Experimental design of in vitro studies
Growing HEp-2 cells
HEp-2 cells were grown on 75 cm2 and/or 300 cm2 tissue culture flasks with a vent screw cap (TPP®) using 12 mL or 45 mL media, respectively, of Dulbecco’s Modification of Eagle’s Medium (DMEM) (Mediatech, Inc. Cat. No. 10-013-CV), 10% fetal bovine serum (FBS) (Atlanta Biologicals Cat. No. S11550), 50 μg/mL of kanamycin sulfate (Invitrogen Cat. No. 11815–032), and incubated at 37°C at 5% CO2 and 93% humidity. Cells were passaged 1:6 every 2–3 days, depending on cell confluence. Twelve-well cell culture plates (Costar®) were seeded with HEp-2 cells at a concentration of 2-3×105 per well containing 6.5 mL of media. Cell concentration was determined prior to passaging by hemocytometer (Bright-Line®) to determine approximate cells per mL. Cells were allowed to grow overnight prior to infectious focus assay.
RSV M37 stock and sucrose
RSV Memphis strain 37 (RSV M37) was frozen at varying sucrose concentrations (0%, 3%, 5%, 8%, 10%, 15%, and 20%) to assess infectivity after cryo-storage and nebulization. HEp-2 cells were grown, as stated above, and infected with RSV M37. After 48 hrs, the virus was isolated from the cells after a freeze-thaw cycle (at −80°C) followed by centrifugation (2500 rpm for 10 min). 5 mL of the supernatant was then added to varying amounts of PBS (Sigma Cat. No. P3813-10PAK; 0.138 M NaCl, 0.0027 M KCl, pH 7.4 @ 25°C), 0% sucrose and 10% FBS solution or PBS, 60% sucrose and 10% FBS solution to attain desired sucrose concentrations of 0%, 3%, 5%, 8%, 10%, 15%, and 20%. The solution of 60% (w/v) sucrose in PBS was made, supplemented with 10% FBS and filtered using a Nalgene filter flask and used to accomplish the various desired final tested concentrations of sucrose mentioned above. A 0.5 mL aliquot was taken from each sucrose concentration and stored in a separate tube; this was used to determine the stock and post freeze-thaw titers of the virus. After preparation, samples were frozen at −80°C for about 3 weeks before use. Upon use, samples were thawed in a 37°C water bath just until ice disappeared.
Infecting HEp-2 cells with RSV M37 for infectious focus assay
Viral titers were assessed prior to freezing, after freezing, and following nebulization in a standard infectious focus assay. Collected condensates of nebulized vapors containing RSV M37 were used to infect HEp-2 cells. Vapors were collected using a nebulization apparatus coupled to a modified cold trap. The nebulization apparatus included: a nebulizer compressor unit, PARI nebulizer tubing, PARI Sprint™ nebulizer (PARI LC™), T-port connector, rubber stopper #3 (22 mm inside diameter), modified garden hose rubber washer (30 mm outer diameter and 22 mm inside diameter), and a 250 mL flask (PYREX®). 6 mL of RSV M37 inoculum containing the varying sucrose concentrations stated above, were individually loaded into the PARI SprintTM nebulizer and nebulized in three 2-mL amounts over a 23- minute period. A new nebulizer was used for each sucrose concentration. The condensed nebulized vapors within the 250 mL flasks were then diluted in duplicate with media in serial dilution fashion from 10−1 to 10−5. HEp-2 cells at a confluency of 75-85% (minimum of 50%) were used for the infectious focus assay. Prior to viral inoculation, the media within pre-seeded 12-well plates was discarded. 200 μL of diluted condensed viral vapor was then transferred to designated HEp-2 cell wells and allowed to incubate for 1 hr at 37°C to allow virus-to-cell contact. After 1 hr, 1 mL of media was added to each well and the plates were again incubated at 37°C for 48 hrs to allow for viral replication. Prior to freezing and post freezing, stock samples were also titered without the nebulization process; samples which came from aliquots of the original virus/sucrose preparations. Titers of these original pre-frozen viral stocks (with and without sucrose) ranged from 1.63 × 106 to 2.91 × 106 FFU/mL. Multiplicity of infection (MOI) for each study was generally between 0.5 and 1. All nebulization and plating was performed in a class II biosafety cabinet (NUAIRE®).
