Cells and viruses
MARC-145 cells, which are a subclone of the African green monkey kidney MA104 cells that is highly permissive to PRRSV , were maintained as described previously . The St. Jude porcine lung (SJPL) epithelial cell line was provided kindly by Dr R.G. Webster (St. Jude Children's Hospital, Memphis, TN, USA) . During this study, karyotyping and genome sequence analyses of the SJPL cells revealed that their species origin was not porcine but was rather monkey . The newborn pig trachea epithelial cell line (NPTr) was provided kindly by Dr. M. Ferrari (Instituto Zooprofilattico Sperimental, Brescia, Italy) . The SJPL and NPTr cell lines were cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Invitrogen Corporation, GibcoBRL, Burlington, ON, Canada) supplemented with 10% fetal bovine serum (FBS) (Wisent Inc, St-Bruno, QC, Canada), 1 mM sodium pyruvate, 2 mM L-glutamine, 1 μM MEM nonessential amino acids, 10 U/mL of penicillin, 10 μg/mL of streptomycin and 250 g/L antibiotic-antimycotic solution (Invitrogen Corporation, GibcoBRL) as described previously [40, 41]. Pulmonary alveolar macrophages (PAMs) were used as a positive controls for the detection of PRRSV receptors. PAMs were harvested from lungs of 2 to 14 weeks old pigs. Pigs were sacrificed following ethic protocol 12-Rech-1640 approved by the Institutional ethic committee following the guidelines of the Canadian Council on Animal Care (CCAC). Briefly, an instillation of the lungs with PBS containing 10 units/mL penicillin, 10 μg/mL streptomycin and 100 mg/L gentamicin (Invitrogen Corporation, GibcoBRL) was realized. Then, PBS was collected and PAMs removed following low speed centrifugation. Cells were washed with medium DMEM complemented with 2 mM L-glutamine, 0,1 mM HEPES, 1 μM Non-essential amino acids (Invitrogen Corporation, GibcoBRL), 250 g/L Amphotericin B (Wisent Inc), 10 units/mL penicillin, 10 μg/mL streptomycin and 100 mg/L gentamicin. Cells were then collected following low speed centrifugation and were resuspended in freezing medium (same as wash medium plus 20% fetal bovine serum (Wisent Inc.) and 10% DMSO (Sigma, St-Louis, MO, USA)) and slowly frozen, than stored in liquid nitrogen until further utilization. PAMs were cultured for 24 hours in complete DMEM prior the immunofluorescence assay. All cell lines were cultured at 37°C in 5% CO2 atmosphere.
The PRRSV strain used to establish the permissivity of the SJPL cells was the MARC-145 cells adapted IAF-Klop North American reference strain  and the Lelystad (LV) European reference strain . The PRRSV virus stocks were obtained following three cycles of freeze-thaw of PRRSV MARC-145 infected cells. Afterward, the virus was purified following a 3.5 hrs period of ultracentrifugation on a 30% sucrose cushion (in a TBS solution: 50mM tris pH7.5, 150mM NaCl) using the SW28 Beckman Coulter rotor at 83,000 relative centrifugal force (rcf). The virus pellets were resuspended in 0.5 mL of PBS and aliquots of the virus stocks were then conserved at –70°C for future use. The infectious dose of the virus stocks was calculated from a 96-well microplate of MARC-145 infected cells by the Kärber method as described previously . Virus titers were expressed in tissue culture infectious dose 50% per mL (TCID50/mL).
Immunofluorescence assay (IFA) for the detection of PRRSV antigen
The presence of PRRSV antigens in infected cells was determined by an immunofluorescence assay (IFA). Briefly, cells infected by PRRSV strains were fixed at different times post-infection (pi) with a 4% paraformaldehyde (PFA) solution prepared as described previously . Mock-infected cells were included as negative controls. After an incubation period of 30 minutes at room temperature, the PFA solution was removed and cells were washed three times with a phosphate buffer saline solution (PBS). Then, cells were incubated during 10 minutes at room temperature with a PBS solution containing 1% Triton X-100. After removing the Triton X-100 solution, the cells were washed three times with a PBS-Tween 20 solution (PBS containing 0.02% Tween 20). After the permeabilization procedure, cells were incubated 30 minutes with PBS containing 0.2% Tween 20 and 1% Fetal Bovine Serum Albumin. Then, the α7 rabbit monospecific antisera (a specific anti-N PRRSV protein antibody)  was diluted 1/200 in the washing buffer and added to the cells and incubated at room temperature for a 30 minutes period. Cells were then washed and incubated for 30 minutes with the washing buffer containing a 1/160 dilution of anti-rabbit specific antisera FITC conjugated (Sigma-Aldrich Inc., St-Louis, USA). Finally, cells were visualized using a DMI 4000B reverse fluorescence microscope, image of the cells were taking with a DFC 490 digital camera and the image were analyzed using the Leica Application Suite Software, version 2.4.0 (Leica Microsystems Inc., Richmond Hill, Canada).
