose dependency across tested concentrations (Figure 3F). This likely reflects its complex properties as a natural polysaccharide immunomodulator—balancing immune network regulation across different doses rather than exhibiting simple linear suppression or toxicity [73]. Pharmacodynamically, effect saturation may exist, with the medium dose (CMP-M) approaching maximum inhibitory efficacy. Additionally, as a crude macromolecular polysaccharide extract, its oral absorption and tissue distribution may exhibit pharmacokinetic saturation, limiting linear increases in target site concentrations at higher doses. Finally, complex interactions among CMP’s multiple components may contribute to non-linear synergistic effects [74]. Future studies should elucidate the mechanism underlying this nonlinear relationship through component purification and determine its optimal therapeutic window. The currently low selectivity indices (EC50/CC50) clearly indicate the direction for future optimization: namely, enriching or purifying single or homogeneous polysaccharide components bearing antiviral activity from CMP through activity-directed stepwise separation (such as ion exchange chromatography and gel filtration chromatography) [75,76]. Expanded in vivo studies—covering toxicity, immunogenicity, durability of protection, and head-to-head comparisons with standard of care—are warranted to support clinical translation. Mechanistic transmission studies (e.g., staged time-of-addition, contact vs. aerosol partitions, and quantification of emitted virus) will be valuable to confirm mode of action within established influenza transmission frameworks [53,66,77]. Collectively, these investigations will clarify CMP’s potential as an effective and safe natural antiviral for influenza and possibly other respiratory viruses.

5. Conclusions

Cnidium monnieri polysaccharides (CMP) showed measurable anti-influenza activity, inhibiting CA04 replication in vitro within non-cytotoxic ranges. In a guinea pig model, pre-exposure CMP reduced nasal shedding and partially blocked airborne transmission, as reflected by decreased seroconversion in exposed animals and reduced tissue viral loads. CMP also attenuated infection-associated inflammatory responses, suppressing TNF-α/IL-1β/IL-6 expression and reducing NF-κB p65 phosphorylation in vivo. Collectively, these findings support CMP as a promising low-toxicity, multi-target candidate for influenza prevention and transmission mitigation, warranting further fractionation/characterization and mechanistic–translational studies.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/v18010086/s1, Table S1 Monosaccharide composition of Cnidium monnieri polysaccharides; Table S2 Molecular Weight Results of Cnidium monnieri polysaccharides; Figure S1 HPGPC Chromatogram of Cnidium monnieri polysaccharides; Table S3 Purity of Cnidium monnieri polysaccharides.

Author Contributions

Conceptualization, Z.W. and L.C.; methodology, H.W., K.L. and Y.J.; software, S.C., Z.L., M.Y., Z.Y. (Zitong Yang) and X.C.; validation, Y.Z. (Yan Zhang) and Z.Y. (Zhixin Yang); formal analysis, Y.L., Y.W. and Y.Z. (Yixin Zhao); investigation, Y.L., Y.W. and Y.Z. (Yixin Zhao); resources, Z.W. and Y.J.; data curation, Z.W. and Y.J.; writing—original draft preparation, H.W., Z.W. and Y.J.; writing—review and editing, H.W., Z.W. and Y.J.; visualization, Z.W. and Y.J.; supervision, Z.W. and L.C.; project administration, Z.W.; funding acquisition, Z.W. All authors have read and agreed to the published version of the manuscript.

Funding

This work was funded by the National Natural Science Foundation of China (General Program: 82472282) and Beijing Nova Program (20250484981).

Institutional Review Board Statement

This study was conducted in strict accordance with the recommendations in the “Guidelines for the Care and Use of Laboratory Animals” by the Ministry of Science and Technology of China. The protocol was approved by the Animal Experiment Ethics Committee of Academy of Military Medical Sciences (approval number: IACUC-SWGCYJS-2024-005, approval date: 10 May 2024). All virus experiments were performed in biosafety laboratories in the Academy of Military Medical Sciences.

Data Availability Statement

The data supporting the findings of this study are available from the corresponding authors upon reasonable request.

Conflicts of Interest

The authors declare no conflicts of interest.

Abbreviations

The following abbreviations are used in this manuscript:
IAVInfluenza A virus
CMPCnidium monnieri polysaccharides
HSVHerpes simplex virus
HBVHepatitis B virus
HIVHuman immunodeficiency virus
DMEMDulbecco’s Modified Eagle Medium
FBSFetal Bovine Serum
CCK-8Cell Counting Kit-8
CC50Median cytotoxic concentration
EC50Median effect concentration
EID50Median infective dose
TCID50Median tissue culture infectious dose
WHOWorld Health Organization
SPFSpecific pathogen free
TCMTraditional Chinese Medicines
HIHemagglutination inhibition

