Material and equipment
Reagents of the highest available grade were used. All experiments and solution preparations were performed using fresh Milli-Q water (LabostarTM TWF, Evoqua Water Technologies LLC, Warrendale, PA, USA) and Ultra-pure water (18.2 MΩ) was obtained from the LaboStar™ 4-DI/UV water system. Fourier transform infrared spectra analysis (FT-IR) was performed on a ATR/FT-IR interspec 200-X spectrometer (Interspectrum OU, Toravere, Estonia). Scanning electron microscopy—energy dispersive X-ray spectroscopy (SEM–EDS) analysis of Chi-S and Chi-C samples was performed in a JEOL JSM-IT300LV microscope (JEOL USA Inc., Peabody, MA, USA), connected to an energy dispersive X-ray detector for elemental analysis with computer-controlled Aztec EDS system software from Oxford Instruments, Abingdon, UK. Real-time PCR reactions were performed using the LightCycler® Nano Instrument (Roche Molecular Systems, Inc). High resolution for PCV2 viral copy number determination and melting curve analysis were achieved with this instrument.
Synthesis of sulfated chitosan
To obtain Chi-S, commercial Chi (Sigma-Aldrich) of two different molecular weights was used. Low molecular weight (LMW) Chi (50–190 kDa) and high molecular weight (HMW) Chi 310–375 kDa were subjected to a sulfation reaction using the modified method described by Naggi et al. [23]. Briefly, 5 g of each Chi was dissolved in 200 mL of cold sulfuric acid (Sigma-Aldrich) (4 °C) and stirred for 2 h. Then the mixture was precipitated dropwise in 1 L of cold ethyl ether (Sigma-Aldrich) (2 °C) under constant stirring. The precipitated product was filtered, washed with abundant cold ethyl ether, and products were collected by adding them to 200 mL of ultra-pure chilled water (4 °C). The water was obtained from first capsule filters with 0.2 µm flow (U.S. Filter), and ultra-pure water (18.2 M Ω) was obtained from the LaboStar TM 4-DI/UV water system. The pH was adjusted to 7.6 with a cool 30% NaOH aqueous solution (Merck) and the mixture was dialyzed against distilled water on Spectra/Por 3 cellulose membranes with 3.5 kDa pore size for 3 days at room temperature. Distilled water was obtained from an automatic water still, model 700-Pobel. The volume of dialysis solution was changed every 24 h. Finally, the product obtained was subjected to evaporation under reduced pressure in a rotary evaporator (Heidolph Laborotta 4001 Efficient), until obtaining 20 mL, which was lyophilized using the Christ Alpha 1–4 equipment, LOC-1M.
Characterization of chitosans
The elemental analysis of the Chi sample was performed in an organic elemental analyzer (INICUBE®, Elementar Americas Inc.) designed for simultaneous carbon, hydrogen, nitrogen, and sulfur analysis in solid and liquid samples. This analysis allows the detection of differences in the presence of sulfur atoms between Chi-S and Chi-C, which indicates sulfation degrees of Chi-C. Furthermore, Fourier transform infrared (FTIR) spectra of Chi-S and Chi-C were obtained with an ATR/FTIR interspec 200-X spectrometer. The FTIR spectra were recorded at 4 cm−1 resolutions and 64 scans. Additionally, surface morphology and micro-analysis of Chi-S and Chi-C samples were observed by scanning electron microscopy (SEM model JSM-IT300LV, JEOL USA Inc.), coupled with energy dispersive X-ray analysis (EDS Aztec, Oxford Instruments); the elemental analysis was performed using a computer-controlled software (Aztec analysis software, Oxford Instruments).
Viruses and cells
PCV2 genotype b (strain 0233) was passed three times and titrated on PK-15 cells as previously described [24]. PK-15 cell line (ATCC CCL-33, passage 10) was maintained in minimal essential medium (MEM) (Corning™ Cellgro™), supplemented with 15% fetal bovine serum (Corning Cellgro), 0.5% antibiotic/antifungal (10,000 IU/mL penicillin, 10,000 µg/mL streptomycin and 25 µg/mL amphotericin B and 1% glutamine (Corning™ Cellgro™). Aliquots of the virus were stored at − 80 °C until required.
Cell seeding and infection of cell cultures
PK-15 cells were seeded at 2.0 × 104 cells /well in 96-well microplates (Corning™ Cellgro™) and incubated for 12 h at 37 °C/5% CO2 until 80% confluence. Before adding Chi samples or the virus, or when quantifying the results, the monolayers were washed twice with phosphate buffered-saline (PBS, pH 7.4 at room temperature). In all the experiments the following controls were included: cell control (cells that were not infected with the virus or treated with Chi samples); virus control (cells that were infected only with the virus but not treated with Chi samples in the antiviral activity assays).
