Materials were obtained from the following manufacturers: Amresco (Solon, OH, USA): Yeast extract (bacterial) and tryptone; Applichem (Darmstadt, Germany): Tris(hydroxymethyl)aminomethane (Tris base); BioRad Laboratories GmbH (Munich, Germany): Oriole™ fluorescent gel stain, Precision Plus Protein™ protein standard (unstained and dual color), TransBlot Turbo RTA Transfer Kit LF PVDF Mini; Biosolve BV (Valkenswaard, Netherlands): Acetonitrile (HPLC gradient grade) and formic acid (≥99%); Biozym Scientific GmbH (Hessisch Oldenburg, Germany): Advansta blocking buffer, Advansta washing buffer, and WesternBright™ Sirius substrate solution; Carl Roth GmbH (Karlsruhe, Germany): Ampicillin, glycerol (99.5%), isopropyl β-D-1-thiogalactopyranoside (IPTG), LB medium (Luria/Miller), phosphate-buffered saline pH 7.4 (PBS), PBS with Tween 20 (PBS-T, pH 7.4), sodium dodecyl sulfate (SDS, ≥99.5%), and urea (≥99.5%); Charles River (Wilmington, MA, USA): CD-1 mouse anti-mammalian orthoreovirus type 3 serum (Lot: S1099); GE Healthcare (Fairfield, CT, USA): HisTrap™ HP (1 mL); Jackson ImmunoResearch Laboratories, Inc. (West Grove, PA, USA): Peroxidase-conjugated AffiniPure goat anti-mouse IgG + IgM (H + L, #115062) and anti-rabbit IgG (H + L, #92797); lysozyme (chicken egg white); Merck Millipore (Burlington, MA, USA): anti-reovirus type-3 antibody, σ-1 hemagglutinin, clone 9BG5 (MAB994); NH Dyeagnostics GmbH (Halle, Germany): Immuno Blue HRP Substrate; Roche Diagnostics International AG (Rotkreuz, Switzerland): DNase I (RNase-free); Serva Electrophoresis GmbH (Heidelberg, Germany): Acrylamide/bis(acrylamide) (30% T, 2.67% C), Albumin bovine (Fraction V, protease-free), TEMED, ammonium persulfate (99%), Coomassie® Brilliant Blue G250, glycine (> 98.5%), protease inhibitor mix B, Tween 20 (pure), and trypsin (sequencing grade, MS approved); Sigma-Aldrich GmbH (Taufkirchen, Germany): 2-mercaptoethanol (BioUltra), ammonium bicarbonate (≥99.5%), carbonate-bicarbonate buffer, imidazole (≥99.5%), magnesium chloride hexahydrate (MgCl2, ≥99%), and Triton™ X-100; SurModics Inc. (Eden Prairie, Minnesota, USA): StabilZyme Select®, Assay Diluent (Protein-free); Thermo Fisher Scientific (Waltham, Massachusetts, USA): SuperBlock® (PBS), 6×-His Epitope Tag Antibody (HIS.H8); Seramun Diagnostika GmbH (Heidesee, Germany): TMB substrate solution. Water was produced in-house using a Purelab Ultra water purification system (resistance > 18.2 MΩ·cm; total organic content < 5 ppb; ELGA LabWater GmbH, Celle, Germany).
Preparation of virus stock
Mammalian orthoreovirus type-3 (strain Dearing) and mammalian orthoreovirus type-2 (strain Jones) were prepared by infection of MA-104 cells (ATCC CRL-2378) grown in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with fetal bovine serum (10%), sodium pyruvate (1 mmol/L) and MEM non-essential amino acids (Life Technologies) at a multiplicity of infection (MOI) of 0.5 for three days. Viral stocks were titrated using an indirect immunofluorescence assay indicating a titer of 4.7 × 107 FFU/ml.
Serum samples
Sera were obtained from experimentally infected animals. Briefly, experimental infections relied on specific-pathogen-free 8 to 10 week old BALB/c (n = 15) and C57BL/6 mice (n = 15) intranasally infected with mammalian orthoreovirus type-3 strain Dearing with a viral load of 1.2 × 106 FFU (manuscript in preparation). Five mice from both groups were euthanized 2, 4 or 6 weeks post infection and blood was collected by cardiac puncture. Field sera obtained from German animal facilities were analyzed by GVG Diagnostics GmbH.
Molecular cloning and protein expression
The coding sequence of mammalian orthoreovirus type-3 strain Dearing segment S1 was codon-optimized for E. coli, synthesized, and cloned (GenScript, Nanjing, China). A DNA-sequence TGGAGCCACCCGCAATTTGAAAAAGGTGGTAGC (corresponding to WSHPQFEKGGS) coding for Strep-tag II (underlined) was added at the 5′ end to the S1 open reading frame. The construct was cloned into pET21b(+) using restriction sites NdeI and XhoI. The plasmid was transformed into E. coli DH5α for propagation and the nucleotide sequence was confirmed by sequencing (Eurofins, Germany). The plasmid was transformed in E. coli Rosetta BL21 (DE3) pLysS. An overnight culture was used to inoculate terrific broth medium (1 L) with ampicillin (100 μg/mL) to obtain a turbidity of 0.1 at 600 nm (OD600). Bacterial cultures were grown (continuous shaking, 37 °C) to an OD600 of 0.7. The temperature was reduced to 30 °C, and protein expression was induced by addition of IPTG (1 mmol/L). After 4 h, cells were harvested by centrifugation (5000 × g, 4 °C, 20 min) and the cell pellet stored at -80 °C until further use.
Solubilization of inclusion bodies and protein purification
Cells were thawed and suspended in lysis buffer (30 mL, 20 mmol/L Tris-HCl, 3 mmol/L MgCl2, pH 8.0). Protease-Inhibitor Mix B (50 μL) and a spatula tip of lysozyme and DNase I were added and the suspension was incubated on ice for 30 min. Cells were lysed on a French® pressure cell press (Thermo Fisher Scientific Inc., Waltham, USA) at 1500 psi and centrifuged (48,000 × g, 4 °C, 1 h). The pellet was suspended in PBS (30 mL, 30 min, 4 °C) and centrifuged (15,000 × g, 4 °C, 30 min). Resuspension (30 mL) and centrifugation were repeated once with Triton X-100 (2%, v/v) in PBS and twice with urea (1.5 and 3 mol/L) in PBS. The remaining inclusion bodies were dissolved in a solution of urea in PBS (8 mol/L, overnight, 4 °C), centrifuged (48,000 × g, 4 °C, 1 h), and adjusted to a final concentration of 25 mmol/L imidazole. The protein solution was sterile-filtered (0.22 μm) and purified by immobilized metal ion affinity chromatography (IMAC) using an ÄKTA purifier FPLC system (GE Healthcare, CT, USA) equipped with a P-900 pump and a P-900 UV detector. