
Cells and viruses
African green monkey kidney cells (Vero cells) were preserved by the Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonosis. The cells were cultured in a disposable 25 cm2 square flask with Dulbecco’s Modified Eagle Media (DMEM) medium containing 5% fetal bovine serum and 1% Penicillin–streptomycin (double antibiotics). The culture conditions were 37 °C and 5% CO2 in an incubator. PEDV strains were isolated and preserved by Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonosis, Yangzhou University. PEDV was subcultured using standard virus adsorption techniques. The viral titers were measured in Vero cells with a TCID50 and stored at − 80 °C until further use.
Selection and design of PEDV interference area
Bioinformatics analyses were used to perform a multiple sequence homology alignment on existing PEDV N and S gene sequences on NCBI, and the conserved sequences were identified as the miRNA target regions of PEDV by a combination analysis (Table 1). AmiRNA interference region selection: the Invitrogen BLOCK-IT™ RNAi (http://www.invitrogen.com/rnai) expression onlinesearch engine design software was applied. According to the optimization principle, sequences with a content of 40–55% were selected for PEDV N and S gene conserved regions, and the scoring principle should be above 4.5 points. The selected sequences were subjected to an online homologous analysis with the porcine source gene sequences in GenBank to minimize the potential non-specific target effect, and two corresponding specific expression sequences were designed. The negative control was provided by Invitrogen, and the single chain oligonucleotide (Oligo) was synthesized by Nanjing Qingke Biotechnology. The plasmid contains an insert that can form hairpin structures and be processed into mature miRNAs; however, it is not expected to target any known vertebrate genes, and the synthesized sequences are all 64 bp in length.
Two sequences were designed using Invitrogen Block iT RNAi Designer targeting sequences of the N and S regions of PEDV.
Construction of miRNA expression vectors, as well as the construction, transformation, and purification of the plasmid and identification of the recombinant plasmid
Each pair of single-chain oligonucleotides was annealed, and the reaction system was: positive DNA oligo (200 μm) 5 μL, negative DNA oligo (200 μm) 5 μL, 10× Annealing Buffer 2 μL (100 mM Tris–HCl pH 8.0, 500 mM NaCl, 10 mM EDTA), and 8 μL deionized water. The reaction system was incubated at 94 °C for 5 min and then slowly cooled to room temperature for annealing. The double-stranded oligonucleotides were cloned into the pcDNA™6.2-GW/ EmGFP-miR expression vector to construct the eukaryotic expression plasmid, and the ligation reaction product was transformed into the TOP10 competent cells. Monoclonal colonies were selected for amplification and culture, and the plasmid was extracted. The restriction analysis was performed by BamHI and XhoI, and the correct plasmid was identified by enzyme digestion. EmGFP forward sequencing primers and miRNA reverse sequencing primers were sent to Nanjing Qingke Biotechnology for sequencing. The selected positive recombinant plasmids were further confirmed by sequencing, and the correctly sequenced plasmids were termed amiRNA-349 and amiRNA-1447, respectively.
Optimization of cell transfection efficiency
The day prior to transfection, Vero cells were digested and seeded into six-well cell plates at a density of 4 × 105 cells per well. When the cell density reached 70–80%. The ratio of plasmid amiRNA-349: Lipofectamine™3000 was 1:0.75–1:1.5, and the plasmid dose was 4 μg, 5 μg, and 6 μg, respectively. Lipofectamine™3000 was used as the transfection reagent for the plasmid transfection. After a 48 h culture for transfection efficiency was observed and photographed.
Cytotoxicity assay
Cell Counting Kit-8 (CCK-8) (Vazyme, China) was performed in accordance with the manufacturer’s instructions. Vero cells were digested and added to 96 well plates at a density of 2 × 104 cells per well and transfected with 100–800 ng/well of plasmids expressing amiRNA in three replicates for each concentration. Cell viability was detected using a Cell Counting Kit-8 (CCK-8) assay after Vero cells were transfected with amiRNAs for 48 h.
