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. 2023 Sep 23;336:199223. doi: 10.1016/j.virusres.2023.199223

Identification of γ-Fagarine as a novel antiviral agent against respiratory virus (hMPV) infection

Jinhua Li a, Yao Zhao c, Ying Dai d, Junning Zhao a,b,⁎
PMCID: PMC10522984  PMID: 37734492

Highlights

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    γ-Fagarine possesses inhibitory activity against human metapneumovirus (hMPV) in vitro and in vivo.

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    γ-Fagarine blocking hMPV binding and infection relates to heparan sulfate proteoglycans.

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    γ-Fagarine influences the lysosome pH to inhibit virus replication.

Keywords: γ-Fagarine, hMPV, Antiviral activity, Lysosome, HSPG

Abstract

Human metapneumovirus (hMPV) causes significant upper and lower respiratory disease in all age groups worldwide. However, there is no licensed drugs or vaccine available against hMPV. γ-Fagarine, an alkaloid isolated from the root of zanthoxylum, has been reported to be effective in the treatment of cancer, inflammatory diseases and antivirals. However, little is known about the inhibitory effect of γ-Fagarine against respiratory virus infection and the mechanism. In this study, we aim to investigate the effect of γ-Fagarine on hMPV infection and explore its underlying molecular mechanisms. Vero-E6 and 16HBE cells were used as cell models. Virus replication and microcosm character were explored in Vero-E6 cells. Then, the antiviral activities were investigated by quantitative real-time PCR (RT-qPCR), western blotting (WB), and indirect immunofluorescence assays (IFAs) in Vero-E6 and 16HBE. Potential mechanisms of γ-Fagarine related to HSPG and lysosome pH were assessed in 16HBE cells. Lastly, a virus-infected mouse model was established and antiviral assay in vivo was conducted. γ-Fagarine showed no toxicity toward Vero-E6 cells and 16HBE cells but demonstrated anti-hMPV activity. Virus titers of γ-Fagarine group were reduced to 33% and 45% of the hMPV groups, respectively. Besides, mechanistic studies revealed that γ-Fagarine could inhibit hMPV by dual mechanisms of direct restraining virus binding with HSPG and influencing lysosome pH. Furthermore, oral delivery of γ-Fagarine to hMPV-infected mice at a dosage of 25 mg/kg reduced the hMPV load in lung tissues. After γ-Fagarine treatment, pathological damage caused by viral infection was also ameliorated. These findings suggest that γ-Fagarine has antiviral effects in vitro and in vivo, which are associated with its ability to restrain virus binding with HSPG and influence lysosome pH, thus indicating that γ-Fagarine has the potential to serve as a candidate to fight against hMPV infection and other respiratory viruses such as influenza viruses and SARS-CoV-2.

Graphical abstract

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1. Introduction

hMPV is a non-segmented negative-sense, single-stranded RNA (ss RNA) virus belonging to the pneumoviridae family containing eight genes and nine proteins identified in 2001 (Hoogen et al., 2001). Globally, hMPV is recognized as the second most common cause of bronchiolitis and pneumonia in children five years of age (Cespedes et al., 2016; Verena Schildgen et al., 2011). hMPV infection results in various disease severities from mild cold-like symptoms to bronchiolitis, pneumonia, and febrile seizures and can potentially lead to death (Kahn, 2006; Marie-E`ve Hamelin, 2006). The SARS-CoV-2 outbreak of 2019 has resulted in severe respiratory tract infections and even deaths during the past three years (CDC, 2023). hMPV has been reported to co-infect with SARS-CoV-2 and other respiratory tract viruses (Scott et al., 2021). However, there is no clinically approved vaccine and drugs for hMPV infection. Ribavirin has been conceived to fight against hMPV in vitro and animals model, but some studies revealed it makes no difference for children (Kroll and Weinberg, 2011). Thus, it is imminent to find novel antiviral compounds against hMPV infection.

The application of traditional Chinese medicine to anti-respiratory viruses has been studied for a long time (Ling et al., 2020; Zhi et al., 2019). It's reported that Lianhuaqingwen exerts anti-viral and anti-inflammatory activity against novel coronavirus (SARS-CoV-2) (Runfeng et al., 2020). Zanthoxylum bungeanum, belonging to the Rutaceae family, is a known medicinal plant widely distributed in China. Zanthoxylum bungeanum pericarp is an available spice in China and is widely used in cooking because of its unique fragrance and taste (Xiang et al., 2016). γ-Fagarine, an alkaloid isolated from the root of zanthoxylum, has been reported to be effective in the treatment of cancer, inflammatory diseases and antivirals (Li et al., 2020; Chen, 2008). However, little is known about the inhibitory effect of γ-Fagarine against respiratory virus infection and the mechanism.

Heparan sulfate proteoglycans (HSPGs), also known as SDC2, are glycoproteins containing one or more covalently attached Heparan sulfate (HS) chains (Essner et al., 2006). HSPGs played diverse roles in cell adhesion and cell communication and was identified as a coreceptor promoting the attachment of SARS-CoV-2 (Liu et al., 2021). Previous studies also revealed that heparan sulfate affected hMPV binding and infection (Chang et al., 2012). Lysosomes, as primary organelles of cells, degrade old and obsolete macromolecules and organelles, as well as extracellular material delivered to them via different forms of autophagy and endocytosis (Holland et al., 2020). hMPV could use endocytosis and membrane fusion pathway for cells entry. It is reported that hMPV membrane fusion is promoted by low pH (Herfst et al., 2008). Previous studies showed virus infection influenced the pH and utilized the acidic atmosphere to infect or synthesize more viruses to facilitate the infection. Influenza virus was reported that viruses experience their initial acidification in the endocytic pathway(Lakadamyali et al., 2003). Human bronchial epithelial cells were used as cell models to study the role of γ-Fagarine during respiratory viral infection (Jones et al., 2021).