References

  1. Neumann, G.; Kawaoka, Y. Transmission of influenza A viruses. Virology 2015, 479–480, 234–246. [Google Scholar] [CrossRef]
  2. Shi, Y.; Li, Y.; Li, H.; Haerheng, A.; Marcelino, V.R.; Lu, M.; Lemey, P.; Tang, J.; Bi, Y.; Pettersson, J.H.; et al. Extensive cross-species transmission of pathogens and antibiotic resistance genes in mammals neglected by public health surveillance. Cell 2025, 188, 6591–6605. [Google Scholar] [CrossRef]
  3. Iuliano, A.D.; Roguski, K.M.; Chang, H.H.; Muscatello, D.J.; Palekar, R.; Tempia, S.; Cohen, C.; Gran, J.M.; Schanzer, D.; Cowling, B.J.; et al. Estimates of global seasonal influenza-associated respiratory mortality: A modelling study. Lancet 2018, 391, 1285–1300. [Google Scholar] [CrossRef] [PubMed]
  4. Yang, J.; Gong, Y.; Zhang, C.; Sun, J.; Wong, G.; Shi, W.; Liu, W.; Gao, G.F.; Bi, Y. Co-existence and co-infection of influenza A viruses and coronaviruses: Public health challenges. Innovation 2022, 3, 100306. [Google Scholar] [CrossRef]
  5. Chang, Y.; Shan, Z.; Shi, W.; Li, Q.; Wang, Y.; Wang, B.; Wang, G.; Chen, H.; Jiang, L.; Li, C. KRT6A Restricts Influenza A Virus Replication by Inhibiting the Nuclear Import and Assembly of Viral Ribonucleoprotein Complex. Viruses 2025, 17, 671. [Google Scholar] [CrossRef]
  6. Meng, F.; Cheng, Z.; Feng, Z.; Zhang, Y.; Zhang, Y.; Wang, Y.; Zhai, Y.; Kuang, P.; Qu, R.; Chen, Y.; et al. H128N Substitution in the Sa Antigenic Site of HA1 Causes Antigenic Drift Between Eurasian Avian-like H1N1 and 2009 Pandemic H1N1 Influenza Viruses. Viruses 2025, 17, 1360. [Google Scholar] [CrossRef]
  7. Han, X.; Ma, R.; Ma, X.; Chen, H.; Zhao, T.; Liu, X.; Zhu, L.; Yuan, L.; Shi, Y.; Zhang, Y.; et al. Development of a broad-spectrum nasal live attenuated influenza vaccine based on mosaic antigen design against influenza B viruses. Emerg. Microbes Infect. 2025, 14, 2576141. [Google Scholar] [CrossRef] [PubMed]
  8. Paules, C.; Subbarao, K. Influenza. Lancet 2017, 390, 697–708. [Google Scholar] [CrossRef] [PubMed]
  9. Moscona, A. Global transmission of oseltamivir-resistant influenza. N. Engl. J. Med. 2009, 360, 953–956. [Google Scholar] [CrossRef]
  10. Ayora-Talavera, G.; López-Martínez, I.; Barrera-Badillo, G.; Aparicio-Antonio, R.; Aréchiga-Ceballos, N.; Aguirre-Barbosa, A.; Wong-Chew, R.M.; Canul-Canul, D.; Solís-Hernández, M. Genetic and Serological Analysis of H7N3 Avian Influenza Viruses in Mexico for Pandemic Risk Assessment. Viruses 2025, 17, 1376. [Google Scholar] [CrossRef]
  11. Duwe, S.C.; Milde, J.; Heider, A.; Wedde, M.; Schweiger, B.; Dürrwald, R. Increase of Synergistic Secondary Antiviral Mutations in the Evolution of A(H1N1)pdm09 Influenza Virus Neuraminidases. Viruses 2024, 16, 1109. [Google Scholar] [CrossRef]
  12. Xu, X.; Wang, S.; Zhang, Y.; Xie, H.; Hu, J.; Wang, Z.; Liu, Y.; Huang, L. Preparation of fucoidan oligosaccharides from Sargassum fusiforme using the “layer by layer” degradation strategy and their binding study with HA proteins of influenza A viruses. Carbohydr. Polym. 2025, 368, 124222. [Google Scholar] [CrossRef]
  13. Xu, Y.; Anirudhan, V.; Gaisina, I.N.; Du, H.; Alqarni, S.; Moore, T.W.; Caffrey, M.; Manicassamy, B.; Zhou, T.; Rong, L.; et al. Mechanistic insights into the small-molecule inhibition of influenza A virus entry. Proc. Natl. Acad. Sci. USA 2025, 122, e2503899122. [Google Scholar] [CrossRef]
  14. Cui, H.; Zhang, C.; Zhang, C.; Cai, Z.; Chen, L.; Chen, Z.; Zhao, K.; Qiao, S.; Wang, Y.; Meng, L.; et al. Anti-Influenza Effect and Mechanisms of Lentinan in an ICR Mouse Model. Front. Cell. Infect. Microbiol. 2022, 12, 892864. [Google Scholar] [CrossRef] [PubMed]
  15. Gan, Y.; Guo, F.; Roy, A.; Wang, X.; Shen, Y. Integrated In Silico, In Vitro, and In Vivo Studies Reveal Mangiferin as a Promising Antiviral Agent Against H1N1/pdm2009 Influenza Virus. Viruses 2025, 17, 873. [Google Scholar] [CrossRef] [PubMed]
