Preparation of A. argyi leaves ethanol extract
A. argyi leaves were harvested in Tangyin country (Henan, China) and were air-dried under shade at room temperature. After drying, the plant samples were ground into powder, and two kilograms (2 kg) of leave powders were extracted three times by maceration with 95% ethanol for 24 h. The combined ethanol extract (252.3 g) was extracted successively with ethyl acetate (1 L), water, dichloromethane (1 L), and petroleum ether (1 L). The ethyl acetate-soluble fraction (12 g) and a dichloromethane-soluble fraction (35 g) were combined, separated by a silica gel column (800 g), and eluted with different ratios of methylene chloride: MeOH solvents (from 100:0 to 1:1) to yield 9 major fractions (fraction 1–9) (Additional file 1: Fig. S1). Fractions were finally freeze-dried and stored at − 20 °C for further usage. The anti-HSV-1 activities of fractions 1–9 were screened by the viral cytopathic effect (CPE) method. Briefly, Vero cells were treated with different concentrations of fraction 1–9 and HSV-1 (MOI = 0.1) for 48 h. The cytopathic changes were observed. No cytopathic lesion was recorded as "−", cytopathic lesions 1% ~ 25% as "+", cytopathic lesions 26%–50% as "++", cytopathic lesions 51% ~ 75% as "+++", and cytopathic lesions 76% ~ 100% as " +++++ ". Fraction-6 exhibited the highest anti-HSV-1 activity, which was further used for antiviral research (hereafter referred to as "AEE or AEE (Fr.6), fraction-6 of A. argyi Ethanol Extract").
Cells, viruses, and antibodies
Kidney epithelial cells MA-104 (CRL-2378.1) and Vero (CCL-81), neuronal cells SH-SY5Y (CRL-2266) and U87-MG (HTB-14) were obtained from the American Type Culture Collection Center (ATCC, USA). U87-MG, Vero, and SH-SY5Y cells were cultured in Dulbecco's modified Eagle's medium (8118305, GIBCO, USA), supplemented with 10% fetal bovine serum (FND500, ExCell Bio, Shanghai, China). MA-104 cells were cultured in modified Eagle's medium (A1451801, GIBCO, USA).
HSV-1 F strain (ATCC, USA) was grown in Vero cells and stored at − 80 °C for further use as previously described . ACV-resistant strains HSV-1/153 and HSV-1/Blue were obtained from the Guangzhou Institutes of Biomedicine and Health (Guangzhou, China). HSV-1 F strain tagged by Green fluorescent protein (GFP) (GFP-HSV-1) was obtained from Professor Yuan Li of Jinan University (Guangzhou, China). HSV-2 and RV virus were obtained from the Wuhan Institute of Virology (Wuhan, China). As previously described, the influenza virus H1N1 was grown and stored at − 80 °C for further use . Antibody anti-ICP0 (ab6513) and anti-gB (ab6506) were purchased from Abcam (Cambridge, UK), and anti-GAPDH (2118) was purchased from Cell Signaling Technology (Danvers, MA, USA).
Different cells were seeded in a 96-well plate (1 × 104 cells/well) for 24 h and were then treated with different concentrations of AEE (0–80 μg/ml) for another 48 h. The cell viability was then evaluated by CCK8-kit (96992, Sigma-Alarich). The cells were added with 5 μl of CCK8 reagent and incubated at 37 °C for 1–2 h. A microplate reader was used to detect the OD value at 490 nm.
Viral plaque assay
A viral plaque assay was used to examine the antiviral effect of AEE as previously described . Briefly, Vero cells were seeded in a 24-well plate for 24 h before being infected with HSV-1 (MOI = 1). The virus was incubated at 37 °C for 2 h to promote viral particle absorption. The maintenance medium containing 1% methylcellulose (SIJIA BIOTECH, Guangzhou, China), with or without AEE, was used to replace the cell culture medium. After 72 h incubation, 10% formalin was used to fix the cells, and 1% crystal violet (Beyotime, Suzhou, China) was subsequently used to stain the cells. Finally, the total plaque numbers of each well were counted to calculate the inhibitory ratio accordingly.
Virus titration assay
Virus titration was determined according to the HSV-1-induced cytopathic effects. Vero cells were treated with a diluted culture medium containing different concentrations of HSV-1 viral particles. The cells were then incubated at 37 °C for 48 h, and the 50% tissue culture infectious dose (TCID50) was measured according to the cellular cytopathic effects. The plaque-forming units (PFU)/ml were further converted from the TCID50 value.
Quantitative real-time PCR (RT-qPCR)
Total RNA was extracted using the TRIZOL reagent (TIANGEN, Beijing, China) under the manufacturer's instructions. The PrimeScript RT Reagent Kit (TAKARA, Dalian, China) then transcribed the sample RNA (1 μg) into cDNA reversely. A Bio-Rad CFX96 Real-time PCR System (Bio-Rad) was used to determine the mRNA expression of different viral genes, and the internal reference gene gapdh was used to normalize gene mRNA level. All primer sequences are shown in Additional file 1: Table S1.