In this study, we investigated the antiviral activity of γ-Fagarine in vitro and in vivo. Firstly, virus replication and microcosm character were explored in Vero-E6 cells. Then, the antiviral activities were investigated by quantitative real-time PCR (RT-qPCR), western blotting (WB), and indirect immunofluorescence assays (IFAs) in Vero-E6 and 16HBE. Antiviral mechanisms of γ-Fagarine related to HSPG and lysosome pH were assessed in 16HBE cells. Lastly, a virus-infected mouse model was established. An antiviral assay in vivo exhibited that γ-Fagarine showed a protective effect in mouse lungs and reduced the virus titers in the lung.

2. Materials and methods

2.1. Drugs, cells and virus

γ-Fagarine (purity >99%) was obtained from Chengdu Pusi Biotechnology Co., Ltd. Ribavirin was purchased from Sigma-Aldrich. Vero-E6 cells were purchased from ATCC. 16HBE cells were obtained from Shanghai Cell Bank (Shanghai, China). Cells were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum and 1% penicillin streptomycin (Gibco, USA). All cells were incubated under 5% CO 2 at 37 °C. hMPV was successfully recovered from full-length cDNA clones of hMPV NL/1/00 by reverse genetics as described previously(Zhang et al., 2011); hMPV was propagated in Vero E6 cells, and the viral supernatant was harvested and centrifuged in a 50 ml tube at 5000 rpm for 30 min at 4 °C (Thermo Fisher Scientific, USA). Then hMPV virions were purified by centrifugation Centricon® Plus-70 Centrifugal Filter Devices (Millipore, USA). Finally, the supernatant was discarded, and the virus particles were suspended in a medium and maintained as stocks at −80 °C. All experiments with infectious hMPV were performed following the approved standard operating procedures of the Biosafety Level 2 facility (BSL-2) at the department of children hospital affiliated with Chongqing medical university.

2.2. Transmission electron microscopy (TEM)

The TEM samples were prepared by Wuhan Servicebio technology CO., Ltd's protocol. hMPV was inoculated in Vero-E6 cells performed in a biosafety level 2 facility, and they were then examined 96 h after infection with the use of transmission electron microscopy. Cells were harvested and then fixed in isoosmotic 2.5% glutaraldehyde diluted in sodium cacodylate buffer (pH 7.3) and post-fixed in 1% osmium tetroxide. Took 20 μL of virus suspension with a pipet-gum and dropped onto the copper grid with carbon film for 3–5 min, and then used filter paper to absorb the excess liquid. Dropped 2% phosphotungstic acid on the copper to stain for 1 min, used filter paper to absorb excess liquid, and dry at room temperature. The cup rum grids are observed under TEM and taken images.

2.3. Cytopathic effect (CPE)

Vero-E6 cells were seeded in 24-well plates 20 mm dishes prior to incubation. Cells were infected with hMPV at the multiplicity of infection (MOI = 1) or 3% FBS for 2 h, washed and then kept in a fresh medium at 37 °C. Morphology Apoptosis of cells was observed and recorded by microscope for up to 4 days.

2.4. Kinetic of infection with hMPV

Vero-E6 cells were seeded on Sterile coverslips into 24-well plates. After 24 h, cells were inoculated with hMPV (MOI=1) for 2 h, 3% FBS medium was added to the infected cells at 24 h, 48 h,72 h and 96 h. To the scheduled times, cells were washed three times and fixed with 4% formaldehyde for 10 min. Then blocked with 0.2% bovine serum albumin (BSA) in PBS and penetrated with 0.1% Triton X-100 diluted by PBS for 30 min at 4 °C. Then, cells were incubated with the indicated primary antibodies (Abcam, ab94802) for 2 h at room temperature. Coverslips were washed three times with PBS and inoculated Alexa Fluor–conjugated secondary antibodies (Abcam, ab150105) for 1 h at room temperature. After three rinses with PBS, incubated with 4,6-diamidino-2-phenylindole (DAPI) for 15 min, Imaging was performed on a Nikon confocal microscopy.

2.5. RNA extraction and RT-qPCR

Cell lysates and supernatants were obtained from scheduled time points of the virus and uninfected groups, then lysed by RNA lysis buffer provided in the RNA isolation kit (Qiagen). RNA isolation was performed as per manufacturer's instructions, and cDNA was prepared using Evo M-MLV RT Premix for qPCR (AG 11,706). RT-qPCR was performed using Pro Taq HS Premix Probe qPCR Kit (AG 11,704) in Bio-Rad Light Cycler 96 system. The thermal cycling conditions comprised a pre-incubation step of 95 °C for 30 s followed by 40 cycles at 95 °C for 5 s and 60 °C for 30 s. And then, the number of hMPV RNA genomes was quantified. The samples were run in duplicate for each data point for an experiment.

2.6. Viral replication kinetics in Vero-E6

Vero-E6 cells in 24-well plates were infected with hMPV. After 2 h of adsorption, the inoculum was removed, and the cells were washed thrice with PBS. Fresh DMEM (supplemented with 3% FBS) was added, and the infected cells were incubated at 37 °C for 24 h, 48 h, 72 h and 96 h. At indicated time points post-infection, the supernatant and cells were harvested by freeze-thaw cycles, followed by centrifugation at 5000 rpm at 4 °C for 30 min. The total RNA of virus was isolated using an RNA extraction kit (Qiagen) and then quantified the gene copies.

2.7. Cell viability

Cell Counting Kit 8 (CCK-8) assays were used to evaluate cell viability. Vero-E6 and 16HBE cells were seeded in 96-well plates at a density of 2.5 × 105 cells /ml. Serial 2-fold dilutions of γ-Fagarine were added to the medium 16 h later. After 24 h incubation, the relative number of surviving cells was measured using the CCK-8(Abmole, China), followed by the manufacturer's instructions. Absorbance was measured at 450 nm wavelength.

2.8. In vitro antiviral assays

2.8.1. Dose response antiviral assays

Drugs suspended in DMSO, series of concentrations γ-Fagarine with two-fold dilutions under test concentrations (200 μM) were prepared respectively 6.25, 12.5, 25, 50, 100 and 200 μM. Vero-E6 cells and 16HBE cells were pre-seeded to 24-well plates (2 × 105 cells/well) for 24 h. Then, the cells were inoculated with hMPV for 2 h. After the supernatant was removed, the cells were washed twice with PBS, and fresh medium was re-added with gradient concentrations of γ-Fagarine at 1 mL/well. The cells were incubated at 37  °C for 24 h. The cell lysis was collected, and antiviral activities were evaluated by quantifying viral F gene copy numbers in the cell lysis via real-time fluorescence quantitative PCR (RT-qPCR). The experiments were done in triplicates, and all the infection experiments were performed at BSL-2.

