Cells and virus
The VerodogSLAM cells (kindly provided by Dr. V. von Messling, Veterinary Medicine Division, Paul-Ehrlich-Institute, Germany) and human embryonic kidney cells (HEK 293) (ATCC CRL-1573) were cultivated in Dulbecco’s modified Eagle’s medium (DMEM High Glucose, Sigma) containing 10% fetal bovine serum (FBS, Gibco), 1% penicillin/streptomycin, and 1 mM L-glutamine (Gibco) at 37 °C in a humidified incubator with 5% CO2. Zeocin (Invitrogen) was added at 1 mg/mL to maintain selection for SLAM (Signaling lymphocytic activation molecule) expression in the VerodogSLAM cell line. The HEK 293 cells were used to generate, grow, and titer the recombinant adenovirus.
A CDV wild-type strain (CDV/LDM-BTU-2, GenBank accession number KJ865300.1), isolated from canine clinical specimens, was used in this study. The CDV/LDM-BTU-2 strain was propagated on the VerodogSLAM cells, and virus stocks were prepared by collecting the infected cells plus supernatant (sup) when CPE (cytopathic effect) was ~ 80%. The virus stock was stored in aliquots at − 80 °C, and virus titers were determined by the method of Reed and Muench (1938) and expressed as log10 TCID50/mL.
Design and construction of shRNA expression plasmids
The CDV N and L genes were defined as virus targets for designing shRNAs because of their high degree of conservation among CDV isolates. We aligned all partial and complete sequences of CDV-selected genes available in GenBank, using the MEGA software v. 6 [54]. We chose three different 20-nt successive stretches with a high degree of similarity for each gene. Top and bottom oligos specific for each gene segment were designed using the web-based BLOCK-iT RNAi Designer (Invitrogen, USA) available at https://rnaidesigner.thermofisher.com/rnaiexpress/ and custom-synthesized by Life Technologies (USA). The chosen shRNA sequences against CDV L and N genes are summarized in the Additional files 1 and 2. Each top strand was designed to include a gene homologous region, a loop, an antisense sequence, and 5′ overhangs for cloning into the pENTR/U6 RNAi entry vector (Invitrogen). A blast search against the canine (Canis lupus familiaris) genome confirmed no homology between the oligo sequences and any other genes. A scrambled sequence (SCR) with no homology to any known CDV gene sequence was also generated as negative control for designed shRNAs (Additional file 1). The predicted folding of the selected shRNAs was done using the mfold web server v. 2.3 [55]. The thermodynamic stability (∆G, Kcal/mol) of the shRNA duplexes (Additional file 2), which determines the asymmetrical RISC assembly, was calculated as previously described [56].
Sense and antisense oligos were mixed, heated at 95 °C for 5 min, and allowed to anneal by slowly decreasing the temperature. Double-stranded oligonucleotide was cloned into the pENTR/U6 vector downstream of the human U6 shRNA promoter (Fig. S1B) following an established protocol (Invitrogen). Recombinant plasmids were verified by colony PCR using M13 primers and Ndel and Xbal double restriction digestion (Thermo Scientific). All constructs were sequenced to ensure the correct design. All seven resulting pENTR/U6-shRNA constructs were used for transient shRNA expression in VerodogSLAM cells and evaluation of antiviral activity.