CCK-8-mediated detection of cytotoxicity
Under optimized transfection conditions, miRNA expression vectors were transfected into Vero cells. The negative control and no-load control were established at the same time. After normal culture, PEDV strains with 0.01 MOI were inoculated in each well. Cell viability was measured 48 h after PEDV infection using CCK-8 Cell Counting Kit according to the manufacturer’s instructions. The absorbance values of amiRNA-treated and virus-infected cells were compared with those uninfected samples without transfection reagents and amiRNA.
Detection of the viral titer
The cell culture, plasmid transfection, and PEDV infection conditions were performed as described above. The cell supernatants were collected at 24 h, 48 h, and 72 h after viral infection, and their TCID50 was determined using the Reed-Muench method.
Real time RT-PCR assay for the detection of PEDV RNA
The cell culture, plasmid transfection, and PEDV infection conditions were performed as described above. The cells were collected at 24 h, 48 h, and 72 h after infection and the total RNA was extracted. Using the 18 s gene as an internal reference, PEDV N gene transcription was detected by relative quantitative RT-PCR according to the instructions of the ChamQ Universal SYBR qPCR Master Mix (Vazyme, China). The positive and reverse primer sequences of the internal reference were as follows:forward primer (18 s F), 5′–TCAGATACCGTCGTAGTTCC–3′;reverse primer (18 s R), 5′–TTCCGTCAATTCCTTTAAGTT–3′.
The specific primer sequences of N gene were as follows:forward primer (PEDV-N F), 5′–CGATGATCTGGTGGCTGCTGTC–3′;reverse primer (PEDV-N R), 5′–TTCCTGCTTAGGCTTCTGCTGTTG–3′.
The 20 μL PCR reaction system consisted of ChamQ Universal SYBR qPCR Master Mix (2×) 10 μL, forward and reverse primers 0.4 μL, reverse transcription cDNA template 2 μL, and ddH2O2 7.2 μL. Amplification procedure 95 °C, pre-denaturation 30 s; 95 °C for 5 s, 60 °C for 34 s, for 40 cycles; 95 °C for 15 s, 60 °C for 60 s, and 95 °C for 15 s. PCR products were used to analyze the relative expression of the PEDV N gene in each group using a 7500 Real-time PCR System software. At the same time, the 18 s gene was set as the internal reference gene, and three replicates were performed in each group.
Western Blot analysis
The cell culture, plasmid transfection, and PEDV infection conditions were performed as described above. The Vero cells were collected PI24h, 48 h, and 72 h, and RIPA (Beyotime, China) was added and placed on ice. The cells were shaken three times once every 10 min, and centrifuged. The supernatant was collected and the α-tubulin protein was used as an internal reference. The cell lysates were dissolved on 12.5% polyacrylamide gel and then electroimprinted on an NC membrane. The membrane was sealed with 5% BSA at 4 °C overnight. The membrane was then incubated with an anti-PEDV-specific polyclonal antibody and an α-tubulin (Cell Signaling Technology, USA) of PEDV was used as an internal sample control.
Chaining of amiRNAs in a single expression construct
This study uses a pcDNA™ 6.2-GW/EmGFP-miR expression vector which permits the co-cistronic expression of multiple amiRNAs in a single expression construct. Briefly, to construct an expression vector which simultaneously expresses two amiRNA, and the donor plasmid is first excised with BamHI/XhoI restriction digestion enzymes to release the insert. The insert was ligated into another amiRNA vector backbone, which is predigested with BglII/XhoI enzymes. A single vector constructs generated by chaining of amiRNA-349 and amiRNA-1447, termed amiRNA-349 + 1447. The effect of concatenated amiRNA-349 + 1447 in PEDV replication inhibition was studied by TCID50, relative qRT-PCR, and Western blot analysis as mentioned above.
Statistical analysis
Results were graphed, with error bars indicating the standard deviation. Statistical analyses were done with Prism 8.4.3(GraphPad Software), and statistical significance was determined using Student’s t test or one-way analysis of variance (*P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001).