Determining RSV M37 infected cells
Immunofluorescence microscopy was used to determine cell infectivity. After the 48-hr incubation period, media within the wells was discarded and wells were rinsed once with TBStw [TBS-0.05% Tween 20, pH 7.4 (TBStw)]. Cells were then fixed with 0.5 mL 60% acetone/40% methanol solution for 1 min. The fixative was then removed and plates were allowed to dry for 2 min. Cells were rehydrated with 0.5 mL per well with TBStw and placed on a rotator (Boekel Scientific, model 260300 F) for 1 min after which well contents were discarded. A 1 mL blocking solution containing 3% bovine serum albumin (BSA) (Fisher Scientific, Hanover, IL) in TBStw was added to each well and plates were placed on the rotator for a minimum of 30 min, after which well contents were discarded. 325 μL of primary antibody [Meridian, MAb to RSV Fusion Protein, Cat. No. C87610M, Clone: RSV 3216 (B016)] containing 3% BSA-TBStw at 1:800 dilution was added to each well and plates were incubated overnight at 4°C on a rocker apparatus, after which well contents were discarded and wells were rinsed with TBStw. 325 μL of the secondary antibody (Invitrogen, Goat anti-Mouse Fab’ conjugated to AlexaFluor 488, Cat. No. A11017) containing 3% BSA-TBStw at 1:800 dilution was then added to each well and plates were placed on the rotator for a minimum of 30 min, after which well contents were discarded. Wells were then rinsed twice with 1 mL TBStw. 1 mL TBStw was then added to each well and fluorescing foci were visualized using an Olympus CKX41 inverted microscope with an external florescence bulb; X-Cite series 120 Q (Lumen Dynamics®). Cell aggregates ≥5 cells were counted as one infectious focus. Infectious foci per well were then converted to focus forming units (FFU) per mL using the following formula: [(arithmetic mean × dilution × 1000 μL/mL) / 200 μL assessed = FFU/mL]. Images were taken using an Olympus DP20 camera-microscope attachment.
Experimental design of in vivo studies
Three groups of 2 to 3-day-old lambs (either sex; Polypay, Suffolk, Dorsett cross breed) were used for the study. RSV strain (Memphis 37) was grown in HEp-2 cells and delivered (1.25 × 106 FFU/mL) to lambs in 6 mL culture medium using a PARI Sprint™ nebulizer (fitted with a cone-shaped face mask fitted with a rubber diaphragm that provided a seal around the mandible and nose) in three 2-mL increments over a 23 minute period. One group (n = 6 lambs) received this amount of RSV in culture media containing 20% sucrose whereas another group (n = 6 lambs) received this same amount of RSV in media lacking sucrose; control lambs (n = 2) received 6 mL culture medium (lacking virus) with 20% sucrose, also by nebulization. After inoculation with RSV, lambs in all groups received a daily antibiotic (Ceftiofur, 1–2 mg/kg body weight, intramuscular) to prevent secondary bacterial infections. Lamb temperatures, body weights, respiratory and heart rates were measured daily. Clinical severity of RSV disease for each lamb was assessed and scored as previously described [11, 17, 18]. Lambs were euthanized by sodium pentobarbital overdose on day 6 post-inoculation (p.i.) when RSV-induced lesions are maximal in this model [11, 17, 18]. Throughout the experiment, lambs received a custom diet (Milk Products Inc., Chilton, WI, USA) that lacked supplemental iodide, and the lack of iodide was verified using anion-exchange chromatography. The purpose of eliminating dietary iodide was to avoid any potential production of hypoiodite (OI−) which is a hypohalide generated by the ovine oxidative mucosal Duox/LPO/halide defense system in the presence of sufficient iodide. Hypoiodite has been demonstrated to be destructive to RSV [19]. All animal experiments were approved by the Institutional Animal Care and Use Committee (IACUC) of Iowa State University.