Cytotoxicity assay
The cytotoxicity of the polymers was determined using the method described by Pourianfar et al. [25] with modifications. Briefly, the media of the 80% confluent PK-15 cells was aspirated and replaced with 100 µl of each polymer solution diluted in MEM/15% FBS at concentrations of 0, 5, 10, 15 and 20 mg/mL. After incubation at 37 °C/5% CO2 for a further 3 days, the results were quantified using CellTiter 96® AQueous One Solution MTS (Promega Corporation), with the absorbance set at 490 nm, according to the manufacturer’s instructions. Cell viability percentages were measured based on the number of living cells in polymer-treated cells relative to cell controls (defined as 100% viability). The cytotoxicity curve was then generated by plotting cell viability percentages against compound concentrations.
Antiviral activity assay
The infectivity of PCV2 on PK-15 cell cultures treated with LMW and HMW Chi-S was evaluated through a plaque infection assay. The monolayers were infected with 10 µL of PCV2 inoculum (106 TCID50 mL−1, MOI = 5), then the microplate was incubated at 37 °C/5% CO2 for 1 h. After the incubation time, the residual viral inoculum was removed by washing the cell monolayers with 1X PBS and immediately covered with 100 µL of MEM supplemented with 15% FBS, 0.5% antibiotic/antifungal and 1% glutamine, which contained five concentrations of Chi-S or Chi-C (0, 5, 10, 15 and 20 mg/mL). The incubation continued for 24 h at 37 °C/5% CO2, after which the supernatant was removed and the cell cultures were treated with D-glucosamine (300 mM) and incubated for additional 48 h. After the incubation period, the monolayers were subjected to 3 freeze–thaw cycles (− 80 °C) and the lysates of all wells were obtained and subsequently centrifuged at 2500 g to store the supernatants for quantification of viral DNA and TCID50 titer.
Viral copy number measuring by quantitative real-time PCR (qPCR)
Viral DNA was extracted according to the manufacturer's instructions (Axigen, Biosciences). PCR was performed using primers that amplify a specific region of PCV2 ORF2 (PCV2qPCR-F 5′-ATGTCCACCGCCCAGGAGG-3′ Position 1523-1541 and PCV2qPCR-R 5′-CCGYTGGAGAAGGAAAAATGGCATC-3′ Position 1603-1627). The SYBR Green system (Kapa Sybr) and a real-time thermal cycler (LightCycler Nano, Roche) were used for real-time PCR, following the manufacturer's instructions. To generate the standard curve, the plasmid pGEM::PCV2 was used, in which the complete PCV2 DNA genome sequence was cloned. The plasmid DNA was extracted from the bacterial strain E. coli DH5α, with a commercial kit (Plasmid Midi Kit, Qiagen), according to the manufacturer's instructions. The quantification was conducted by measuring the OD260 using NanoDrop spectrophotometer and DNA copies by using the online NEBio calculator (https://nebiocalculator.neb.com/#!/ligation). Serial dilutions (101–105) were prepared in duplicate of known molar concentrations of plasmid DNA. The viral DNA copies were interpolated from the standard curve obtained. The degree of inhibition of the virus was recorded by quantify the genomic copies of PCV2 in log10 DNA copies/mL.
Quantification of virus titer
The quantification of PCV2 titer was determined using the method described by Zhu et al. [26] with modifications. Total virus yield (supernatants from cell lysates) were determined by inoculating tenfold serial dilutions into confluent PK-15 cells in 96-well culture plates (Corning™ Cellgro™). After 72 h of incubation, supernatant was removed, cells were fixed with 80% cold acetone and the viral antigen was detected using immunofluorescence (IFA) with mouse anti-Cap PCV2-specific monoclonal antibody (isotype IgG2a, Jeno Biotech Inc.) and FITC-conjugated goat anti-mouse IgG (H + L) (Kirkegaard & Perry Laboratories Inc.). The 50% tissue culture infective dose (TCID50) was calculated according to the Reed-Muench method and expressed as TCID50/mL.
Detection of PCV2 Cap protein by dot blot and Western blot
Samples of the supernatant (10 μl) were taken and the amount of PCV2 Cap antigen was determined using the dot blot and Wester blot as described by Bucarey et al. [27] with modifications. Briefly, samples were transferred onto a nitrocellulose membrane using a Biodot™ microfiltration apparatus (Bio-Rad, CA, USA) or resolved by 12% SDS-PAGE electrophoresis under reducing conditions (1% glycerol, 0.4% SDS, 0.1% mercaptoethanol, 12.5 mM Tris–HCl, pH 6.6) and electrotransferred onto a nitrocellulose membrane (BioRad) using a TransBlotTM Semi-Dry Transfer Cell (BioRad, USA). The nitrocellulose membrane was then blocked overnight in 5% skim milk at 4 °C and then incubated overnight at 4 °C with a mouse anti-Cap PCV2-specific monoclonal antibody (isotype IgG2a, Jeno Biotech Inc.) or anti-β-actin antibody (Sigma Aldrich®) diluted 1:100 in PBS/0.1% Tween20 (PBST). After washing with PBST, the membrane was incubated with horseradish peroxidase-conjugated goat anti-mouse IgG (H + L) (1:1000 dilution; Kirkegaard & Perry Laboratories Inc.) for 1 h. After further washing, the signal was detected using 4-chloro-1-naphthol/H2O2 as directed by the manufacturer (Pierce, Rockford, IL, USA). The concentration of PCV2 Cap protein was estimated by comparing the signal intensities of the Western blots with those of of β-actin as loading control or by comparing the signal intensities of the dot blots with those of known concentrations of a highly purified recombinant 6xhis-Cap fusion protein, using an image analysis program (ImageJ) [28].