  16. Zhang, C.; Yuan, M.; Rong, W.; Du, H.; Li, X.; Ji, T.; Li, J.; Dai, B.; Ma, Z.; Qi, H.; et al. Synergistic effects of Lianhuaqingwen in combination with Oseltamivir and Baloxavir against seasonal influenza virus: In vitro and in vivo assessment. J. Ethnopharmacol. 2025, 338, 119091. [Google Scholar] [CrossRef] [PubMed]
  17. Krammer, F.; Smith, G.J.D.; Fouchier, R.A.M.; Peiris, M.; Kedzierska, K.; Doherty, P.C.; Palese, P.; Shaw, M.L.; Treanor, J.; Webster, R.G.; et al. Influenza. Nat. Rev. Dis. Primers 2018, 4, 3. [Google Scholar] [CrossRef]
  18. Fan, P.; Lv, P.; Zhang, S.; Zhu, Z.; Qian, K.; Han, J.; Cui, Y.; Feng, Y.; Li, Z.; Qiang, L.; et al. Licoflavone B Suppresses Influenza A Virus by Targeting the Viral RNA-Dependent RNA Polymerase (RdRp). Viruses 2025, 17, 1157. [Google Scholar] [CrossRef]
  19. Cui, X.R.; Guo, Y.H.; Liu, Q.Q. Cangma Huadu granules, a new drug with great potential to treat coronavirus and influenza infections, exert its efficacy through anti-inflammatory and immune regulation. J. Ethnopharmacol. 2022, 287, 114965. [Google Scholar] [CrossRef]
  20. Aldridge, J.R., Jr.; Moseley, C.E.; Boltz, D.A.; Negovetich, N.J.; Reynolds, C.; Franks, J.; Brown, S.A.; Doherty, P.C.; Webster, R.G.; Thomas, P.G. TNF/iNOS-producing dendritic cells are the necessary evil of lethal influenza virus infection. Proc. Natl. Acad. Sci. USA 2009, 106, 5306–5311. [Google Scholar] [CrossRef] [PubMed]
  21. Flory, E.; Kunz, M.; Scheller, C.; Jassoy, C.; Stauber, R.; Rapp, U.R.; Ludwig, S. Influenza virus-induced NF-κB-dependent gene expression is mediated by overexpression of viral proteins and involves oxidative radicals and activation of IκB kinase. J. Biol. Chem. 2000, 275, 8307–8314. [Google Scholar] [CrossRef] [PubMed]
  22. Ma, Q.H.; Ren, M.Y.; Luo, J.B. San Wu Huangqin decoction regulates inflammation and immune dysfunction induced by influenza virus by regulating the NF-κB signaling pathway in H1N1-infected mice. J. Ethnopharmacol. 2021, 264, 112800. [Google Scholar] [CrossRef] [PubMed]
  23. Li, J.; Jie, X.; Liang, X.; Chen, Z.; Xie, P.; Pan, X.; Zhou, B.; Li, J. Sinensetin suppresses influenza a virus-triggered inflammation through inhibition of NF-κB and MAPKs signalings. BMC Complement. Med. Ther. 2020, 20, 135. [Google Scholar] [CrossRef]
  24. Meng, F.; Yang, H.; Qu, Z.; Chen, Y.; Zhang, Y.; Zhang, Y.; Liu, L.; Zeng, X.; Li, C.; Kawaoka, Y.; et al. A Eurasian avian-like H1N1 swine influenza reassortant virus became pathogenic and highly transmissible due to mutations in its PA gene. Proc. Natl. Acad. Sci. USA 2022, 119, e2203919119. [Google Scholar] [CrossRef]
  25. Wang, C.C.; Prather, K.A.; Sznitman, J.; Jimenez, J.L.; Lakdawala, S.S.; Tufekci, Z.; Marr, L.C. Airborne transmission of respiratory viruses. Science 2021, 373, 6558. [Google Scholar] [CrossRef] [PubMed]
  26. Zhang, Y.; Li, J.; Jiang, L.; Chen, Q.; Fu, Y.; Jin, Y.; Chen, Z.; Tang, F.; Zeng, X.; Wen, H.; et al. Comparison of SARS-CoV-2 aerosol emission from patients with Omicron BA.1 or BA.2 subvariant infection. J. Infect. 2022, 85, e37–e39. [Google Scholar] [CrossRef]
  27. Guo, W.; Fu, Y.; Jia, R.; Guo, Z.; Su, C.; Li, J.; Zhao, X.; Jin, Y.; Li, P.; Fan, J.; et al. Visualization of the infection risk assessment of SARS-CoV-2 through aerosol and surface transmission in a negative-pressure ward. Environ. Int. 2022, 162, 107153. [Google Scholar] [CrossRef]
  28. Wang, Z.; Li, J.; Qian, L.; Liu, L.; Qian, J.; Lu, B.; Guo, Z. Composition and Distribution Analysis of Bioaerosols Under Different Environmental Conditions. J. Vis. Exp. 2019, 143, e58795. [Google Scholar] [CrossRef]
  29. Shi, J.; Kong, H.; Cui, P.; Deng, G.; Zeng, X.; Jiang, Y.; He, X.; Zhang, X.; Chen, L.; Zhuang, Y.; et al. H5N1 virus invades the mammary glands of dairy cattle through ‘mouth-to-teat’ transmission. Natl. Sci. Rev. 2025, 12, nwaf262. [Google Scholar] [CrossRef]
  30. Giugliano, R.; Ferraro, V.; Chianese, A.; Della Marca, R.; Zannella, C.; Galdiero, F.; Fasciana, T.M.A.; Giammanco, A.; Salerno, A.; Cannillo, J.; et al. Antiviral Properties of Moringa oleifera Leaf Extracts against Respiratory Viruses. Viruses 2024, 16, 1199. [Google Scholar] [CrossRef]