2.8.2. Kinetic of anti-viral activity

Vero-E6 cells and 16HBE cells in 24-well plates were infected with hMPV at an MOI of 1. After 2 h of adsorption, the virus was removed, and the cells were washed thrice with PBS. Fresh DMEM (supplemented with 3% FBS) or γ-Fagarine solution (200 μM) was added, and the infected cells were incubated at 37 °C for 6 h, 12 h, 24 h and 48 h. Then the supernatant was discarded, and cells’ total RNA isolated using a total RNA extraction kit (Magen), and then the F gene copies were quantified at indicated time points.

2.8.3. Antiviral immunofluorescence assays

Vero-E6 cells and 16HBE cells were seeded on Sterile coverslips into 24-well plates. After 24 h, cells were inoculated with hMPV (MOI=1), and the control group was added 3%FBS for 2 h. And then, the control group and hMPV group were added 1 ml 3%FBS, positive group was treated with 1 ml ribavirin (200 μM), and the experimental group was treated with γ-Fagarine. After 24 h infection, all cells were washed three times, fixed with 4% PFA (paraformaldehyde) for 30 min at room temperature, permeabilized with 0.2% Triton X-100 for 30 min, and blocked with 1% BSA. To detect virally infected cells, the Anti-Metapneumovirus Fusion protein antibody (hMPV 24) was used as a primary antibody with staining at room temperature followed by staining with a secondary antibody Alexa Fluor 488. After three rinses with PBS, incubated with DAPI for 15 min, Imaging was performed on a Nikon confocal microscopy.

2.8.4. Western blotting

Vero-E6 cells and 16HBE (7 × 105 cells) plated into six-well plates were incubated with hMPV or treated with 3%FBS. And then, the control group and hMPV group were added 1 ml 3%FBS, the positive group was treated with 1 ml ribavirin (200 μM) and the experimental group was treated with γ-Fagarine. At the indicated time points (24 h), cells were washed with PBS and lysed in radioimmunoprecipitation assay (RIPA) buffer supplemented with protease inhibitor cocktail (Beyotime Biotechnology) for 30 min. Upon centrifugation at 12,000 rpm for 20 min at 4 °C, clarified cell lysates were incubated with 5 × sample buffer at 100 °C for 10 min. Proteins were separated on 8% SDS–polyacrylamide gel electrophoresis gels and transferred to Immobilon-P polyvinylidene difluoride transfer membranes (Millipore). The protein concentration is 1 μg/μL and loaded 20 μL/well. The membranes were blocked with 5% milk in tris-buffered saline with 0.1% Tween 20 (TBST) for 1 h at room temperature and incubated with primary antibodies (hMPV 57, Abcam, ab94802) overnight at 4 °C. After three rinses with TBST, the membranes were incubated with horseradish peroxidase (HRP)–conjugated secondary antibodies for 1 h at room temperature. Membranes were then developed with ECL Western blotting reagents (Beyotime Biotechnology). Western blotting signals were visualized using Bio-Rad Imager.

2.9. Anti-attachment assays

16HBE cells were seeded on 24-well plates to investigate the virus binding on the membrane. After 24 h in culture, cells were respectively pre-incubated DMEM. And then, the control group and hMPV group were added 1 ml 3%FBS, the positive group was treated with 1 ml ribavirin (200 μM) and experimental group was treated with γ-Fagarine diluted by DMEM for 2 h. Then cells were inoculated with hMPV (MOI =1) except the control group, which was treated with 3% FBS at 4 °C for 30 min. Finally, the supernatants were discarded, and cells were washed three times with PBS to be fixed with 4% PFA for IFAs.

16HBE cells seeded on 24-well plates for 24 h were segmented into 8 groups, uninfected, hMPV, γ-Fagarine, ase, γ-Fagarine+ase, γ-Fagarine +ase+SDC2, SDC2 and heparin group, respectively. Before incubation with hMPV (MOI=1), ase, SDC2, ase+SDC2 and heparin were added to corresponding group for 2 h. Next, the control group were added 1 ml 3% FBS, and other groups were infected with hMPV for 2 h. After 2 h incubation, the control group, hMPV group, ase group, SDC2 and heparin were added 1 ml 3%FBS. And γ-Fagarine group, γ-Fagarine+ase, γ-Fagarine +ase+SDC2 were added 1 ml γ-Fagarine suspension (200 μM). After infection for 24 h, one plate cells were used to conduct IFAs and calculate the MFI of F protein, another was used to extract total RNA and quantify viral F gene copies.

2.10. Lysosome fluorescence determination

16HBE cells were seeded on the confocal dish at a density of 2 × 105/ml, then infected with hMPV for 2 h and treated with γ-Fagarine or ribavirin. After 24 h, uninfected and hMPV-infected cells were all incubated with Lysotracker Red DND-99 (100 nM) and Hoechst 33,258 for 1 h and washed with PBS three times according to manufacturer instructions. And then, the lysosome fluorescence intensity was monitored under a laser confocal microscope. In parallel, another experiment was conducted to extract RNA and quantify the viral F gene copies.

2.11. hMPV mouse model

Female BALB/c mice age of 6–8 weeks and body weight of 18–20 g were used. The mice were housed under a 12 h dark-light cycle at a constant temperature. Animal experiments were executed by certified staff in Center for Animal Experiments of Chongqing medical University, approved by the Institutional Animal Care and Use Committee.