The DNA copy numbers of HSV-1 viral genes were collected and analyzed as before . Briefly, cells were infected with HSV-1 with or without AEE for 24 h, and the cell pellets and supernatant were collected. The viral samples were frozen at − 80 °C and thawed three times. A UNIQ-10 viral DNA extraction kit (Sangon, China) was used to isolate total virus genomic DNA, which was further subjected to RT-qPCR to determine the DNA copy numbers of viral genes.
Viral attachment, penetration, and inactivation assay
To evaluate the effect of AEE on viral attachment, Vero cells were pre-cooled at 4 °C for 1 h, then treated with AEE and HSV-1 (30 PFU/well) at 4 °C to facilitate viral particle attachment. After 80 min incubation, the culture medium was removed, and the cells were then incubated with a cover layer at 37 °C for another 72 h. Finally, the cells were fixed and stained to calculate the plaque numbers.
To evaluate the effect of AEE on viral penetration, Vero cells pre-cooled at 4 °C were incubated with HSV-1 (30 PFU/well) for 2 h at 4 °C. The culture medium was then replaced with an AEE-containing medium and cultured at 37 °C for another 10 min. The cells were washed with PBS buffer (pH = 3) and alkaline PBS (pH = 11.0) to remove viral particles not entering the cells. The cells were cultured with a cover layer at 37 °C for 72 h, and a plaque assay was performed.
To evaluate the effect of AEE on viral inactivation, HSV-1 viral particles (30–50 PFU/well) were treated with AEE for 2 h at 37 °C. Vero cells in a 24-well plate were then incubated with the virus-AEE mixture at 37 °C for another 2 h. A cover layer was used to replace the mixture and cultured at 37 °C for 72 h, which was then analyzed by plaque assay.
To evaluate the effect of AEE on the entry of RV, pre-cooled MA-104 cells were treated with RV (MOI = 1) and AEE (10 μg/ml) at 4 °C for 80 min. The culture medium was removed, and the cells were incubated at 37 °C for 24 h. Total RNA was extracted, and the mRNA expression levels of RV gene VP4 and VP7 were detected by RT-qPCR.
Western blot assay
RIPA buffer (P0013B, Beyotime, China) containing 1 mM phenylmethylsulfonyl fluoride was used to extract total proteins. After measuring concentration, protein samples were separated by SDS-PAGE (10%-15% gradient) and were transferred to the appropriate polyvinylidene fluoride membrane (Millipore). The protein membrane was then blocked by 5% nonfat milk at room temperature for 1 h, incubated with different primary antibodies at 4 °C overnight, and then incubated with appropriate secondary antibodies for 1 h at room temperature. Viral proteins were further detected by ECL solutions, and protein bands were captured by a 5200-image analysis system (Tannon, Shanghai, China).
An ultra-high-performance liquid chromatograph (UltiMate 3000 UPLC system, Thermo, USA)/mass spectrometry (TripleTOF™ 5600 LC/MS, AB SCIEX, USA) was used to isolate the possible component of AEE. Software Analyst TF 1.7 and MS-DIAL 4.24 were used to acquire and analyze data. An Acquity UPLC HSS T3 analytical column (100 × 2.1 mm, 1.8 μm) was used for chromatographic separation, with gradient elution consisting of water with 0.1% formic acid (v/v, solvent A) and 0.1% formic acid in acetonitrile (solvent B) (positive ion mode), or consisting of water with 2 mM ammonium acetate (solvent A) and acetonitrile (solvent B) (negative ion mode).
Auto Dock 4.2.6 software (The Scripps Research Institute) was used to perform molecular docking assay. Briefly, the two-dimensional structures of 12 AEE components were obtained from the PubChem database (https://pubchem.ncbi.nlm.nih.gov/) and were further transformed into the PDB format by Chem3D software (Chem3D 220.127.116.11). The X-ray crystal structures of gD (PDB ID: 3U82), nectin-1 (PDB ID: 3SKU), gB (PDB ID: 4BOM), and PILRα (PDB ID: 5XO2) were downloaded from the PDB database (http://www.rcsb.org/). After the ligands and receptor molecule files were prepared, a grid box was set for the movement and rotation of the ligand via Glide (Maestero version 12.5, Schrodinger). AutoDock4 program was then run to process molecular docking. PyMOL 2.5 (DeLano Scientific LLC, CA, USA) was used to visualize the simulation. Finally, the PLIP (https://plip-tool.biotec.tu-dresden.de/) was used to analyze the ligand and receptor binding of hydrophobic bonds and hydrogen.
Transmission electron microscopy
The morphology and shape of HSV-1 particles were examined by FEI Tecnai G2 Spirit TWIN transmission electron microscopy (TEM). Isolated viral particles in a volume of 1 ml medium were incubated with or without AEE (10 μg/ml) at 37 °C for 2 h, and the virus-AEE mixtures were prepared by dripping on a carbon-coated copper grid for subsequent procedures according to the instructions.
Data were presented as mean ± SD. At least 3 independent experiments were performed. The GraphPad Prism 7 software was used for statistical analysis. One-way ANOVA or Student's t-test was carried out, with significance as ns, not significant, *P < 0.05, **P < 0.01, and ***P < 0.001.