  31. Wang, L.; Didelot, X.; Bi, Y.; Gao, G.F. Assessing the extent of community spread caused by mink-derived SARS-CoV-2 variants. Innovation 2021, 2, 100128. [Google Scholar] [CrossRef]
  32. Sun, Y.; Yang, A.W.H.; Lenon, G.B. Phytochemistry, Ethnopharmacology, Pharmacokinetics and Toxicology of Cnidium monnieri (L.) Cusson. Int. J. Mol. Sci. 2020, 21, 1006. [Google Scholar] [CrossRef] [PubMed]
  33. Su, F.; Zhao, Z.; Ma, S.; Wang, R.; Li, Y.; Liu, Y.; Li, Y.; Li, L.; Qu, J.; Yu, S. Cnidimonins A-C, Three Types of Hybrid Dimer from Cnidium monnieri: Structural Elucidation and Semisynthesis. Org. Lett. 2017, 19, 4920–4923. [Google Scholar] [CrossRef]
  34. Tamura, S.; Fujitani, T.; Kaneko, M.; Murakami, N. Prenylcoumarin with Rev-export inhibitory activity from Cnidii Monnieris Fructus. Bioorganic Med. Chem. Lett. 2010, 20, 3717–3720. [Google Scholar] [CrossRef]
  35. Huang, R.L.; Chen, C.C.; Huang, Y.L.; Hsieh, D.J.; Hu, C.P.; Chen, C.F.; Chang, C. Osthole increases glycosylation of hepatitis B surface antigen and suppresses the secretion of hepatitis B virus in vitro. Hepatology 1996, 24, 508–515. [Google Scholar] [CrossRef] [PubMed]
  36. Chatterjee, D.; Mondal, P.P.; Bhattacharya, A.; Chakrabarti, A.K. Role of the Chaperone Protein 14-3-3η in Regulation of the Infection Dynamics of the Influenza A (H1N1) Virus. Viruses 2025, 17, 1337. [Google Scholar] [CrossRef]
  37. Voronina, D.V.; Vavilova, I.V.; Zubkova, O.V.; Ozharovskaia, T.A.; Popova, O.; Chugunova, A.S.; Goldovskaya, P.P.; Zrelkin, D.I.; Savina, D.M.; Favorskaya, I.A.; et al. Single-Dose Intranasal or Intramuscular Administration of Simian Adenovirus-Based H1N1 Vaccine Induces a Robust Humoral Response and Complete Protection in Mice. Viruses 2025, 17, 1085. [Google Scholar] [CrossRef] [PubMed]
  38. Zhang, B.; Liu, M.; Huang, J.; Zeng, Q.; Zhu, Q.; Xu, S.; Chen, H. H1N1 Influenza A Virus Protein NS2 Inhibits Innate Immune Response by Targeting IRF7. Viruses 2022, 14, 2411. [Google Scholar] [CrossRef]
  39. David, C.; Verney, C.; Si-Tahar, M.; Guillon, A. The deadly dance of alveolar macrophages and influenza virus. Eur. Respir. Rev. Off. J. Eur. Respir. Soc. 2024, 33, 240132. [Google Scholar] [CrossRef]
  40. Bi, Y.; Yang, J.; Wang, L.; Ran, L.; Gao, G.F. Ecology and evolution of avian influenza viruses. Curr. Biol. 2024, 34, R716–R721. [Google Scholar] [CrossRef]
  41. Stannard, H.; Koszalka, P.; Deshpande, N.; Desjardins, Y.; Baz, M. Pre-Clinical Evaluation of the Antiviral Activity of Epigalocatechin-3-Gallate, a Component of Green Tea, against Influenza A(H1N1)pdm Viruses. Viruses 2023, 15, 2447. [Google Scholar] [CrossRef]
  42. Zhang, J.; Wu, B.; Yang, N.; Lin, X.; Chen, M.; Chang, H.; Shi, Y.; Lin, X.; Li, G.; Dai, W.; et al. The mechanism of action in Mussaenda pubescens (Yuye Jinhua) against influenza A virus: Evidence from in vitro and in vivo studies. Phytomed. Int. J. Phytother. Phytopharm. 2025, 145, 157070. [Google Scholar] [CrossRef]
  43. Su, J.; Lai, J.; Li, J.; Liu, X.; Chen, H.; Li, C.; Zhu, B.; Jia, X.; Li, Y. Carambolaside W Inhibited H1N1 Influenza Virus-Induced Oxidative Stress through STAT-3/BCL-XL Signaling Pathway. Viruses 2023, 15, 1858. [Google Scholar] [CrossRef] [PubMed]
  44. Kwon, E.B.; Kim, Y.S.; Hwang, Y.H.; Kim, B.; Lee, S.B.; Park, S.K.; Choi, M.S.; Ha, H.; Choi, J.G. Antiviral activity of soybean GL 2626/96 (Glycine max) ethanolic extract against influenza A virus in vitro and in vivo. Biomed. Pharmacother. Biomed. Pharmacother. 2022, 156, 113780. [Google Scholar] [CrossRef] [PubMed]
  45. Zhang, C.; Li, J.; Cui, H.; Jin, Y.; Chen, Z.; Zhang, L.; Song, S.; Lu, B.; Wang, Z.; Guo, Z. Lentinan Reduces Transmission Efficiency of COVID-19 by Changing Aerodynamic Characteristic of Exhaled SARS-CoV-2 Aerosols in Golden Hamsters. Microorganisms 2025, 13, 597. [Google Scholar] [CrossRef]
  46. Guo, Z.; Zhang, C.; Zhang, C.; Cui, H.; Chen, Z.; Jiang, X.; Wang, T.; Li, Y.; Liu, J.; Wan, Z.; et al. SARS-CoV-2-related pangolin coronavirus exhibits similar infection characteristics to SARS-CoV-2 and direct contact transmissibility in hamsters. iScience 2022, 25, 104350. [Google Scholar] [CrossRef] [PubMed]