Immunofluorescence assay for PRRSV receptors CD151, CD163, and Sialoadhesin detection
The presence of CD151, CD163 and Sialoadhesine (Sn) proteins in MARC-145, SJPL and PAM cells was determined by an IFA. Briefly, cells were fixed with a 4% PFA solution as described previously . After an incubation period of 30 minutes at room temperature, the PFA solution was removed and cells were washed three times with PBS. Then, cells were incubated with a permeabilization and blocking solution, PBS solution containing 0.1% Triton X-100, 7% normal sheep serum (NSS) and 5% non fat dry milk (NFDM), during 30 minutes at room temperature. After removing the permeabilization/blocking solution, the cells were washed three times with a PBS. Then, cells were incubated overnight with primary antibodies. All those antibodies were diluted 1/100 in antibody solution containing PBS, 0.1% Triton X-100, 1.4% NSS, and 1% NFDM. The antibodies used were: rabbit polyclonal anti-human CD151 (Santa Cruz Biotechnology, CA, USA); mouse monoclonal anti-pig CD163 (AbD Serotec, Oxford, United Kingdom) for PAMs cells or goat polyclonal anti-human CD163 (Santa Cruz Biotechnology, CA, USA) for MARC-145 and SJPL cells; mouse monoclonal anti-pig CD169 (synonym: Sialoadhesin, Siglec-1) (AbD Serotec, Oxford, United Kingdom) for PAMs cells or goat polyclonal anti-human Siglec-1 (Santa Cruz Biotechnology, CA, USA) for MARC-145 and SJPL cells. The anti-CD163 and anti-CD169 polyclonal antibodies are known to react against several animal species. Their reactivity was confirmed against the PAMs control positive cells (data not shown). Thereafter, cells were washed three times and incubated for 60 minutes with the antibody buffer containing a 1/160 dilution of anti-rabbit specific antisera FITC conjugated (Sigma-Aldrich Inc., St-Louis, USA) or 1/200 dilution of anti-mouse specific antisera FITC conjugated (ICN Immuno Biological, CA, USA). Nuclei were stained with 4',6-diamidino-2-phenylindole DAPI (Sigma-Aldrich Inc., St-Louis, USA) as recommended by the manufacturer. Negative controls were obtained from cells where only the primary antibody was omitted. Cells were visualized the same way as described above.
Virus production during multiple cell passages
An amount of 106 MARC-145 or SJPL cells were infected with 0.005 MOI of IAF-Klop strain. Then, cells with their supernatants were subjected to three cycles of freeze-thaw at -70°C and the virus stock solutions were kept at -70°C for future use. Four subsequent viral passages in MARC-145 and SJPL cells were done as described above except that a dilution of 1/20 of the previous viral stock solutions was used for cell infection. Mock-infected cells were included as controls in each passage. The amount of virus production at each passage was calculated from a 96-well microplate of MARC-145 infected cells by the Kärber method and the results were expressed in tissue culture infectious dose 50% per 106 infected cells (TCID50/106 cells).
Virus replication kinetics assay
105 MARC-145 and SJPL cells were infected with IAF-Klop and LV strains using an MOI of 1. At different times pi (0, 4, 9, 12, 18, 24, 48, 72, 96 and 120 hrs), whole cell cultures were collected and were processed by low speed centrifugation to separate the cell pellet (cell fraction) from the culture medium (supernatant fraction). Both fractions were stored at -70°C until used. Afterwards, virus titration was performed in MARC-145 cells as described above. Mock-infected cells were included in each experiment as controls. All experiments were repeated two times in triplicate.