BALB/c mice were infected with hMPV (1 × 107,60 μL) and incubated with 1 day, 3 days, 5 days, 7 days, 11 days and 15 days. At indicated time points, the mice were euthanized. Each mouse’ left lung was removed, weighed, and homogenized in 1 ml of PBS solution using a 24 tissues homogenizer (Qiagen) following the manufacturer's recommendations. And the hMPV virus gene copies of all the mice were determined. The right lung of each mouse was removed, inflated and fixed with 4% PFA. Fixed tissues were embedded in paraffin and sectioned at 5 μm. Slides were then stained with hematoxylin-eosin (H&E) and IFA to examine histological changes by light microscopy. Histopathological changes were evaluated based on the extent of interstitial inflammation, edema, and peribronchiolar inflammation.

2.12. hMPV challenge and treatment in mouse model

γ-Fagarine was dissolved in saline and diluted to 25 mg/ml final concentrations for animal experiments. Ribavirin was diluted to 100 mg/ml. Firstly, Nasal drip infected mice with hMPV(1 × 107,60 μL) and infected with 24 h; Mice were intragastrically administered γ-Fagarine (25 mg/kg) or Ribavirin (100 mg/kg) with the volume of 0.1 ml/10 g of body weight. Saline, γ-Fagarine and ribavirin were administered every 24 h for the remainder of the study. Body weight were measured daily. At 5 days after infection, mice were euthanized, and viral titers were determined via RT-qPCR, the right lungs were fixed with 4% PFA for HE and IFA.

2.13. Statistical analysis

Statistical analysis and data visualization Analyses were performed using GraphPad Prism 8 (La Jolla, CA, USA). All the individual data points are presented and compared using one-way analysis of variance (ANOVA). *, P<0.05, **, P<0.01, ***, P<0.001, P values of <0.05 are denoted as statistically significant throughout.

3. Results

3.1. Characterization of hMPV in Vero-E6 cells

To confirm the virus's characteristics, Vero-E6 cells infected with hMPV were harvested by centrifuge and detected by TEM. TEM results revealed virus were pleomorphic particles in supernatants and measuring 250 nm (Fig. 1A). Virus were both found on the apical surface and inside the cell (Fig. 1B). Obviously, the control group incubated with 3% FBS was not found the CPE, still, CPE increased with the incubation time prolonging in hMPV groups (Fig. 1C). Moreover, IFAs were conducted to examine the expression of F protein during virus infection, it is worth noting that both the infection rate and the fluorescence intensity were strengthened. But mean fluorescence intensity increased to 72 h and then decreased (Fig. 1D and E). In addition, the virus F gene copies increased continuously within three days and then declined by RT-qPCR detection (Fig. 1F). Those results showed that hMPV could infect Vero-E6 cells, replicate and induce CPE.

Fig. 1.

Fig 1

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Characterization of hMPV in Vero-E6 cells. (A) hMPV virus particle in the supernatant (72 hpi) and (B) Vero-E6 cells (72 hpi). arrows: virus particles, Negative-stained TEM image of hMPV. (C) CPE of hMPV in Vero-E6 cells at the indicated time points. (D) Staining of hMPV F protein (green) in Vero-E6 cells, Nuclei were stained with DAPI (blue). (E) Fluorescence intensity of F protein in Vero-E6 cells. (F) hMPV F gene replication kinetics in Vero-E6 cells (n = 3).

3.2. Cell viability and antiviral activity

Firstly, molecular structure of γ-Fagarine is shown in Fig. 2A.γ-Fagarine showed no obvious cytotoxicity at concentrations under 200 μM (Fig. 2B and C). Then the efficacy of γ-Fagarine against hMPV infection was further tested by detecting viral F gene copies in Vero-E6 and 16HBE cells. With γ-Fagarine concentrations increased, the viral gene copies decreased dose-dependently and displayed great difference (p<0.0001) in 50, 100 and 200 μM. At a concentration of 200 μM, hMPV F gene copies decrease by 67% and 55% in Vero-E6 and 16HBE, respectively (Fig. 2D and F). Next, hMPV viral replication in Vero-E6 and 16HBE cells was measured in 6 h, 12 h, 24 h and 48 h. It seems that γ-Fagarine treatment could decrease the expression and showed great difference at indicated four times course (p<0.0001), and ribavirin did not display an inhibitory effect after infection 48 h (Fig. 2E). Meanwhile, there was no apparent inhibitory effect observed in 16HBE cells after 6 h infection. but after 12 h or 24 h infection, RT-qPCR results showed significant difference (p<0.001) (Fig. 2F). At 48 h, the γ-Fagarine group significantly lower the virus replication, but only slight change of the ribavirin group was observed (Fig. 2G). Here, we observed that γ-Fagarine showed no cell viability and significant inhibitory effect in Vero-E6 and 16HBE cells.

Fig. 2.

Fig 2

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Cell viability and Assessment of antiviral activity in cell models. (A) γ-Fagarine molecular structure. Cell viability of Vero-E6 cells (B) and 16HBE (C) cells were infected with hMPV (MOI =1) for 2 h and treated for 24 h with increasing concentrations γ-Fagarine. (D) Vero-E6 cells were infected with hMPV and treated with different concentrations γ-Fagarine. At 24 dpi, viral RNA copies were quantified from cell culture by RT-qPCR. Data are means ± SD; n = 3 biological replicates. (E) Vero-E6 cells were infected with hMPV and treated with 200 μM concentration γ-Fagarine for 6 h, 12 h, 24 h and 48 h. (F) 16HBE cells were infected with hMPV and treated with different concentrations of γ-Fagarine. At 24 dpi, viral RNA copies (per ml) were quantified from cell culture by RT-qPCR. Data are means ± SD; n = 3 biological replicates. (G) 16HBE cells were infected with hMPV and treated with 200 μM concentration γ-Fagarine for 6 h, 12 h, 24 h and 48 h. Data are mean ± SEM. n = 3 biological replicates. One-way ANOVA followed by Dunnett's post-test. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, and ****P ≤ 0.0001, ns, not significant.