  47. Zhang, C.; Wang, Z.Y.; Cui, H.; Chen, L.G.; Zhang, C.M.; Chen, Z.L.; Dong, S.S.; Zhao, K.; Fu, Y.Y.; Liu, J.X.; et al. Emergence of H5N8 avian influenza virus in domestic geese in a wild bird habitat, Yishui Lake, north central China. Virol. Sin. 2023, 38, 157–161. [Google Scholar] [CrossRef]
  48. Jin, Y.; Cui, H.; Jiang, L.; Zhang, C.; Li, J.; Cheng, H.; Chen, Z.; Zheng, J.; Zhang, Y.; Fu, Y.; et al. Evidence for human infection with avian influenza A(H9N2) virus via environmental transmission inside live poultry market in Xiamen, China. J. Med. Virol. 2023, 95, e28242. [Google Scholar] [CrossRef]
  49. Jiang, L.; Li, J.; Cui, H.; Zhang, C.; Jin, Y.; Fu, Y.; Ma, N.; Tang, F.; Zhang, Y.; Zheng, J.; et al. Etiologic characteristics of avian influenza H11 viruses isolated from the live poultry market in southeast coastal region in China. Front. Microbiol. 2022, 13, 1002670. [Google Scholar] [CrossRef]
  50. Zhang, C.; Jin, Y.; Cui, H.; Wang, Z.; Chen, Z.; Zhang, L.; Song, S.; Lu, B.; Guo, Z. Cross-Species Transmission Risks of a Quail-Origin H7N9 Influenza Virus from China Between Avian and Mammalian Hosts. Viruses 2025, 17, 1402. [Google Scholar] [CrossRef]
  51. Tang, J.; Gao, R.; Liu, L.; Zhang, S.; Liu, J.; Li, X.; Fang, Q.; Feng, Z.; Xu, C.; Huang, W.; et al. Substitution of I222L-E119V in neuraminidase from highly pathogenic avian influenza H7N9 virus exhibited synergistic resistance effect to oseltamivir in mice. Sci. Rep. 2021, 11, 16293. [Google Scholar] [CrossRef]
  52. Taubenberger, J.K.; Morens, D.M. The pathology of influenza virus infections. Annu. Rev. Pathol. 2008, 3, 499–522. [Google Scholar] [CrossRef]
  53. Lowen, A.C.; Mubareka, S.; Steel, J.; Palese, P. Influenza Virus Transmission Is Dependent on Relative Humidity and Temperature. PLoS Pathog. 2007, 3, e151. [Google Scholar] [CrossRef]
  54. Park, C.; Jang, J.; Jang, J. Airborne Influenza Virus Surveillance Platform Using Paper-Based Immunosensors and a Growth-Based Virus Aerosol Concentrator. Environ. Sci. Technol. 2025, 59, 6502–6511. [Google Scholar] [CrossRef]
  55. Guo, Z.; Wang, Z.; Qian, L.; Zhao, Z.; Zhang, C.; Fu, Y.; Li, J.; Zhang, C.; Lu, B.; Qian, J. Biological and chemical compositions of atmospheric particulate matter during hazardous haze days in Beijing. Environ. Sci. Pollut. Res. Int. 2018, 25, 34540–34549. [Google Scholar] [CrossRef]
  56. Li, J.; Zheng, J.; Chen, P.; Wang, B.; Zhang, Y.; Xiong, J.; You, L.; Jin, Y.; Jiang, L.; Tang, F.; et al. Higher SARS-CoV-2 shedding in exhaled aerosol probably contributed to the enhanced transmissibility of Omicron BA.5 subvariant. J. Med. Virol. 2023, 95, e28365. [Google Scholar] [CrossRef]
  57. Li, J.; Zhang, Y.; Jiang, L.; Cheng, H.; Li, J.; Li, L.; Chen, Z.; Tang, F.; Fu, Y.; Jin, Y.; et al. Similar aerosol emission rates and viral loads in upper respiratory tracts for COVID-19 patients with Delta and Omicron variant infection. Virol. Sin. 2022, 37, 762–764. [Google Scholar] [CrossRef] [PubMed]
  58. Wang, Z.; Fu, Y.; Guo, Z.; Li, J.; Li, J.; Cheng, H.; Lu, B.; Sun, Q. Transmission and prevention of SARS-CoV-2. Biochem. Soc. Trans. 2020, 48, 2307–2316. [Google Scholar] [CrossRef]
  59. Guo, Z.D.; Wang, Z.Y.; Zhang, S.F.; Li, X.; Li, L.; Li, C.; Cui, Y.; Fu, R.B.; Dong, Y.Z.; Chi, X.Y.; et al. Aerosol and Surface Distribution of Severe Acute Respiratory Syndrome Coronavirus 2 in Hospital Wards, Wuhan, China, 2020. Emerg. Infect. Dis. 2020, 26, 1583–1591. [Google Scholar] [CrossRef] [PubMed]
  60. Zhu, H.; Lu, X.; Ling, L.; Li, H.; Ou, Y.; Shi, X.; Lu, Y.; Zhang, Y.; Chen, D. Houttuynia cordata polysaccharides ameliorate pneumonia severity and intestinal injury in mice with influenza virus infection. J. Ethnopharmacol. 2018, 218, 90–99. [Google Scholar] [CrossRef] [PubMed]
  61. Li, Z.; Li, L.; Zhou, H.; Zeng, L.; Chen, T.; Chen, Q.; Zhou, B.; Wang, Y.; Chen, Q.; Hu, P.; et al. Radix isatidis Polysaccharides Inhibit Influenza a Virus and Influenza A Virus-Induced Inflammation via Suppression of Host TLR3 Signaling In Vitro. Molecules 2017, 22, 116. [Google Scholar] [CrossRef]
