Evaluation and extraction of plant materials
Extracts made by boiling the herb in water. The voucher specimen of the plant material was deposited in the CAS Key Laboratory of Pathogenic Microbiology and Immunology (CASPMI), Institute of Microbiology, Chinese Academy of Sciences. Dandelion, purchased from a medicine store, was dissolved in sterile H2O (100 mg/ml) at room temperature for 2 h and then extracted twice with water at 100°C for 1 h. The aqueous extracts were filtered through a 0.45 μm membrane. This aqueous dandelion extract lyophilized, and the resulting light yellow powder (17% w/w yield) was dissolved with cell culture medium when needed.
Viruses, cells and viral infections
Human influenza virus A/Puerto Rico/8/34 (H1N1) (PR8) and A/WSN33 (WSN) were grown in 10-day old fertilized chicken eggs. After incubation at 37°C for 2 days, the allantoic fluid was harvested and used for infection.
All cell lines were purchased from ATCC (Rockville, MD, USA). Madin-Darby canine kidney (MDCK) cells or Human lung adenocarcinoma cell line (A549) were cultured in Dulbecco's modified eagle medium (DMEM) or RPMI-1640 medium, respectively, with 10% fetal bovine serum (FBS, Gibco, USA), penicillin 100 U/ml, and streptomycin 10 μg/ml. Prior to infection, the cells were washed with phosphate-buffered saline (PBS) and were cultured in infection medium (DMEM without FBS, 1.4% BSA) supplemented with antibiotics and 2 μg/ml of trypsin (Gibco; Invitrogen, Carlsbad, CA).
Hemagglutination inhibition test
Influenza viruses are characterized by their ability to agglutinate erythrocytes. This hemagglutination activity can be visualized upon mixing virus dilutions with chicken erythrocytes in microtiter plates. The chicken erythrocytes were supplemented with 1.6% sodium citrate (Sigma, USA) in sterile water, separated by centrifugation (800 × g, 10 min, room temperature) and washed three times with sterile PBS. Serial two-fold dilutions of dandelion extracts were made in 25 μl of PBS in 96-well V-bottom plates. Influenza viruses in 25 μl of PBS (4 HAU) were added to each dilution, and the plates were incubated for 1 h at room temperature. 25 μl of 1% (v/v) chicken erythrocytes in PBS was added to each well. The hemagglutination pattern was read following the incubation of the plates for 0.5 h at room temperature. The highest dilution that completely inhibited hemagglutination was defined as the hemagglutination inhibition (HI) titer.
Cell viability assay
A549 or MDCK cells were left untreated or treated with the indicated amounts of dandelion extracts ranging from 20 to 0.1563 mg/ml, and oseltamivir ranging from 12.5 to 0.098 mg/ml for 48 h; MDCK cells were left untreated or treated with 0.1 mg/ml oseltamivir, 2.5 mg/ml and 15 mg/ml dandelion extracts for 72 h. All drugs were multiproportion diluted in serum-free medium. Cell-proliferation and metabolism were measured using the CCK8-assay. Briefly, the cells were treated with CCK-8 solution (dojindo, 10 μl/well) and incubated for 4 h at 37°C. The absorbance was measured using a microplate reader (DG5032, Huadong, Nanjing, China) at 450 nm. The untreated control was set at 100%, and the treated samples were normalized to this value according to the following equation: Survival rate (%) = optical density (OD) of the treated cells - OD of blank control/OD of negative control - OD of blank control × 100.
Plaque titrations and antiviral assays
Plaque titrations: MDCK cells grown to 90% confluency in 96-well dishes were washed with PBS and infected with serial dilutions of the supernatants in PBS for 1 h at 37°C. The inoculum was aspirated and cells were incubated with 200 μl DMEM (medium containing 1.4% BSA, 2 μg/ml of trypsin and antibiotics) at 37°C, 5% CO2 for 2-3 days. Virus plaques were visualized by staining with trypan blue.
Antiviral assay: MDCK cells were infected with the influenza A virus strain PR8 or WSN (1 × 106 PFU) and were left untreated or treated with dandelion extracts (0.0782-5 mg/ml), oseltamivir (0.0047-0.3 mg/ml) (Sigma), or suxiaoganmaojiaonang (0.069-4.375 mg/ml). At 16 h post infection supernatants were taken. This procedure was repeated two times in triplicate. Supernatants were assayed for progeny virus yields by standard plaque titrations. Virus yields of mock-treated cells were arbitrarily set as 100%.
