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Baby hamster kidney cells BHK-21 (preserved in "Chinese general virus collection center" (CGVCC)) were cultivated in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS) in 5% CO2 at 37 ℃. The JEV strain SA14 was recovered from pACYC-JEV (kindly donated by Prof. Bo Zhang, Wuhan Institute of Virology, China) as previously described and stored at -80 ℃ (Li et al., 2014). JEV-specific antibodies and anti-EDIII and anti-NS1 were used as previously described (Du et al., 2015).
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The full-length JEV infectious cDNA clone pACYC-JEV was modified (Li et al., 2014). Firstly, the 5′-terminal nucleotides (1–172 bp) of the virus genome were introduced immediately downstream of the CMV promoter of pEGFP-C1 (Clontech, CA, USA) by overlap-extension polymerase chain reaction (OE-PCR) to generate pJEV-5UTR, and the nucleotides corresponding to the enhanced green fluorescent protein (egfp) gene was removed. Then, the nucleotides of the virus genome from nucleotide 2560 to the 3′-terminus as well as nucleotides encoding the ribozyme of hepatitis delta virus (HDVr) and the polyadenylation (poly A) sequence of Simian virus 40 (SV40) at the 3′-terminus of the genome were inserted downstream of the 5′-terminal nucleotides of pJEV-5UTR, generating the pJEV-infu backbone, which lacks nucleotides 173–2559 corresponding to the entire sequences of the prM and E proteins, as well as small portions of the C and NS1 proteins. Note that the engineered site where nucleotide 172 was ligated to nucleotide 2560 directly generates a new unique endonuclease Sma I site, which could be used for the linearization of pJEV-infu.
To generate mutated viruses using the pACYC-JEV infectious clone system, mutations were introduced into the pACYC-JEV plasmid by OE-PCR as described previously (Li et al., 2014).
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The JEV-complement fragment was generated using pACYC-JEV as a template and the primers infusion-F and infusion-R (Table 1). The resulting DNA fragment encodes nucleotides 158 to 2574 of JEV SA14, corresponding to the entire missing sequence region in pJEV-infu as well as 15 nucleotides extending at the 5′-terminus and 3′-terminus, respectively.
Table 1. Primer sequences used in this study.
OE-PCR was performed to introduce mutations into the JEV-complement fragment (Lin et al., 2012). Briefly, each desired mutation was introduced into two partially overlapping "inner primers." A left-PCR was performed using infusion-F as a common forward "outer" primer and the antisense inner primer, whereas a right-PCR was performed including a sense inner primer and a common reverse "outer" primer, infusion-R. After gel purification, these two first-round PCR products were used together as a template for a second PCR, employing the forward and reverse "outer" primers described above.
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The backbone plasmid pJEV-infu was linearized by Sma I digestion, and the JEV-complements were ampli fied by PCR. The products were then gel-purified and in vitro recombination reactions were performed by incubating the linearized backbone (0.2 μg) and the complementary fragment (0.2 μg) with 2 μL of 5× In-Fusion HD Enzyme Premix (Clontech, CA, USA) in a final volume of 10 μL according to the manufacturer's instructions. The entire recombination reaction mixture was directly transfected into BHK-21 cells (5 × 105 cells/well) in a six-well plate using Lipofectamine 2000 (Invitrogen, CA, USA) according to the manufacturer's instructions. Transfected cells were cultured, and virus-containing supernatants were harvested at 2 to 5 days post transfection (p.t.). After centrifugation at 500 × g for 10 min to remove cell debris, the supernatants were frozen at -80 ℃ until use.
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BHK-21 cells (1 × 105 cells/well) grown on 24-well plates were infected with the supernatants of transfected cells obtained at 5 days p.t. as described above. At 36 h post infection (p.i.), the cells were fixed with paraformaldehyde (4% in phosphate-buffered saline [PBS]) for 15 min and then treated with PBS containing 0.5% Triton X-100 for a further 15 min at room temperature. Permeabilized cells were then stained with a JEV NS1-specific primary antibody (anti-NS1) and goat-anti-rabbit (fluorescein isothiocyanate [FITC]-conjugated; Proteintech, Chicago, USA) immunoglobulin.
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BHK-21 cells (2 × 105 cells/well) grown on 12-well plates were infected with the supernatants of the transfected cells obtained as described above. At 36 h p.i., the cells were lysed and prepared for sodium dodecyl sulfate-polyacrylamide gel electrophoresis, followed by transfer to a poly (vinylidenefluoride) membrane using a Trans-Blot apparatus (Bio-Rad, CA, USA). Western blot was performed with JEV-specific antibodies (anti-EDIII and anti-NS1) as primary antibodies and horseradish peroxidase-conjugated goat anti-rabbit antibody (Proteintech, Chicago, USA) as a secondary antibody. The antibody–protein complexes were visualized with SuperSignal West Pico Chemiluminescent Substrate (Fermentas, CA, USA) and the MicroChemi Bio-imaging System (DNR, IN, USA).
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Monolayer cultures of BHK-21 cells (2 × 105 cells/ well) grown in 12-well plates were inoculated with sequentially diluted JEV. After 2 h of incubation at 37 ℃, the cells were washed with 1 mL DMEM three times and then coated with a layer of 1% SeaPlaque agarose (Lonza, Switzerland) in 2% FBS-DMEM. At 3 days p.i, 0.1% crystal violet solution was added to visualize the plaques.
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Virus titers were determined based on the 50% tissue culture infectious dose (TCID50) using standard cell culture procedures. Briefly, BHK-21 cells were grown in 96-well plates, and six replicates were infected with 100 μL of 10-fold serial dilutions of the virus sample. After incubation at 37 ℃ for 3 days, the cytopathic effect (CPE) was monitored using an inverted microscope, and infectivity titers were expressed as TCID50/mL according to the method previously described (Reed and Muench, 1938).
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BHK-21 cells were infected with the indicated viruses at a multiplicity of infection (MOI) of 0.01. Cell-free media were collected at 12, 24, 36, 48, and 60 h p.i., and virus titers were determined as described above. Each virus infection was performed in triplicate.
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BHK-21 cells transfected with ligation reaction mixtures were harvested at 5 days p.t., and total RNA was extracted using TRIzol reagent (Invitrogen, CA, USA). One-step qRT-PCR was then carried out using One Step SYBR PrimeScriptTM PLUS RT-PCR Kit (Takara, Japan) following the manufacturer's instructions. Primers targeting the NS1 regions of JEV were used, including the forward primer JEV qRT-F and the reverse primer JEV qRT-R (Table 1). The amount of JEV genome was normalized to the β-actin gene using the comparative cycle threshold values determined in parallel. Primer sets targeting β-actin included the forward primer actin-F and reverse primer actin-R (Table 1). PCR specificity was verified by melting curve analysis.
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In vitro RNA transcription was performed as previously described (Li et al., 2014). Briefly, the wild-type or mutated infectious clone pACYC-JEV plasmids were digested with Xho I and purified, and were then subjected to in vitro transcription using a MEGAscript T7 kit (Ambion, TX, USA) according to the manufacturer's protocols. The RNA was resolved in RNase-free water and stored at -80 ℃. BHK-21 cells (1 × 105 cells/well) growing in a 24-well plate were transfected with 200 ng wild type or mutated transcripted viral RNA. At 48 h p.t., the cells were fixed and stained with anti-NS1 antibody.