  62. Kataoka, K.; Muta, T.; Yamazaki, S.; Takeshige, K. Activation of macrophages by linear (1→3)-β-D-glucans. Impliations for the recognition of fungi by innate immunity. J. Biol. Chem. 2002, 277, 36825–36831. [Google Scholar] [CrossRef]
  63. Qi, J.; Lv, C.; Guo, J.; Li, Y.; Sima, M.; Luo, R.; Xiang, H.; Xia, X.; Zhou, Y.; Wang, T. Schisandra chinensis (Turcz.) Baill. polysaccharide inhibits influenza A virus in vitro and in vivo. FEBS Open Bio 2023, 13, 1831–1843. [Google Scholar] [CrossRef] [PubMed]
  64. Ison, M.G.; Hayden, F.G. 154—Antiviral Agents Against Respiratory Viruses. In Infectious Diseases, 4th ed.; Cohen, J., Powderly, W.G., Opal, S.M., Eds.; Elsevier: Amsterdam, The Netherlands, 2017; pp. 1318–1326.e2. [Google Scholar]
  65. Zheng, J.; Wang, Z.; Li, J.; Zhang, Y.; Jiang, L.; Fu, Y.; Jin, Y.; Cheng, H.; Li, J.; Chen, Z.; et al. High amounts of SARS-CoV-2 in aerosols exhaled by patients with Omicron variant infection. J. Infect. 2022, 84, e126–e128. [Google Scholar] [CrossRef]
  66. Leung, N.H.L.; Chu, D.K.W.; Shiu, E.Y.C.; Chan, K.H.; McDevitt, J.J.; Hau, B.J.P.; Yen, H.L.; Li, Y.; Ip, D.K.M.; Peiris, J.S.M.; et al. Respiratory virus shedding in exhaled breath and efficacy of face masks. Nat. Med. 2020, 26, 676–680. [Google Scholar] [CrossRef]
  67. Jiang, L.; Jin, Y.; Li, J.; Zhang, R.; Zhang, Y.; Cheng, H.; Lu, B.; Zheng, J.; Li, L.; Wang, Z. Respiratory Pathogen Coinfection During Intersecting COVID-19 and Influenza Epidemics. Pathogens 2024, 13, 1113. [Google Scholar] [CrossRef] [PubMed]
  68. Zhang, C.; Guo, K.; Cui, H.; Chen, L.; Zhang, C.; Wang, X.; Li, J.; Fu, Y.; Wang, Z.; Guo, Z.; et al. Risk of Environmental Exposure to H7N9 Influenza Virus via Airborne and Surface Routes in a Live Poultry Market in Hebei, China. Front. Cell. Infect. Microbiol. 2021, 11, 688007. [Google Scholar] [CrossRef] [PubMed]
  69. Dong, N.; Li, X.; Xue, C.; Zhang, L.; Wang, C.; Xu, X.; Shan, A. Astragalus polysaccharides alleviates LPS-induced inflammation via the NF-κB/MAPK signaling pathway. J. Cell. Physiol. 2020, 235, 5525–5540. [Google Scholar] [CrossRef]
  70. Xie, M.; Cai, J.; Zhong, X.; Liang, J.; Liang, S.; Xian, M.; Yan, C.; Wang, S. Extraction and structural profiling of polysaccharides from Rehmannia glutinosa and anti-inflammatory action via the NF-κB/IκBα pathway. Ind. Crops Prod. 2024, 208, 117874. [Google Scholar] [CrossRef]
  71. He, J.; Lu, J.; Zhan, L.; Zheng, D.; Wang, Y.; Meng, J.; Li, P.; Zhao, J.; Zhang, W. An Alkali-extracted polysaccharide from Poria cocos activates RAW264.7 macrophages via NF-κB signaling pathway. Arab. J. Chem. 2023, 16, 104592. [Google Scholar] [CrossRef]
  72. Kuo, C.L.; Ting, B.; Tseng, R.J.; Liu, S.P.; Liou, J.Y. Comparative Efficacy of Antrodia cinnamomea on Liver Function Biomarkers in Mice and Rats: A Network Meta-Analysis. Antioxidants 2025, 14, 660. [Google Scholar] [CrossRef] [PubMed]
  73. Zhang, B.; Zeng, M.; Zhang, Q.; Wang, R.; Jia, J.; Cao, B.; Liu, M.; Guo, P.; Zhang, Y.; Zheng, X.; et al. Ephedrae Herba polysaccharides inhibit the inflammation of ovalbumin induced asthma by regulating Th1/Th2 and Th17/Treg cell immune imbalance. Mol. Immunol. 2022, 152, 14–26. [Google Scholar] [CrossRef] [PubMed]
  74. Ren, D.D.; Chen, K.C.; Li, S.S.; Zhang, Y.T.; Li, Z.M.; Liu, S.; Sun, Y.S. Panax quinquefolius polysaccharides ameliorate ulcerative colitis in mice induced by dextran sulfate sodium. Front. Immunol. 2023, 14, 1161625. [Google Scholar] [CrossRef] [PubMed]
  75. Ridgway, H.; Apostolopoulos, V.; Moore, G.J.; Gadanec, L.K.; Zulli, A.; Swiderski, J.; Tsiodras, S.; Kelaidonis, K.; Chasapis, C.T.; Matsoukas, J.M. Computational Evidence for Bisartan Arginine Blockers as Next-Generation Pan-Antiviral Therapeutics Targeting SARS-CoV-2, Influenza, and Respiratory Syncytial Viruses. Viruses 2024, 16, 1776. [Google Scholar] [CrossRef]