Post-mortem analysis of RSV disease severity
After euthanasia, the thorax was opened, lungs were removed, gross lesions scored as described previously [11, 17, 18] and photographed in situ and ex vivo. Tissue samples were collected from all lung lobes of each animal in a standardized manner. Multiple samples from each lobe were placed into cryovials and snap-frozen in liquid nitrogen for subsequent use in RNA isolation for hydrolysis probe-based reverse transcription real-time quantitative polymerase chain reaction (RT-qPCR). Two samples from each lobe were placed into tissue cassettes and fixed in 10% neutral-buffered formalin (NBF) for histological and immunohistochemical analysis, and two lung samples from each animal were placed into cryomolds and covered with CRYO-OCT Compound (Tissue-Tek, Torrance, CA) then stored at −80°C until cryosectioning. Immediately after lung removal, percentage parenchymal involvement was estimated for each lobe. Group averages were calculated to obtain the gross lesion score for each lobe [17, 18].
In addition to the lung samples collected for RNA isolation and histological samples, bronchoalveolar lavage fluid (BALF) was collected from each lamb for determination of viral titers and cytology. BALF for viral titer assessment was obtained by flushing the right caudal lung lobe through a major bronchus with 5 mL of ice-cold modified Iscove’s media (42.5% Iscove’s modified Dulbecco’s medium, 7.5% glycerol, 1% heat-inactivated FBS, 49% DMEM, and 5 μg/mL kanamycin sulfate). 1 mL of each recovered BALF sample was then used immediately (never being frozen) for infectious focus assay. BALF for cytology (~0.5 mL from each lamb) was obtained by flushing a major bronchus of the accessory lobe with 1 mL PBS, pH 7.4. These BALF samples were submitted to the clinical pathology laboratory (Iowa State University, College of Veterinary Medicine) for total nucleated cell counts and slide preparation for cytology. An ADVIA120™ automated hematology analyzer was used to perform cell counts. Cytospin preparations of BALF were performed using a Shandon Cytospin 3 set at 800 rpm (samples were spun for 10 minutes with low acceleration). Slides were stained with modified Wright’s using a Hematek automated staining system. Differential cell counts (based on a 300 cell differential) were performed by a board-certified veterinary clinical pathologist blind to the identity of the samples.
Histologic scores were determined by evaluating percent consolidation and converting the observed percentage ranges to a simple integer based on a consolidation scale used by our laboratory previously (35, 36): 0% consolidation = 0, 1-9% consolidation = 1, 10-39% consolidation = 2, 40-69% consolidation = 3, 70-100% consolidation = 4 [17, 18]. Group averages were calculated to obtain the alveolar consolidation score.
RNA isolation and RT-qPCR
Tissues from right and left cranial, right and left middle, and right and left caudal lung lobes (0.3-0.4 g of each) were homogenized in TRIzol (Invitrogen), pooled in equal (w/v) portions to create representative slurries for each animal, each of which was adjusted to 0.091 g/mL with additional TRIzol. Total RNA isolation continued as per manufacturer’s guidelines (Invitrogen) followed by DNase treatment (Ambion, TURBO DNase, Austin, TX) and diluting the isolates 1:10 with a combination of RNaseOUT (Invitrogen) and nuclease-free water (Ambion/Invitrogen). A NanoDrop (Thermo Fisher Scientific) was used to assess each sample for general RNA purity and quantity (A260nm/A280nm all >1.98). In addition, Agilent Bioanalyzer 2100 analysis of the RNA routinely showed RIN values >8.0. RT-qPCR was carried out using One-Step Fast qRT-PCR Kit mastermix (Quanta, BioScience, Gaithersburg, MD) in a GeneAmp 5700 Sequence Detection System (Applied Biosystems, Carlsbad, CA) employing PREXCEL-Q for all set-up calculations [20–23]. Primers and probe for the RSV M37 nucleoprotein mRNA sequence targeted by the RT-qPCR in this study were designed using ABI Primer Express 2.0 based on RSV accession number M74568. Forward primer: 5’-GCTCTTAGCAAAGTCAAGTTGAACGA; reverse primer: 5’-TGCTCCGTTGGATGGTGTATT; hydrolysis probe: 5’-6FAM-ACACTCAACAAAGATCAACTTCTGTCATCCAGC-TAMRA. Prior to RT-qPCR, each 1:10-diluted total RNA sample was further diluted so that each final reaction contained 0.784 ng total RNA/μL; a dilution and sample concentration determined to be optimal by PREXCEL-Q [19, 20]. The GeneAmp 5700 Sequence Detection System thermocycling conditions were 5 minutes at 50°C; 30 seconds at 95°C; and 45 cycles of: 3 seconds at 95°C and 30 seconds at 60°C. Samples and standards were assessed in duplicate wells and each quantification cycle (Cq) was converted to a relative quantity (X0r) based on a relative dilution standard curve using the equation: Xor = EAMP(b ‒ Cq), where EAMP and b are the exponential PCR amplification efficiency and the y-intercept values, respectively, obtained from a sample mixture-derived standard curve for RSV M37 nucleoprotein. The efficiency-corrected delta Cq (EAMPΔCq) method was employed for final quantification calculations. Results were normalized to total tissue RNA loaded per reaction; identical for all reactions as per the PREXCEL-Q protocol. No-RT control reactions all proved negative for RSV and the assays were determined to be free of inhibition based on the PREXCEL-Q method [20–23].