3.3. γ-Fagarine potently inhibits hMPV infection and replication

The anti-hMPV effects studies of γ-Fagarine were performed on Vero-E6 cells and 16HBE cells. A schematic diagram of cells infected with hMPV is shown in Fig. 3A. Firstly, both Vero-E6 and 16HBE showed similar results that F protein expression was noticeably decreased by the treatment of γ-Fagarine and ribavirin (200 μM) at 24 h post-infection. γ-Fagarine showed a better inhibitory effect than ribavirin (Fig. 3B). The F protein MFI of γ-Fagarine group and RBV group decreased significantly contrast with control group (Fig. 3C and D). WB results showed that the expression levels of N protein treated with γ-Fagarine and RBV was significantly inhibited compared with hMPV group in Vero-E6 cells (Fig. 3E). Next, the N protein level in 16HBE were investigated in 24 h, γ-Fagarine group treated for 24 h decreased significantly (Fig. 3F). These results further verified that γ-Fagarine showed an inhibitory effect and exhibited a stronger antiviral effect than RBV.

Fig. 3.

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Antiviral research in cell models. (A) Overview of in vitro study design. (B) γ-Fagarine reduce the expression of virus F protein in Vero-E6 and 16HBE cells. Mean fluorescence intensity of F protein in Vero-E6 cells (C) and 16HBE cells (D). (E) Impacts of γ-Fagarine on N protein expression of Vero-E6 cells. (F) Impacts of γ-Fagarine on N protein expression of 16HBE cells. The experiment was repeated twice.

3.4. HSPG influence hMPV infection

To identify the mechanism of γ-Fagarine during hMPV inhibition, we used 16HBE cells as cell model treated with γ-Fagarine for 2 h before infection, confocal images showed that virus F protein binding decreased (Fig. 4A), this result suggested γ-Fagarine inhibited virus attachment with cell membranes. To investigate whether the reduction of virus binding on the cell membrane surface is related to HSPG. Pre-incubation of hMPV with heparinase and heparin significantly reduced hMPV F protein expression in 16HBE cells (Fig. 5A). Adding γ-Fagarine and heparinase did not display additive effects. Meanwhile, pre-incubation SDC2 proteins can mildly improve the mean MFI of F protein. Pre-incubation SDC2 proteins and heparinase and then adding γ-Fagarine after cells infected with hMPV, which decreased hMPV F protein expression (Fig. 5A and B). As detailed in Fig. 5C, RT-qPCR assays demonstrated a similar inhibitory effect, and viral gene copies displayed obvious decrease when the heparin and heparinase were added to the 16HBE cells. In addition, Pre-incubation SDC2 significantly increased F gene expression. It can be inferred that γ-Fagarine effectively reduced viral binding to HSPG and then inhibited the virus replication.

Fig. 4.

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Confocal images illustrated the attachment of virus with membrane. Pre-treatment of γ-Fagarine and ribavirin for 2 h and, then cells were inoculated with hMPV (MOI =1) except the control group, which was treated with 3% FBS at 4 °C for 30 min. Finally, the supernatants were discarded, and cells were washed three times with PBS to be fixed with 4% PFA for IFAs. Staining of hMPV F protein (green) in 16HBE cells, Nuclei were stained with DAPI (blue).

Fig. 5.

Fig 5

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HSPG affected the virus attachment to cell membrane. (A) Adding γ-Fagarine, heparin and HSPG protein to cells, IFAs demonstrated the inhibition of heparin and the promotion of HSPG protein. (B) Expression of virus F protein in different groups. (C) Viral load examined by RT-qPCR.

3.5. γ-Fagarine affected the lysosome pH and hMPV infection

Lysosome pH influenced virus infection. Whether γ-Fagarine inhibitory effects were related to lysosome acidification was assessed. Compared with uninfected cells, a stark increase of lysotracker red fluorescence intensity was observed in the hMPV group and the γ-Fagarine group showed less fluorescence intensity, but the ribavirin group did not have the change (Fig. 6A and B). Then, the RT-qPCR results showed both γ-Fagarine and the RBV group F gene copies substantially decreased by 53% and 47% in Fig. 6C, respectively. Those results indicated γ-Fagarine might alter the cell lysosome acidification to inhibit replication.

Fig. 6.

Fig 6

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γ-Fagarine affect the lysosome pH and influence the virus infection. (A) Confocal fluorescence images of 16HBE cells incubated with lyso-Tracker Red DND-99. Scale bars, 10 μm. (B) Mean fluorescence intensity of F protein in 16HBE cells incubated with lyso-tracker. (C) viral genome copies examined by RT-qPCR.

3.6. hMPV mouse models

In order to investigate the in vivo antiviral effect, the mouse model was established by previously reported the protocol (Marie-E`ve Hamelin, 2006). The mice were intranasally inoculated with hMPV on day 0 and then euthanized to harvest the mice lung and detect the RNA copies. Eventually, histological analysis and IFAs were further examined (Fig. 7A). The mice lung gene copies were continually rising in 1–5 days, and the viral gene copies decreased quickly (9–15 days) (Fig. 7B). Histopathological analyses revealed that hMPV infection induced obvious pathological damage such as alveolar structure destruction and the damages remained existed for day 15 when F gene copies can be ignored (Fig. 7C). Meanwhile, IFA results indicated the existence of F protein, but the F protein was not disappeared up to 15 days (Fig. 7D). The evidence exhibited hMPV infected mouse model was successfully established and obvious damages in mouse lung were detected in protein and RNA level. Surprisingly, damage caused by hMPV infection existed for more time than gene copies displayed.

Fig. 7.

Fig 7

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Establishment of infection mouse model. (A) Overview of in vivo study design. (B) Viral load in the lung was examined by RT-qPCR at the indicated time points. (C) Representative images of lung histopathological changes from hMPV infected BALB/c mice (1 × 107 TCID 50) at indicated time, Scale bar, 200 μm. (D) F protein of virus in the lung was stained by green, Scale bar, 50 μm.

3.7. In vivo antiviral activity

Mice infected with hMPV were used to evaluate the antiviral activity in vivo according to the schematics illustrated in Fig. 8A. Apparently, the mice infected with hMPV showed weight loss compared with the saline group and decreased to 83% of original weight. However, weight loss was not so significantly in γ-Fagarine and ribavirin group (Fig. 8B). γ-Fagarine treatment reduced the viral titers in lung tissues compared to the hMPV group and showed an obvious difference (p<0.05) and the ribavirin fairly decreased the viral titers (Fig. 8C). Similarly, after γ-Fagarine treatment, the yield of viral RNA in lung tissues was reduced, indicating a superior therapeutic effect. Histopathological analyses revealed that hMPV infection resulted in apparent pathological damage, including alveolar structure destruction, hemorrhage, and inflammatory cell infiltration in the lung parenchyma, while γ-Fagarine treatment mitigated these phenomena (Fig. 8D). These findings suggested that -fagarine could reduce viral loads in lung tissues while also alleviating histopathological changes and protecting mice from hMPV infection.