Virus isolation was attempted from 22 swine samples (lung and lymph nodes tissues) submitted from October 2007 to September 2008 to the Veterinary virology diagnostic laboratory of the Veterinary College of the Université de Montréal. Those samples originated from 3 to 10 weeks old animals housed in different Canadian farms and they were submitted for different reasons such as PRRSV outbreaks, porcine circovirus associated disease outbreaks, or others health problems. Three of the submitted samples were PRRSV negative by a real-time PCR diagnostic assay (Tetracore Inc., Rockville, MD, USA) and the amount of infectious PRRSV contained in the 19 real-time PCR positives cases was determined using the same assay as described previously . For virus isolation, about 1-2 cm3 of pool of tissue samples were homogenized and resuspended in 9 mL of culture medium without FBS. Then, three cycles of freeze-thaw at -70°C were performed and tissues homogenates were centrifuged and the supernatants were filtered (with a filter size of 0.2 micron). Following the sample treatment, 1 mL of filtered sample was used to inoculate cells and cells were incubated for 5 days. Then, three cycles of freeze-thaw were performed at -70°C and cell lysates were centrifuged at 4000 rpm for 10 min. Supernatants of cell lysates were collected and used for a subsequent cell infection cycle. One mL of the cell lysate supernatants was inoculated to freshly prepared cell cultures and cells were incubated for 5 days. This new infection step was done for three consecutive times. At the fourth passage, the virus isolation status was confirmed by the presence of CPE and a positive IFA result. To further characterize the PRRSV strains that were isolated from both cell lines, PCR products encompassing the ORF5 gene were obtained from tissues and fourth virus isolation cell passages, and sequenced subsequently. Sequences were analyzed using the CLUSTAL W alignment method of the BioEdit sequence alignment editor version 7.0.9 software (Ibis Therapeutics, Carlsbad, CA, USA).
MARC-145 and SJPL cells were infected with PRRSV IAF-Klop strain at 0.5 MOI or were incubated with a mix of apoptotic inducers (500 μg/mL actinomycin D, 60 nM vinblastine sulfate, 100 μg/mL cycloheximide and 40 μg/mL puromycin 2HCl; Biomol Research Laboratories Inc., Plymouth meeting, PA, USA) as positive controls. Cellular changes associated with the infection or the inducers were visualized respectively at 72 hrs pi and 24 hrs post-incubation, respectively, under a light microscope (Leica Microsystems Inc.). At this time, cells were disrupted in a lysis buffer (50 mM HEPES, pH 7.4, 100 mM NaCl, 0.1% CHAPS, 1 mM DTT and 100 μM EDTA) for 5 minutes followed by sonication (Sonifier S-450A, Branson, Danbury, CT, USA). Then, protein concentrations were measured by a Bradford assay following the manufacturer’s instructions (Bio-Rad Laboratories Ltd, Mississauga, ON, Canada). Subsequently, apoptosis was assessed by detecting the activation of procaspases 3/7 as described by Gagnon et al. (2003), with minor modifications . Briefly, a volume of cell lysate corresponding to 50 μg of total cell protein was added to the assay buffer (50 mM HEPES, pH 7.4, 100 mM NaCl, 0.1% CHAPS, 1 mM DTT, 100 μM EDTA and 10% glycerol). Then, specific substrate for caspases 3/7, the Ac-DEVD-AFC fluorogenic substrate (Biomol Research Laboratories Inc.), was added at a final concentration of 200 μM and the rate of fluorescence released was monitored with a 96-well plate fluorometer (Synergy HT, Biotek, Winooski, VT, USA). The results were expressed as relative fluorescence released (relative fluorescence units or RFU) per second per μg of cell lysates.
Inhibition of PRRSV replication
To determine the amount of porcine IFNα that is able to inhibit the replication of PRRSV in permissive cell lines, 104 MARC-145 and SJPL cells were incubated overnight. The cells were then infected with the PRRSV IAF-Klop strain at an infectious dose of 0.5 MOI in a culture medium without FBS and incubated during 4 hrs. The culture medium was then removed and replaced by a complete medium (i.e. with 10% FBS) with different serially diluted concentrations of porcine IFNα (PBL, New Jersey, USA) and incubated during 5 days. Then, the development of CPE was monitored and an IFA was performed. All the experiments were done in duplicate.