  76. Li, Q.; Jiang, L.; Wang, Y.; Liu, X.; Wang, B.; Shan, Z.; Wang, Y.H.; Wang, Y.; Chen, H.; Li, C. HACD3 Prevents PB1 from Autophagic Degradation to Facilitate the Replication of Influenza A Virus. Viruses 2024, 16, 702. [Google Scholar] [CrossRef]
  77. Richard, M.; van den Brand, J.M.A.; Bestebroer, T.M.; Lexmond, P.; de Meulder, D.; Fouchier, R.A.M.; Lowen, A.C.; Herfst, S. Influenza A viruses are transmitted via the air from the nasal respiratory epithelium of ferrets. Nat. Commun. 2020, 11, 766. [Google Scholar] [CrossRef]
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Figure 1. In vitro anti-influenza activity of CMP: (A) Cytotoxicity of CMP in A549 cells determined by the CCK-8 assay; (B) Antiviral efficacy of CMP within the non-cytotoxic concentration range assessed by CCK-8 following influenza virus infection; (C) Antiviral activity of CMP under three different treatment conditions: (i) Pre-treatment; (ii) Co-treatment; (iii) Post-treatment; * p < 0.05, ** p < 0.01, *** p < 0.001. (D) Microscopic observation of CPE changes under different treatment conditions 36 h post-infection. The scale bar represents 200 μm.
Figure 1. In vitro anti-influenza activity of CMP: (A) Cytotoxicity of CMP in A549 cells determined by the CCK-8 assay; (B) Antiviral efficacy of CMP within the non-cytotoxic concentration range assessed by CCK-8 following influenza virus infection; (C) Antiviral activity of CMP under three different treatment conditions: (i) Pre-treatment; (ii) Co-treatment; (iii) Post-treatment; * p < 0.05, ** p < 0.01, *** p < 0.001. (D) Microscopic observation of CPE changes under different treatment conditions 36 h post-infection. The scale bar represents 200 μm.
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Figure 2. Guinea pig transmission efficiency and viral tissue distribution: (A,B) Nasal-wash viral titers of donors in CMP-pretreated versus untreated control groups; (C) Individual serum HI titers of guinea pigs in CMP-pretreated versus untreated control groups; (D) Lung index of guinea pigs; (E,F) Distribution of influenza virus in the respiratory tract (nasal turbinates, trachea, and lung).
Figure 2. Guinea pig transmission efficiency and viral tissue distribution: (A,B) Nasal-wash viral titers of donors in CMP-pretreated versus untreated control groups; (C) Individual serum HI titers of guinea pigs in CMP-pretreated versus untreated control groups; (D) Lung index of guinea pigs; (E,F) Distribution of influenza virus in the respiratory tract (nasal turbinates, trachea, and lung).
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Figure 3. The immunomodulatory effects of CMP in vivo: (A) Viral titer in lung tissue. The mRNA levels of inflammatory cytokines TNF-α (B), IL-1β (C) and IL-6 (D) in lung tissue were detected using RT-qPCR. (E) Western blot electrophoresis bands of NF-κB p65 and p-NF-κB p65 in lung tissue from 5 dpi mice. NC: NC group; PC: PC group; OS: OS group; L: CMP-L group; M: CMP-M group; H: CMP-H group. Same legend applies below. (F) Phosphorylation levels of NF-κB p65 protein in mouse lung tissue post-infection (pp65/p65). ### p < 0.001, vs. the NC group; * p < 0.05, ** p < 0.01, *** p < 0.001, vs. the PC group.
Figure 3. The immunomodulatory effects of CMP in vivo: (A) Viral titer in lung tissue. The mRNA levels of inflammatory cytokines TNF-α (B), IL-1β (C) and IL-6 (D) in lung tissue were detected using RT-qPCR. (E) Western blot electrophoresis bands of NF-κB p65 and p-NF-κB p65 in lung tissue from 5 dpi mice. NC: NC group; PC: PC group; OS: OS group; L: CMP-L group; M: CMP-M group; H: CMP-H group. Same legend applies below. (F) Phosphorylation levels of NF-κB p65 protein in mouse lung tissue post-infection (pp65/p65). ### p < 0.001, vs. the NC group; * p < 0.05, ** p < 0.01, *** p < 0.001, vs. the PC group.
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Figure 4. Proposed mechanism of CMP in mitigating influenza aerosol transmission and airway inflammation.
Figure 4. Proposed mechanism of CMP in mitigating influenza aerosol transmission and airway inflammation.
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MDPI and ACS Style

Wang, H.; Jin, Y.; Li, Y.; Wang, Y.; Zhao, Y.; Cheng, S.; Li, Z.; Yan, M.; Yang, Z.; Chen, X.; et al. Cnidium monnieri Polysaccharides Exhibit Inhibitory Effect on Airborne Transmission of Influenza A Virus. Viruses 2026, 18, 86. https://doi.org/10.3390/v18010086

AMA Style

Wang H, Jin Y, Li Y, Wang Y, Zhao Y, Cheng S, Li Z, Yan M, Yang Z, Chen X, et al. Cnidium monnieri Polysaccharides Exhibit Inhibitory Effect on Airborne Transmission of Influenza A Virus. Viruses. 2026; 18(1):86. https://doi.org/10.3390/v18010086

Chicago/Turabian Style

Wang, Heng, Yifei Jin, Yanrui Li, Yan Wang, Yixin Zhao, Shuang Cheng, Zhenyue Li, Mengxi Yan, Zitong Yang, Xiaolong Chen, and et al. 2026. "Cnidium monnieri Polysaccharides Exhibit Inhibitory Effect on Airborne Transmission of Influenza A Virus" Viruses 18, no. 1: 86. https://doi.org/10.3390/v18010086

APA Style

Wang, H., Jin, Y., Li, Y., Wang, Y., Zhao, Y., Cheng, S., Li, Z., Yan, M., Yang, Z., Chen, X., Zhang, Y., Yang, Z., Wang, Z., Liu, K., & Chen, L. (2026). Cnidium monnieri Polysaccharides Exhibit Inhibitory Effect on Airborne Transmission of Influenza A Virus. Viruses, 18(1), 86. https://doi.org/10.3390/v18010086

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