Attachment assay
The ability of Chi-S to inhibit viral attachment to PK15 cell line was evaluated as previously described [25] with modifications. The 80% confluent monolayers were pre-cooled at 4 °C for 1 h followed by (1) Cellular infection with 10 µL/well of PCV2 inoculum (106 TCID50 ml−1, MOI = 5), supplemented with 100 µl of MEM containing LMW Chi-S at the following concentrations (0, 5, 10, 15 and 20 mg/mL), (2) infection with 10 µL/well with virus inoculum that had been mixed and pre-incubated with 100 µL/well of each compound at 4° C for 30 min. All plates were kept at 4 °C for another 3 h, after which the monolayers were washed twice with PBS to remove excess compounds and any unbound viruses. The plates were then filled with 100 μl/well MEM and incubated for 24 h at 37 °C/5% CO2, at which point the supernatant was removed and the cultures were treated with D-glucosamine (300 mM), after which the incubation was continued in the above-mentioned conditions until completing 48 h. After incubation, the monolayers were subjected to 3 freeze–thaw cycles (− 80 °C) and the lysates of all wells were obtained and centrifuged at 2500 g to store the supernatants and quantifying the genomic copies and titers of PCV2.
Viral penetration assay
The viral penetration assay was conducted using the modified penetration test as previously described [25], in order to evaluate the ability of the compounds to inhibit viral entry to the cells. Confluent cells (80%) were pre-cooled and infected with 10 µL of PCV2 inoculum (106 TCID50 ml−1, MOI = 5) and incubated at 4 °C for 3 h to allow viral attachment. After this step, the cells were treated at room temperature with 100 µL/well of LMW Chi-S using the same concentrations (0, 5, 10, 15 and 20 mg/mL), followed by incubation at 37 °C/5% CO2 for 20, 40, and 60 min (separate plates) to allow the compound to interrupt the penetration of virus into cells. Each time point was tested separately and independently. The supernatant was aspirated followed by washing the cells with alkaline PBS (pH 11) for 1 min to inactivate viruses that did not penetrate the cell, after which, acidic PBS (pH 3) was added to neutralize the pH. Then, the PBS was aspirated and neutralized by washing the cells with PBS (pH 7.4) and the cells covered with 100 µL of MEM. The plates were incubated at 37 °C with 5% CO2 for 24 h at 37 °C/5% CO2, where the supernatant is removed and the cultures are treated with D-glucosamine (300 mM), continuing with incubation in the above-mentioned conditions until completing 48 h. After incubation, the monolayers were subjected to 3 freeze–thaw cycles (− 80 °C) and the lysates of all wells obtained, centrifuged at 2500 g. Supernatants were kept for quantifying genomic copies and titers of PCV2.
Statistical analysis
Each Chi concentration was tested in triplicate. Data were expressed as mean ± standard deviation (SD). The reduction in viral DNA copies and titers were determined by the difference between the average value of DNA copies or TCID50 of PCV2 obtained in the control (without polymer) and the average value of viral DNA copies and titers obtained with all 5,10, 15, and 20 mg/mL concentrations of Chi-C or Chi-S polymers. The reduction in the genomic copies and titers of PCV2 were expressed as a percentage, using Eq. (1).
$$\begin{aligned} & \frac{{10^{{\left( {\text{LC Concentration}} \right)}} { }}}{{10^{{\left( {\text{LV Control}} \right)}} }} \times 100 = {\text{X}} \\ & 100 - {\text{X}} = {{\% }}\;{\text{PCV}}2\;{\text{reduction}} \\ \end{aligned}$$
(1)
where LC concentration and LV control correspond to the average of log10 DNA copies/mL or log10 TCID50/mL obtained at each polymer concentration and the average value obtained in the control (without polymer), respectively. The results of the antiviral activity tests were previously analyzed with an ANOVA test using a multivariate regression model. Tukey's honest significant difference test (HSD) was then used to compare the antiviral activity between the type and concentration of Chi. A significance level (p value) set to ≤ 0.05 and ≤ 0.005 were considered to compare the group means. All the graphs, calculations, and statistical analyses were performed using GraphPad Prism software (GraphPad Software, San Diego, CA).