Analysis of cytokine mRNAs expression by real time reverse transcriptase-quantitative PCR
SJPL and MARC-145 cells were infected as described above or transfected with Polyinosinic–polycytidylic acid potassium salt (Poly (I:C) [50 μg/mL] (Sigma-Aldrich Inc., St-Louis, USA) as a positive control for innate immunity induction, using polyethylenimine (PEI) [1 μg/μL] (Sigma-Aldrich Inc., St-Louis, USA), for 72 hours or treated with 1μg/ml of lipopolysaccharide (LPS) (Sigma-Aldrich Inc., St-Louis, USA) for 20 hours, as an IFNγ inducer. Total cellular RNA was extracted from cells using Trizol reagent (Invitrogen, Burlington, ON, Canada) according to the manufacturer’s protocol. Quantification of RNA was performed with a Nanodrop (NanoDrop Technologies, Inc., Wilmington, Delaware, USA). 1 μg of total RNA was reverse-transcribed using the QuantiTect reverse transcription kit (Qiagen, Mississauga, ON, Canada). The cDNA was amplified using the SsoFast™ EvaGreen® Supermix kit (Bio-rad, Hercules, CA, USA). The PCR amplification program for all cDNA consisted of an enzyme activation step of 3 min at 98°C, followed by 40 cycles of a denaturing step for 2 sec at 98°C and an annealing/extension step for 5 sec at 58°C. The primers used for amplification of the different target cDNA are for IFNα: F-GCCCTTTGCTTTACTGATGG; R-TCTGCTCATTTGTTTCAGGAG, IFNβ: F-TCCTGTGGCAATTGAATGG; R-AATAGCGAAGATGTTCTGGAG, IFNγ: F-ACTCGAATGTCCAACGCAAAGCAG; R-TCGACCTCGAAACATCTGACTCCT, TNF-α: F-TCTGTCTGCTGCACTTTGGAGTGA; R-TTGAGGGTTTGCTACAACATGGGC. All primers were tested to achieve amplification efficiency between 90% and 110%. The primer sequences were all designed from the NCBI GenBank mRNA sequences using web-based software primerquest from Integrated DNA technologies . The Bio-Rad CFX-96 sequence detector apparatus was used for the cDNA amplification. The quantification of differences between the different groups was calculated using the 2-ΔΔCt method. Beta-2 microglobulin (B2M) was used as the normalizing gene to compensate for potential differences in cDNA amounts. The B2M primers from monkey origin used are F-GTGCTATCTCCACGTTTGAG and R-GCTTCGAGTGCAAGAGATTG. The non-infected MARC-145 and SJPL cells were used as the calibrator reference in the analysis.
A two-way ANOVA model, followed by Bonferroni post-hoc tests (Graphpad PRISM Version 5.0 software) were used to determine if a statistically significant difference exists between MARC-145 and SJPL cell lines in regards to the amount of PRRSV produced after multiple cell passages, procaspases 3/7 activation and cytokines mRNA up regulation in PRRSV infected cells and in Poly (I:C) transfected cells. One-way ANOVA model, followed by Tukey's Multiple Comparison Test (Graphpad PRISM) were used to determine if a statistically significant difference exists between treatments within MARC-145 or SJPL cells in procaspases 3/7 activation and their mRNA relative expression of IFNα, IFNβ, IFNγ, and TNF-α. Differences were considered statistically significant with a p<0.05. For the viral replication kinetic experiment, the time-course of TCID50 measured from the cell pellets and supernatants was analyzed with SAS Version 9.1 software. The following linear mixed-effect model for repeated measurements was solved using restricted maximum-likelihood estimation : Y
= μ + α
. Where Y
is the measured TCID50; μ is the grand mean; cell line (α
i), type of analytical matrix (β
), and sampling time (τ
) are fixed factors; the experiment replicate (R
) is a random effect; and e
is the random error term. As indicated in the equation above, this statistical model included all dual and triple interactions between the fixed-effect factors, and the random-effect factor R
was nested within cell and analytical matrix. The strategy for covariance structure modeling proposed by Littell et al (2000) was used . Briefly, the model was estimated first with a free covariance structure. After inspecting the estimated covariance matrix, the model was estimated anew with more parsimonious covariance models (e.g., compound symmetry, first-order autoregressive), which structure resembled that of the unstructured covariance matrix. The heterogeneous first-order autoregressive covariance model was selected because it was the best fit to the empirical covariance matrix, as determined with the Akaike information criterion . Least-square means were used to assess differences between the two cell lines at each time and for each type of analytical matrix (i.e., cell or supernatant fractions), using Bonferroni-adjusted significance thresholds. The areas under the time-TCID50 curves (AUC) were calculated for each cell*matrix*replicate in order to obtain estimates of total viral production for each cell line following the 120 hrs duration of the experiment.