Fig. 8.

Fig 8

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Therapeutic oral efficacy of γ-Fagarine in the hMPV mouse model. (A) Overview of in vivo study design. (B) Body-weight changes after 5 day's treatment, n = 4 for all groups. (C) Viral load in the lung examined by RT-qPCR, γ-Fagarine reduce lung viral load in hMPV infected mouse model. Mouse were intranasally inoculated with 1 × 107 PFU hMPV, followed by virus challenge where mice were orally administrated 25 mg/kg (n = 4) γ-Fagarine for consecutive days. The vehicle group was given saline as a control. (D) Representative images of lung histopathological changes from hMPV infected BALB/c mice at 5 dpi, Scale bar, 200 μm and representative images of fluorescence staining. Staining of hMPV F protein (green) in BALB/c lung, Nuclei were stained with DAPI (blue), Scale bar, 100 μm.

4. Discussion

Several studies have noted hMPV resulted in large damage to people's health (Djamin et al., 2015; Lu et al., 2011). It has been estimated that about 10–12% of the respiratory illnesses in children are associated with hMPV, which is considered as one of the most prevalent viruses causing hospitalization in young children (Feuillet et al., 2012). Although extraordinary efforts have been underway globally to identify safe and effective treatments, there is approved drug to inhibit the hMPV infection. Thus, identification of new candidate therapies would enable the development of antivirus drugs. γ-Fagarine (also named as 4,8-Dimeth-oxy-furo [2,3-b] quinoline) is one of alkaloids isolated from Zanthoxylum, which exhibits diverse pharmacological and biological activities(Goodman et al., 2019). However, until now, the bioactivity of γ-Fagarine in respiratory virus infection has not been reported. In the current study, γ-Fagarine's antiviral activity was estimated in vitro and in vivo and potential mechanism was conducted.

The TEM results suggested the virus particle is round and enveloped, which was semblable with previous studies (Schildgen et al., 2011). The replication kinetics indicated that the virus infected Vero-E6 and replicated rapidly. According to virus titers detected at different time points, we chose to detect the virus titer after 24 h viral infection to judge its antiviral effect in Vero-E6 and 16HBE (Qiao, 2020; Sourimant, 2022). F protein and N protein were the essential proteins of hMPV, which located in cell membrane and cell nucleus (Kroll and Weinberg, 2011), respectively. According to detecting the two proteins expression and RNA expression level, γ-Fagarine's antiviral activity was determined (Jeong et al., 2020; Leyrat et al., 2014). Ribavirin has been safely used in humans to treat multiple pathologies, including respiratory syncytial virus (RSV), Lassa fever, and, most notably, hepatitis C virus (HCV) infections (Casaos et al., 2019). In this study, ribavirin was chosen as a positive drug. One unexpected finding was that γ-Fagarine treatment reduced viral titers and lasted longer than ribavirin. Therefore, it is definitely worth investigating whether γ-Fagarine could have a better antiviral effect with a long-term administration.

Several studies have suggested that HSPG is an important attachment factor influencing virus infection (Cox et al., 2012). Apical HSPG in the airway serves as a binding factor during infection and HS modulating compounds may serve as a platform for potential antiviral development (Chang et al., 2012; Klimyte et al., 2016). Currently, HSPG analogues are being studied for use in anti-COVID-19 drugs (Guimond et al., 2022). In our findings, adding heparinase and heparin limit hMPV infection. γ-Fagarine exhibited inhibitory effect and have no addictive antiviral effect with heparinase probably by inhibiting the virus from binding to HSPG proteins. Besides, γ-Fagarine was firstly confirmed to possess the ability to inhibit virus binding with HSPG, which potentially be developed into a broad spectrum of antiviral drugs. hMPV efficiently replicate and transcript by inducing formation of inclusion bodies (Nicolás Cifuentes-Muñoz, 2017). Previous research reflected that lysosome pH changed after virus infection (Ghosh et al., 2020; Yang et al., 2016). Our findings indicated hMPV infection altering lysosome pH and resulted in more virus producing. And adding γ-Fagarine influenced acidification of lysosome and inhibited the infection of hMPV. However, there is no change happened when ribavirin was added to the cell culture, and ribavirin would not influencing the pH. This finding might it possible to use lysosomes as a new target for antiviral drugs development (Lan et al., 2022).

In this study, a mouse model was established using BALB/c mice infected with hMPV, which have been reported to be more susceptible to hMPV than other mice. Currently, ribavirin was chose as positive drugs (Witkowski, 1972). oral administration γ-Fagarine at 25 mg/kg safely effectively protected mice from hMPV infection, inhibited weight loss of mice, and reduced hMPV viral load in the lung tissues. γ-Fagarine has been previously used to treat bacterial and HCV (Chen, 2008), but not respiratory viruses. Our results suggested that γ-Fagarine could be used to develop new anti-hMPV drugs.

In summary, we demonstrate that γ-Fagarine possesses dose–activity relationships and strong anti-hMPV properties in vitro and in vivo, which are probably attributed to limiting virus binding to HSPG in cell membrane and influencing lysosome pH. Importantly, our findings reveal that γ-Fagarine administration has medicinal feasibility in hMPV-infected mouse models, including effective viral infection suppression and corresponding hMPV-related tissue pathology remission. Therefore, our study highlights that γ-Fagarine is an innovative and efficient anti-hMPV agent which provides a potent therapeutic and prophylactic implementation option for treating reemerging hMPV outbreaks and other respiratory virus infection.

CRediT authorship contribution statement

Jinhua Li: Methodology, Investigation, Writing – original draft, Data curation. Yao Zhao: Project administration, Writing – review & editing. Ying Dai: Methodology, Resources. Junning Zhao: Project administration, Writing – review & editing, Funding acquisition, Supervision.