Transfection of shRNA constructs
The VerodogSLAM cells were seeded 1 day prior to transfection in 24-well tissue culture plates at a density of 2 × 105 cells/well in DMEM supplemented with 10% FBS, so that the cell monolayer was fully confluent by the time of the transfection. After 24 h, cells were inoculated with CDV/LDM-BTU-2 at a multiplicity of infection (MOI) of 0.01. Following an incubation period of 2 h at 37 °C, the inoculum was removed and the cells were washed twice with 500 μL of DMEM. Four different amounts of plasmids (0.25, 0.5, 0.75, and 1 μg) were each complexed with 2 μl Lipofectamine 2000 (Invitrogen) per well in 100 μL of DMEM serum-free medium. Plasmid DNA and Lipofectamine 2000 were mixed and incubated for 20 min at room temperature according to the manufacturer’s instructions. The infected cells were washed, and the medium was replaced with 200 μL of fresh DMEM serum-free medium, followed by the addition of 100 μL of plasmid-lipofectamine complexes. Six hours later, the transfection medium was replaced by 1 mL of DMEM containing antibiotics and 2% FBS. All assays were performed in triplicate. At 72 h post-infection, RNAi silencing efficacy was evaluated by observing and scoring the cytopathic effects (CPE). The CPE scoring scale was defined as follows: 1 – CPE < 25%, 2 - CPE between 25 and 50%, 3 - CPE between 51 and 75%, and 4 – CPE > 75%, as exemplified in Additional file 3. At the same time point, the plates were frozen/thawed 1X, and cell lysates plus supernatant were harvested and stored at − 80 °C until use for virus titration and real-time quantitative RT-PCR analysis. The virus titer was determined according to the method of Reed and Muench, [57] and expressed as log10 TCID50/mL. Target-specific shRNAs constructs with the highest silencing effects were co-transfected to determine whether gene suppressive activity would be enhanced.
Construction of multiple shRNA-encoding adenoviral vector
Multiple shRNA-expression cassettes were designed with three N-specific shRNAs under control of the U6 promoter. The target gene was determined according to the suppressive activity of plasmid-based shRNAs previously mentioned. The shRNA sequences (Ni1, Ni2, and Ni3) selected for construction of the recombinant adenovirus are summarized in Additional file 1. The multi-shRNA fragment was synthesized (GenScript Corporation, Piscataway, NJ USA) with restriction enzyme sites Clal and Xbal as sticky ends and inserted into a shuttle plasmid (pUC57), using standard cloning procedures. Likewise, a scrambled shRNA control cassette containing sh-SCR (Additional file 1) was synthesized and transferred into a pUC57 vector.
To generate the recombinant replication-defective Human adenovirus type 5 (Ad5) vector, each shuttle pUC57 construct was digested with Clal and Xbal (New England Biolabs), and the fragment of interest was ligated into the corresponding sites of the pAd5-Blue vector. The DNA ligation mix was transformed into electrocompetent TOP10 cells, and the obtained colonies were screened by PCR with the primers pAd5 forward 5′-AAGTGTGGCGGAACACATGTAAGC-3′ and pAd5 reverse 5′-AAGCAAGTAAAACCTCTACAAATGTGGTATGG-3′, which flank the transgene. Plasmid DNA minipreps were digested with Hindlll (New England Biolabs) and compared with the vector control pAd5-Blue to verify the integrity of the Ad5 constructs and to identify which minipreps contained the desired transgene. Plasmids were also digested with BsrGl (New England Biolabs) and sequenced in an ABI PRISM® 3100 Genetic Analyzer to confirm their correctness. The Ad5 recombinant plasmids were linearized with Pacl (New England Biolabs), purified by ethanol precipitation, and transfected into low-passage HEK 293 cells that had been plated the previous day. The HEK 293 cells cultured in 6-well plates at 30% confluence were transfected with the linearized adenoviral plasmids using PolyFect Transfection Reagent (Qiagen) and visually monitored for CPE (cell rounding and detachment). Recombinant viruses were harvested 7–10 days after transfection upon the appearance of CPE, amplified in HEK 293 cells, and confirmed by PCR (pAd5 forward and reverse) and restriction analyses (Hindlll digestion). The Ad5 vector constructs expressing N-specific multi-shRNAs and scrambled shRNA were defined as Ad5Ni(1–3) and Ad5SCR, respectively. A schematic representation of the recombinant viruses is represented in Fig. 7. Virus titration was performed with serial 10-fold dilutions of recombinant adenoviruses and expressed in log10 TCID50/mL as described by Reed and Muench [57].
