Cell, virus, antibody and protein
Vero-1008 was grown and maintained in Dulbecco’s Modified Eagle’s Medium (DMEM, Sigma-Aldrich, MO, USA) supplemented with 10% heated-inactivated fetal bovine serum (Gibco, Life Technologies Corp., Calif., USA), 100 μg/ml streptomycin and 100 IU/ml penicillin (Sigma-Aldrich, MO, USA) at 37 °C with 5% CO2. A BrCr strain of EV71 was provided by Dr. H Wang in Wuhan Institute of Virology (WIV), Chinese Academy of Sciences (CAS). A mouse-adapted EV71 (EV71/MAV-VR) used in this study was kindly provided by Dr. Z Huang in Key Laboratory of Molecular Virology and Immunology, Institut Pasteur of Shanghai, Shanghai Institutes for Biological Sciences, CAS, China [21]. The BrCr and MAV-VR EV71 strains have the same 3Dpol amino acid sequence. For clarification, EV71/MAV-VR was only used in the challenge experiment, while the rest EV71 mentioned in this study represent EV71 BrCr strain. A purified recombinant 3Dpol protein was provided by Dr. P Gong in WIV, CAS [22].
Animals
BALB/c aged 6–8 wks and pregnant ICR mice were purchased from Beijing Laboratory Animal Research Center and housed under specific pathogen free (SPF) conditions in the Animal Center of WIV, CAS, China.
Generation of mAbs against EV71 3Dpol
IgG mAbs against EV71 3Dpol were developed by traditional hybridoma technique as previously described [23]. In brief, 5-wk-old female SPF BALB/c mice were immunized subcutaneously with 100 μg of 3Dpol at 2-wk interval. Four wks after the last booster and 3 days before cell fusion, the mice were boosted with 200 μg of 3Dpol. Three days later, murine splenocytes were harvested and fused with SP2/0 using 50% polyethyleneglycol (Sigma-Aldrich, MO, USA). Hybridoma culture supernatants were preliminarily screened by EV71 3Dpol protein using ELISA. The positive hybridoma cells were cloned by a limiting dilution and the stable hybridoma clones were injected into liquid paraffin-pretreated abdominal cavities of BALB/c mice. Subsequently, the mAbs were harvested and purified from the ascite with an antibody purification kit according to the manufacturer’s specifications (NAb™ Protein A/G Spin Kit, Thermo Scientific, IL, USA). This mouse study was approved by the ethics committee of life science and research in Wuhan Institute of Virology (WIV), Chinese Academy of Sciences (CAS) (No. WIVA09201502).
Immunofluorescence assay (IFA)
Vero-1008 cells were seeded into 24-well tissue culture plates (Costar Corning Inc., NY, USA) at a concentration of 1 × 105 cells per well. When the cells reached approximately 80% confluence, culture medium was removed and then cells were washed three times with sterile PBS (pH 7.4) and incubated with EV71 (MOI = 0.1) for 1 h at 37 °C. After removal of supernatant, fresh medium was added and cultures were incubated at 37 °C. At 24 h of post infection, the infected cells were fixed with absolute methanol and processed for indirect immunofluorescence assay (IFA) using purified mAbs, followed by fluorescein isocyanate-conjugated goat anti-mouse IgG for confirming antibody specificity against EV71. Fluorescent images were examined under a fluorescent microscope.
Immunoblotting
The EV71-infected Vero-1008 were separated by SDS-PAGE and then electrophoretically transferred to PVDF membrane (GE Healthcare, PA, USA). The membrane was blocked for 1 h at RT with blocking solutions containing 5% Bovine Serum Albulmin (BSA) in TBS (20 mM Tris-HCl (pH 7.5), 150 mM NaCl), and then incubated overnight with purified mAbs at 4 °C. After washing with T-TBS (TBS + 0.05% Tween 20), the membrane was incubated for 45 min with HRP-conjugated anti-mouse IgG (Jackson ImmunoResearch Laboratories, PA, USA). After washing with T-TBS, the membrane was developed by treatment with ECL Western Blotting Detection Reagents (Boster, Wuhan, China). A house keeping protein β-actin was also detected as control.
EV71 infection and mAbs IgG transfection
Vero-1008 cell was seed in 24-well plate 1 × 105 cell per well culturing for 24 h, and was infected with EV71 (MOI = 0.1). Three hrs later, EV71-infected were transfected with IgG using Liposomal Transfection Reagent DOTAP (Roche, Basel, Switzerland) as previously described [24]. Briefly, 15 μl of DOTAP was incubated with 10 μl of purified IgG (5 μg/well) for 20 min in serum-free medium. After incubation, 500 μl DMEM medium and mixture was added. Twenty-four hrs later, virus-infected cell were taken pictures and undergone three freeze-thaw circles, and were subject to determine the viral titers.
Plaque assay
A series of 1:10 dilutions were made by mixing 15 μl of EV71-infected cell with 135 μl of DMEM. One hundred microliters of each dilution were seeded to individual wells of 24-well plate containing confluent Vero-1008 cells (2 × 105 cells/well). The plate was incubated at 37 °C with 5% CO2 for 1 h before the layer of 2% methyl cellulose was added. After 4 days of incubation at 37 °C with 5% CO2, the cells were fixed in 3.7% formaldehyde and then stained with 1% crystal violet. Plaque numbers were recorded after washing the plates with tap water [25].
RdRp-mediated RNA elongation assay in vitro
In 10 μl reaction system containing 50 mM HEPES, 75 mM KCl, 5 mM MgCl2, 4 mM TCEP, 300 μM NTP, and 4 μM RNA Complex, the 5 μg functional 3Dpol was added, and the system maintained at 22.5 °C for 30 min, finally the reaction was terminated with RAB. RNA species were resolved by 15% polyacrylamide/7 M Urea gel electrophoresis and visualized by Stains-All staining [26]. RNA elongation activity of 3Dpol could be assessed based on the appearance of elongated RNA band.
In vivo evaluation of antiviral efficacy of mAbs
The protective efficacy of the mAbs was evaluated by an in vivo assay as previously described [21]. Pregnant ICR mice maintain under SFP condition. Five groups of neonatal ICR mice at 1 day of age were inoculated i.p. with 50 μg of mAb/mouse (50 μl) respectively, followed by i.p. inoculation with 3.55 × 106 TCID50 of EV71/MAV-VR (50 μl) at 24 h later. The challenged mice were monitored daily for survival and clinical score for 16 days. Clinical scores were graded as follows: 0, healthy; 1, reduced mobility; 2, limb weakness; 3, paralysis; and 4, death. Mice that lost more than 35% of the control mice (PBS treated group) body weight were euthanized and counted as dead. All the mice were observed for 16 days and euthanized by injection with ketamine mixture. Mice were euthanized by injection (i.m.) with 15 μg/50 μl of ketamine mixture (79.3% Ketaject, 17.5% Atropine, and 3.2% Acepromazine) per gram mice body weight. The EV71/MAV-VR challenge study was approved by the ethics committee of life science and research in Wuhan Institute of Virology (WIV), Chinese Academy of Sciences (CAS) (No. WIVA09201504).
Statistical analyses
Kaplan-Meier survival curves were used to display mortality data, and log rank analyses were performed to determine statistical significance between different groups. * indicates value of p < 0.05, ** indicates value of p < 0.01.