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgments

The study was supported by grants from The Project of Sichuan Provincial Administration of Traditional Chinese Medicine (2020YSHB01) and Major Scientific and Technological Projects of Sichuan Provincial Administration of Traditional Chinese Medicine (2021XYCZ001).

Data availability

  • Data will be made available on request.

References

  1. Casaos J., Gorelick N.L., Huq S., Choi J., Xia Y., Serra R., Skuli N. The use of ribavirin as an anticancer therapeutic: will it go viral? Mol. Cancer Ther. 2019;18(7):1185–1194. doi: 10.1158/1535-7163.MCT-18-0666. [DOI] [PubMed] [Google Scholar]
  2. CDC . Centers for Disease Control and Prevention; 2023. COVID Data Tracker Weekly Review. [Google Scholar]
  3. Cespedes P.F., Palavecino C.E., Kalergis A.M., Bueno S.M. Modulation of host immunity by the human metapneumovirus. Clin. Microbiol. Rev. 2016;29(4):795–818. doi: 10.1128/CMR.00081-15. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Chang A., Masante C., Buchholz U.J., Dutch R.E. Human metapneumovirus (HMPV) binding and infection are mediated by interactions between the HMPV fusion protein and heparan sulfate. J. Virol. 2012;86(6):3230–3243. doi: 10.1128/JVI.06706-11. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Cifuentes-Muñoz N., Branttie J., Slaughter K.B., Dutch R.E. Human metapneumovirus induces formation of inclusion bodies for efficient genome replication and transcription. J. Virol. 2017;91(24):e01282. doi: 10.1128/JVI.01282-17. -01217. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Cox R.G., Livesay S.B., Johnson M., Ohi M.D., Williams J.V. The human metapneumovirus fusion protein mediates entry via an interaction with RGD-binding integrins. J. Virol. 2012;86(22):12148–12160. doi: 10.1128/JVI.01133-12. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Djamin R.S., Uzun S., Snelders E., Kluytmans J.J., Hoogsteden H.C., Aerts J.G., Van Der Eerden M.M. Occurrence of virus-induced COPD exacerbations during four seasons. Infect. Dis. (Lond.) 2015;47(2):96–100. doi: 10.3109/00365548.2014.968866. [DOI] [PubMed] [Google Scholar]
  8. Essner J.J., Chen E., Ekker S.C. Syndecan-2. Int. J. Biochem. Cell Biol. 2006;38(2):152–156. doi: 10.1016/j.biocel.2005.08.012. [DOI] [PubMed] [Google Scholar]
  9. Feuillet F., Lina B., Rosa-Calatrava M., Boivin G. Ten years of human metapneumovirus research. J. Clin. Virol. 2012;53(2):97–105. doi: 10.1016/j.jcv.2011.10.002. [DOI] [PubMed] [Google Scholar]
  10. Ghosh S., Dellibovi-Ragheb T.A., Kerviel A., Pak E., Qiu Q., Fisher M., Altan-Bonnet N. Beta-Coronaviruses use lysosomes for egress instead of the biosynthetic secretory pathway. Cell. 2020;183(6):1520–1535. doi: 10.1016/j.cell.2020.10.039. e1514. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Goodman C.D., Hoang A.T., Diallo D., Malterud K.E., McFadden G.I., Wangensteen H. Anti-plasmodial effects of Zanthoxylum zanthoxyloides. Planta Med. 2019;85(13):1073–1079. doi: 10.1055/a-0973-0067. [DOI] [PubMed] [Google Scholar]
  12. Guimond S.E., Mycroft-West C.J., Gandhi N.S., Tree J.A., Le T.T., Spalluto C.M., Turnbull J.E. Synthetic heparan sulfate mimetic pixatimod (PG545) potently inhibits SARS-CoV-2 by disrupting the spike-ACE2 interaction. ACS Cent. Sci. 2022;8(5):527–545. doi: 10.1021/acscentsci.1c01293. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Hamelin M.E., Prince G.A., Gomez A.M., Kinkead R., Boivin G. Human metapneumovirus infection induces long-term pulmonary inflammation associated with airway obstruction and hyperresponsiveness in mice. J. Infect. Dis. 2006;193:1634–1642. doi: 10.1086/504262. [DOI] [PubMed] [Google Scholar]
  14. Herfst S., Mas V., Ver L.S., Wierda R.J., Osterhaus A.D., Fouchier R.A., Melero J.A. Low-pH-induced membrane fusion mediated by human metapneumovirus F protein is a rare, strain-dependent phenomenon. J. Virol. 2008;82(17):8891–8895. doi: 10.1128/JVI.00472-08. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Holland L.K.K., Nielsen I.O., Maeda K., Jaattela M. SnapShot: lysosomal functions. Cell. 2020;181(3):748. doi: 10.1016/j.cell.2020.03.043. -748 e741. [DOI] [PubMed] [Google Scholar]
  16. Hoogen B.G.V.D., Jong J.C.D., Groen J., Kuiken T., Osterhaus A.D.M.E. A newly discovered human pneumovirus isolated from young children with respiratory tract disease. Nat. Med. 2001;7(6):719–724. doi: 10.1038/89098. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Jeong S., Park M.J., Song W., Kim H.S. Advances in laboratory assays for detecting human metapneumovirus. Ann. Transl. Med. 2020;8(9):608. doi: 10.21037/atm.2019.12.42. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Jones J.E., Le Sage V., Lakdawala S.S. Viral and host heterogeneity and their effects on the viral life cycle. Nat. Rev. Microbiol. 2021;19(4):272–282. doi: 10.1038/s41579-020-00449-9. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Kahn J.S. Epidemiology of human metapneumovirus. Clin. Microbiol. Rev. 2006;19(3):546–557. doi: 10.1128/CMR.00014-06. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Klimyte E.M., Smith S.E., Oreste P., Lembo D., Dutch R.E. Inhibition of human metapneumovirus binding to heparan sulfate blocks infection in human lung cells and airway tissues. J. Virol. 2016;90(20):9237–9250. doi: 10.1128/JVI.01362-16. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Kroll J.L., Weinberg A. Human metapneumovirus. Semin. Respir. Crit. Care Med. 2011;32(4):447–453. doi: 10.1055/s-0031-1283284. [DOI] [PubMed] [Google Scholar]
  22. Lakadamyali M., Rust M.J., Babcock H.P., Zhuang X. Visualizing infection of individual influenza viruses. Proc. Natl. Acad. Sci. USA. 2003;100(16):9280–9285. doi: 10.1073/pnas.0832269100. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Lan Y., He W., Wang G., Wang Z., Chen Y., Gao F., Song D. Potential antiviral strategy exploiting dependence of SARS-CoV-2 replication on lysosome-based pathway. Int. J. Mol. Sci. 2022;23(11):6188. doi: 10.3390/ijms23116188. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Leyrat C., Paesen G.C., Charleston J., Renner M., Grimes J.M. Structural insights into the human metapneumovirus glycoprotein ectodomain. J. Virol. 2014;88(19):11611–11616. doi: 10.1128/JVI.01726-14. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Li R.L., Zhang Q., Liu J., Sun J.Y., He L.Y., Duan H.X., Wu C.J. Hydroxy-alpha-sanshool possesses protective potentials on H(2)O(2)-stimulated PC12 cells by suppression of oxidative stress-induced apoptosis through regulation of PI3K/Akt signal pathway. Oxid. Med. Cell. Longev. 2020;2020 doi: 10.1155/2020/3481758. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Ling L.J., Lu Y., Zhang Y.Y., Zhu H.Y., Tu P., Li H., Chen D.F. Flavonoids from Houttuynia cordata attenuate H1N1-induced acute lung injury in mice via inhibition of influenza virus and Toll-like receptor signalling. Phytomedicine. 2020;67 doi: 10.1016/j.phymed.2019.153150. [DOI] [PubMed] [Google Scholar]
  27. Liu L., Chopra P., Li X., Bouwman K.M., Tompkins S.M., Wolfert M.A., Boons G.J. Heparan sulfate proteoglycans as attachment factor for SARS-CoV-2. ACS Cent. Sci. 2021;7(6):1009–1018. doi: 10.1021/acscentsci.1c00010. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Lu G., Gonzalez R., Guo L., Wu C., Wu J., Vernet G., Hung T. Large-scale seroprevalence analysis of human metapneumovirus and human respiratory syncytial virus infections in Beijing, China. Virol. J. 2011;8:62. doi: 10.1186/1743-422X-8-62. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Qiao J., Li Y.S., Zeng R., et al. SARS-CoV-2 M pro inhibitors with antiviral activity in a transgenic mouse model. Science. 2020;371:1374–1378. doi: 10.1126/science.abf1611. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Runfeng L., Yunlong H., Jicheng H., Weiqi P., Qinhai M., Yongxia S., Zifeng Y. Lianhuaqingwen exerts anti-viral and anti-inflammatory activity against novel coronavirus (SARS-CoV-2) Pharmacol. Res. 2020;156 doi: 10.1016/j.phrs.2020.104761. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Schildgen V., van den Hoogen B., Fouchier R., Tripp R.A., Alvarez R., Manoha C., Schildgen O. Human metapneumovirus: lessons learned over the first decade. Clin. Microbiol. Rev. 2011;24(4):734–754. doi: 10.1128/CMR.00015-11. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Scott S.J., Pfotenhauer B., Weiner J.J., Hilleshiem J., Khubbar M., Bhattacharyya S. Respiratory pathogen coinfections in SARS-CoV-2-positive patients in Southeastern Wisconsin: a retrospective analysis. Microbiol. Spectr. 2021;9(2) doi: 10.1128/Spectrum.00831-21. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Sourimant J., Lieber C.M., Aggarwal M., et al. 4′-Fluorouridine is an oral antiviral that blocks respiratory syncytial virus and SARS-CoV-2 replication. Science. 2022;361:161–167. doi: 10.1126/science.abj5508. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Witkowski J.T., Robins R.K., Sidwell R.W., Simon L.N. Design, synthesis, and broad spectrum antiviral activity of 1 -β-D-ribofuranosyl- 1,2,4-triazole-3-carboxamide and related nucleoside. J. Med. Chem. 1972;15(11):1150–1154. doi: 10.1021/jm00281a014. [DOI] [PubMed] [Google Scholar]
  35. Xiang L., Liu Y., Xie C., Li X., Yu Y., Ye M., Chen S. The chemical and genetic characteristics of szechuan pepper (Zanthoxylum bungeanum and Z. armatum) cultivars and their suitable habitat. Front. Plant Sci. 2016;7:467. doi: 10.3389/fpls.2016.00467. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Yang G., Chen D. Alkaloids from the roots of zanthoxylum nitidum and their antiviral and antifungal effects. Chem. Biodivers. 2008;5:1718–1722. doi: 10.1002/cbdv.200890160. [DOI] [PubMed] [Google Scholar]
  37. Yang H., He H., Tan B., Liu E., Zhao X., Zhao Y. Human metapneumovirus uses endocytosis pathway for host cell entry. Mol. Cell. Probes. 2016;30(4):231–237. doi: 10.1016/j.mcp.2016.06.003. [DOI] [PubMed] [Google Scholar]
  38. Zhang J., Dou Y., Wu J., She W., Luo L., Zhao Y., Zhao X. Effects of N-linked glycosylation of the fusion protein on replication of human metapneumovirus in vitro and in mouse lungs. J. Gen. Virol. 2011;92(7):1666–1675. doi: 10.1099/vir.0.030049-0. Pt. [DOI] [PubMed] [Google Scholar]
  39. Zhi H.J., Zhu H.Y., Zhang Y.Y., Lu Y., Li H., Chen D.F. In vivo effect of quantified flavonoids-enriched extract of Scutellaria baicalensis root on acute lung injury induced by influenza A virus. Phytomedicine. 2019;57:105–116. doi: 10.1016/j.phymed.2018.12.009. [DOI] [PubMed] [Google Scholar]

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Data Availability Statement

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