Assessment of cell viability after Ad5 vector infection
Cell toxicity of recombinant Ad5Ni(1–3) and Ad5SCR was determined using a colorimetric method which is based on the mitochondrial reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) (Sigma, St. Louis, MI). Briefly, VerodogSLAM cells were plated at a density of 1 × 104/well in 96-well microplates and inoculated 24 h later with DMEM-diluted Ad5 constructs at MOI values of 10, 20, 30, 40, 50, 100, 200, and 300. After a 1-h adsorption period at 37 °C, the inoculums were removed and cells were washed twice with DMEM. Subsequently, 200 uL of DMEM with 2% FBS were added and cells were incubated for 72 h at 37 °C. At 72 h post-transduction, the culture medium was removed and 50 μL of MTT working solution (1 mg/mL) were added to each well; the microplate was incubated for 4 h. The MTT formazan crystals were solubilized by adding DMSO, and the optical densities were determined by an absorbance microplate reader (ELX800 Absorbance Microplate Reader, BioTek, Winooski, Vermont, USA) with a 540-nm optical filter. Cell viability was calculated by subtracting the optical density fraction of treated cells from the untreated cells. At least three independent experiments were performed, with eight replicates per treatment.
Pre-treatment with the recombinant Ad5 construct
The VerodogSLAM cells were seeded 1 day prior to the experiment in 24-well tissue culture plates at a density of 105 cells/well in DMEM supplemented with 10% FBS. Cells were transduced with Ad5Ni(1–3) at MOI values of 5, 10, 20, and 30, and after a 1-h adsorption period at 37 °C, the inoculums were removed, cells were washed twice with DMEM, and the medium was replaced by culture medium containing 2% FBS. After an incubation time of 12 h, cells were infected with CDV/LDM-BTU-2 at the MOI of 0.01 and incubated for 2 h at 37 °C and 5% CO2. The viral inoculum was removed and cells were washed twice with DMEM and replaced by culture medium with 2% FBS. Mock and CDV infection controls (no Ad5 construct pre-infection) were set up in parallel. Recombinant adenovirus with scrambled shRNA sequence was also evaluated under the same conditions. At 72 h post-inoculation, 24-well plates were frozen/thawed 1X, and cell lysates plus supernatant were harvested and stored at − 80 °C until use for virus titration by TCID50, plaque-forming unit reduction assay, and real-time quantitative RT-PCR analysis. All assays were performed in triplicate.
Post-treatment with recombinant Ad5 construct
The VerodogSLAM cells were seeded in 24-well plates (105 cells/well in 500 μL DMEM + 10% FBS) and incubated for 24 h (37 °C, 5% CO2). Cells were first infected with CDV/LDM-BTU-2 at the MOI of 0.01. Two hours later, viral inoculum was removed, cells were washed twice with DMEM, and the medium was replaced by 2% FBS culture medium. At 12 h post-inoculation, cells were transduced with Ad5Ni(1–3) at MOI values of 5,10, 20, and 30. After a 1-h adsorption period at 37 °C, the inoculums were removed, cells were washed twice with DMEM, the medium was replaced by 2% FBS culture medium, and the plates were placed in the incubator. After 72 h, the plates were frozen/thawed once, and cell lysates plus supernatant were harvested and stored at − 80 °C until use for viral analysis and measurement procedures as described under 2.6. Mock infections and CDV infections (no Ad5 construct pre-infection) were included in the experiment; Ad5SCR was evaluated under the same conditions. All assays were performed in triplicate.
Ad5-shRNA treatment at various time intervals
The VerodogSLAM cells were first infected with CDV/LDM-BTU-2 at the MOI of 0.01 and then transduced with Ad5Ni(1–3) vector at either MOI 2.5 or 5 at 2, 12, and 24 h post-infection. The experiments were done in triplicates and repeated independently. Plates were incubated at 37 °C for 72 h. All subsequent steps were performed as explained earlier under 2.7. Mock infection and non-treated CDV infection controls were set up in parallel. Virus reduction analysis read out was based on viral titer (TCID50/mL), RNA copy number, and inhibition percentage, according to the methods described above.
