Cells, viruses, and plasmids
DEF cells were obtained from 9 to 11 days specific pathogen-free duck embryos, as described previously [24], and cultured in Dulbecco’s modified Eagle’s medium (cat. no. 8116176; Gibco, Grand Island, NY, USA) supplemented with 5% fetal bovine serum (cat. no. 1722658; Gibco) and antibiotics (0.1 mg/ml of streptomycin and 0.1 mg/ml penicillin) at 37 °C under an atmosphere of 5% CO2/95% air. DEV strain CSC was kept in our laboratory.
To construct a GFP-LC3 recombination plasmid, the LC3 gene was amplified from DEF cells with the primer pair LC3F 5`-ATG CAA CCG CCT CTG-3` and LC3R 5`-TCG CGT TGG AAG GCA AAT C-3`, corresponding to the GenBank sequence for duck LC3B gene (NW_004676873.1), and cloned into the pEGFP-C1 vector, to express LC3B protein with the GFP protein.
Virus infection and drug or small interfering RNA (siRNA) treatment
DEF cells were infected with DEV for 2 h at 37 °C, washed three times with sterile phosphate-buffered saline (pH 7.4), then maintained in 2% in culture medium supplemented with fetal bovine serum for various time points until samples were harvested. The cells were then cultured in 2% culture medium supplemented with fetal bovine serum with or without pre-treatment with the same drug for the indicated times. The optimal concentrations of chemicals used in this experiment were 10 mM 1,2-Bis(2-aminophenoxy)ethane-N,N,N′,N-tetraacetic acid (BAPTA-AM; Abcam, Cambridge, UK), 5 μM STO-609 (Merck-Millipore, Darmstadt, Germany),4 μM ionomycin and 2.5 μM Fluo-3 AM (Beyotime Institute of Biotechnology, Haimen, China). The toxicities of both drugs and siRNAs were tested using the WST-1 Cell Proliferation and Cytotoxicity Assay Kit (Beyotime), according to the manufacturer’s instructions. At 36, 48, and 60 h post-infection (hpi), DEF cells were collected for subsequent analysis.
Western blot analysis
Proteins from cells treated with either drugs or siRNAs, or infected with DEV were extracted using immunoprecipitation lysis buffer (Beyotime) with the protease inhibitor phenylmethylsulfonyl fluoride (Beyotime), then boiled for 10 min in 5× loading buffer, separated by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and transferred onto nitrocellulose membranes (GE Healthcare Life Sciences, Little Chalfont, UK), according to manufacturers’ instructions. The membranes were blocked with 3% bovine serum albumin (Sigma-Aldrich Corporation, St. Louis, MO, USA) for 2 h at room temperature and then incubated with the following primary antibodies for 2 h at room temperature: rabbit anti-LC3B antibody (Sigma-Aldrich Corporation), mMouse anti-CaMKKβ antibody (Sigma-Aldrich Corporation), rabbit anti-p-AMPK antibody (Thermo Fisher Scientific, Waltham, MA, USA), mouse anti-AMPK antibody (Thermo Fisher Scientific), mouse anti-β-actin antibody (Sigma-Aldrich Corporation).Then, the membranes were incubated with IRDye 800 CW goat anti-mouse or goat anti-rabbit immunoglobulin IgG as secondary antibodies for 1 h at room temperature. Antibody detection was conducted using an Odyssey Infrared Fluorescence Scanning Imaging System (LI-COR Biosciences, Lincoln, NE, USA). Quantitation from western blot image intensity was achieved by adding rectangle to the image to gain data directly using the Odyssey Infrared Fluorescence Scanning Imaging System Application Software Version3.0.
Confocal fluorescence microscopy
For the detection of autophagosomes, DEF cells at 70–80% confluence in culture dishes were transfected with 2.5 μg of the GFP-LC3 plasmid using the Calcium Phosphate Transfection Kit (cat. no. K2780–01; Invitrogen Corporation, Carlsbad, CA, USA). At 24 hpi, chemical-treated or virus-infected DEF cells at different time points were fixed with absolute ethanol for 30 min and the cell nuclei were stained with 4′-6-diamidino-2-phenylindole (cat. no. D1306; Beyotime). The green fluorescence of GFP-LC3 was observed by confocal laser microscopy using a Leica SP2 confocal system (Leica Microsystems, Wetzlar, Germany).
CaMKKβ siRNA
In order to further study the effects of cell autophagy on viral replication, siRNA targeting the autophagy-related gene beclin-1 was synthesized (Shanghai GenePharma Co., Ltd., Shanghai, China). The sequence of the siRNA was GCC UAC AAC GAG GAC GAU ATT (sense) and UAU CGU CCU CGU UGU AGG CTT (antisense). Six-well plates were transfected with siRNA and negative control (NC)-siRNA using transfection reagents for 24 h and then infected with DEV. Cell samples were collected to detect the effects of siRNA.
Median tissue culture infective dosed (TCID50)
DEF cells were cultured in covered 96-well plates and then infected with DEV virus diluted to 10− 1 to 10− 8. At 72 hpi, the cells were observed and pathological changes were recorded. Viral titers were determined according to the Reed–Muench method.
Intracellular Ca2+ detection by flow cytometry
Cytosolic free Ca2+ ions were detected by using Fluo-3 AM. Fluo-3 AM itself is not combined with Ca2+ ions, but once dye is added to the cells, it can hybridize with Fluo-3 AM, and Fluo-3 AM will fluorescence upon binding to Ca2+. DEF cells were infected with DEV or treated with BAPTA-AM for the indicated times, then incubated with Fluo-3 AM in the dark at 37 °C for 1 h. Afterward, the cells were suspended in phosphate-buffered saline. To observe fluorescence, as an indicator of intracellular Ca2+ ions, the cells were monitored using a flow cytometer (BD FACSAria™; BD Biosciences, San Jose, CA, USA) at an excitation wavelength of 488 nm.
Statistical analysis
All experimental results are expressed as the mean ± standard deviation (SD) of three independent experiments. The Tukey’s test was used for statistical analysis. A probability (p) value of < 0.05 was considered statistically significant.