Simultaneous treatment assay: dandelion extracts (2.5 mg/ml), oseltamivir (0.1 mg/ml) or suxiaoganmaojiaonang (4.375 mg/ml) was mixed with virus individually and incubated at 4°C for 1 h. The mixture was inoculated onto near confluent MDCK cell monolayers (1 × 105 cells/well) for 1 h with occasional rocking. The solution was removed, the cells were washed twice with PBS and the inoculum was aspirated, and then the cells were incubated with 2 ml of DMEM supplemented with 1.4% BSA, antibiotics, 2 μg/mL trypsin at 37°C under 5% CO2 atm.
Post treatment assay: Influenza viruses (1 × 106 PFU) were inoculated onto near confluent MDCK cell monolayers (1 × 105 cells/well) for 1 h with occasional rocking. The media was removed and replaced by DMEM containing 1.4% BSA, antibiotics, 2 μg/mL trypsin and dandelion extracts (2.5 mg/ml), or oseltamivir (0.1 mg/ml), or suxiaoganmaojiaonang(4.375 mg/ml). The cultures were incubated at 37°C under 5% CO2 atm.
After 6, 12, 24, 36 and 48 h incubation in all antiviral assays, the supernatant was analyzed for the production of progeny virus using the hemagglutinin test and was compared with the untreated control cells. Cell proliferation and metabolism were analyzed by the CCK8-assay at 48 h post-treatment. Virus yields from the mock-treated cells were normalized to 100%.
Real-time reverse transcription-PCR analysis
MDCK cells were grown to about 90% confluence infected with influenza virus (1 × 106 PFU). Medium was removed after 1 h, and cultured in the presence of dandelion extracts (2.5 mg/ml) 13 h. The inoculum was aspirated after 13 h. Cells were scraped off, washed twice with PBS, and collected by centrifugation (500 g for 5 min). Total RNA was prepared using the RNApure total RNA fast isolation kit (Shanghai Generay Biotech Co., Ltd). The primer sequence used for quantitative real-time PCR of viral RNA were 5' -TGTGTATGGACCTGCCGTAGC - 3' (sense) and 5' - CCATCCACACCAGTTGACTCTTG - 3' (antisense). The Canis familiaris beta-actin was used as internal control of cellular RNAs, with primer sequences of 5' -CGTGCGTGACATCAAGGAAGAAG - 3' (sense) and reverse: 5' -GGAACCGCTCGTTGCCAATG - 3' (antisense). The primer sequences used in real-time PCR were designed using Beacon Designer 7 software.
Real-time reverse transcription-PCR was performed using 100 ng of RNA and the One-step qPCR kit (RNA-direct SYBR Green Real-time PCR Master Mix, TOYOBO). Cycling conditions for real-time PCR were as follows: 90°C for 30 s, 61°C for 20 min, and 95°C for 1 min, followed by 45 cycles of 95°C for 15 s, 55°C for 15 s and 74°C for 45 s. As the loading control, we measured the level of Canis familiaris beta-actin mRNA. Real-time PCR was conducted using the ABI Prism 7300 sequence detection system, and the data were analyzed using ABI Prism 7300 SDS software (Applied Biosystems).
Minigenome assay and transient transfection
To test the transcription efficiency of the influenza virus polymerases after drug treatment, a minigenome assay was performed in Human embryonic kidney (293T) cells. Briefly, ambisense plasmids encoding PB2, PB1, PA and NP were cotransfected together with the influenza virus replicon reporter plasmid pPOLI-luciferase. The reporter plasmid pPOLI-luciferase was constructed by inserting the luciferase protein open reading frame (ORF) flanked by the noncoding regions of the M gene of influenza A virus between the BamHIand NotI site of the pPOLI vector (a generous present from Dr. Edward Wright). Calcuim phosphate transfection was used. Briefly, the cell culture was replaced by Opti-medium; 0.5 μg of each plasmid was mixed, incubated at room temperature for 15 min, and added over 80% confluent 293T cells seeded the day before in six-well plates. Six hours later, the DNA-transfection mixture was replaced by DMEM containing 10% FBS. At 48 h posttransfection, the cells were treated with cell lysis buffer, centrifuged, and supernatant was collected. Add 5 μl aliquots of cell lysate to individual luminometer tubes containing 180 μl of luciferase assay buffer at room temperature. To start the assay, inject 100 μl of luciferin solution into the luminometer tube and measure the light output in the luminometer.
Data were presented as mean ± SD. The data were statistically evaluated using a one-way ANOVA to compare differences between the groups. A p-value of < 0.05 was considered to be significant. The IC50 and CC50 values were calculated using GraphPad Prism programme.