Plaque-forming unit reduction assay for Ad5-expressed shRNA treatment
The gene suppressive activity of Ad5Ni(1–3) was determined by measuring the reduction in the number of CDV infectious plaques relative to the untreated control. Briefly, confluent monolayers of VerodogSLAM cells by 2 × 105 were plated in each well of 24-well plates and incubated for 24 h. Samples of the above Ad5 construct treatments were added to the cells, and after 2 h of incubation, the inoculums were removed and the cells were overlaid with 2.5% carboxymethyl cellulose (CMC) with 10% FBS and incubated for 72 h. After that, cell monolayers were fixed and stained with crystal violet solution, and the plaque numbers were counted. The Ad5SCR treatment samples were also evaluated under the same conditions. The percentage of plaque reduction (PR %) compared to untreated infected cells was calculated using the following formula: PR (%) = (C - T) × 100/C, where C is the mean of the number of plaques from triplicate untreated control wells and T is the mean of the number of plaques from triplicate treated wells.
Quantification of transcripts and viral genome
Total RNA was extracted from cell lysate plus supernatant (500 μL), using Trizol reagent (Invitrogen) according the manufacturer’s instructions. The RNA was eluted in 20 μL and stored at − 80 °C. The concentration and quality of RNA were checked using a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific). For each real-time assay, 100 ng of RNA template were analyzed by RT-qPCR, following the protocols of the GoTaq 1-Step RT-qPCR System (Promega) (final volume 20 ul). Primers for the CDV N gene (Additional file 4) were designed based on previously published sequences [58, 59]. Selection of primers for the L protein-encoding gene was conducted by multiple sequence alignment using the MEGA software v. 6. A conserved region of the CDV L gene was selected for designing the primers with a product length of 136 bp (Additional file 4). Primers sets were synthesized by Integrated DNA Technologies (IDT). The RNA measurement by SYBR green incorporation was carried out in an Applied Biosystems 7500 Real-Time PCR system (Applied Biosystems), using the following thermal cycling profile: reverse transcription at 50 °C for 30 min, activation of Taq polymerase at 95 °C for 10 min and 40 cycles consisting of denaturation at 95 °C for 10 s, annealing at 60 °C for 30 s, and polymerization at 72 °C for 30 s. At the end of the amplification, a melt curve was generated from 70 to 95 °C, and fluorescence data were collected every 0.3 °C during melting. Real-time RT-PCR data were analyzed with the Applied Biosystems 7500 Software v. 2.0.6 (Applied Biosystems). The RNA transcript levels of the genes of interest in treated and control cells were determined by absolute quantification using the standard curve method. Briefly, a 287-bp amplified N gene fragment resulting from conventional PCR with P1 and P2 primers (Additional file 4), described by Frisk et al. [59], was cloned into the pGEM-T Easy (Promega). The pGEM-inserted N fragment contained the amplicon sequence chosen for real-time qRT-PCR. Moreover, another pGEM-T Easy ligation was made with a 136-bp PCR product amplified from the CDV L gene with L1 and L2 primers (Additional file 4). After confirming and linearizing both pGEM-T Easy constructs with Spel restriction enzyme (Promega), linearized plasmids were used as templates for in vitro transcription with the MEGAshortscript T7 Transcription Kit (Ambion) according to the manufacturer’s instructions. Turbo DNase-treated transcripts were ethanol-precipitated and resuspended in RNAse-free water. Synthetic RNAs were quantified using a Qubit 2.0 Fluorometer (Thermo Fischer Scientific), and the copy number was determined using the following formula: (X g/μL DNA / [transcript length in base pairs × 340]) × 6.022 × 1023 = Y ssRNA molecules/μL. Standard curve was constructed with five points in triplicate from serial 10-fold dilutions of transcripts with an amplification efficiency E = 98.5% (slope = − 3.286, R2 = 0.998). The RNA samples were tested in duplicate, and the inhibition of CDV replication was expressed as RNA copy number.
Statistical analysis
Statistical analysis was performed by two-way ANOVA (analysis of variance) using the GraphPad Prism Software v. 5.01 for Windows (GraphPad Software, La Jolla, California, USA). The Tukey test was used for pairwise comparisons among means. Values of cell viability were calculated from a linear regression equation. All graphs were produced based on the means ± standard errors from three independent experiments. A p-value < 0.05 was considered statistically significant.