Immunohistochemistry for RSV
Immunohistochemistry for localization, and relative quantification of RSV antigen was performed on formalin-fixed paraffin-embedded (FFPE) tissues [11, 17, 18]. After heating for 15 minutes at 58°C and standard deparaffinization through xylene and graded alcohols, antigen retrieval was performed using TE-0.05% Tween 20, pH 9.0 and a Decloaking Chamber™ Plus (Biocare Medical, Concord, CA). A temperature of 125°C was reached in about 18 minutes, after which the system cooled to 80°C after another 22 minutes. Blocking for 15 minutes with 3% bovine serum albumin (BSA) (Fisher Scientific, Hanover, IL) in TBS-0.05% Tween 20, pH 7.4 (TBStw) and blocking with 20% normal swine serum (NSS) (Gibco/Invitrogen) in TBStw for 15 minutes was followed by addition of a primary polyclonal goat anti-RSV (all antigens) antibody (EMD/Millipore/Chemicon, Billerica, MA) which was applied for 1.5 hours at room temperature (~22°C) at a dilution of 1:500 (8–10 μg/mL) in TBStw containing 10% NSS and 3% BSA. After TBStw rinses, biotinylated rabbit anti-goat secondary antibody (Kirkegaard-Perry Labs, Gaithersburg, MD) diluted 1:300 in TBStw containing 10% NSS and 3% BSA was applied for 45 minutes. Slides were rinsed, blocked for endogenous peroxidase activity (using 3% peroxide in TBStw) for 25 minutes, and streptavidin-conjugated HRP (Invitrogen) diluted 1:200 in TBStw was applied for 30 minutes. After rinsing slides with TBStw, Nova Red (Vector, Burlingame, CA) was applied for about 90 seconds followed by water rinses, counterstaining in Harris’ hematoxylin for 2 minutes, bluing with alkaline Scott’s water for 1 minute, standard dehydration through graded alcohols and xylene, and cover-slipping using Permount (Sigma, St. Louis, MO). Slides (containing two pieces of lung tissue each) were scored using the following procedure: 20 unique 10X fields on each slide were assessed for antigen staining and immunoreactive cells were counted within bronchioles and alveoli. The number of cells immunoreactive for RSV per field was then scored as percent lobular involvement based on the method used in our previous studies [17, 18] without using the non-parametric 1–4 scale assignment.
Statistical analysis
All analyses were performed using GraphPad Prism 6 (GraphPad Software Inc, La Jolla, CA). RT-qPCR, IHC and gross lesion data was assessed by one-way ANOVA followed by Tukey’s post-test. All clinical data were assessed by two-way ANOVA, and cumulative weight change was additionally assessed by one-way ANOVA followed by Tukey’s post-test. Scored parameters were assessed using non-parametric (Kruskal-Wallis) and/or unpaired, two-tailed, non-parametric Mann–Whitney test analyses. Significance was accepted at P < 0.1 in these evaluations.
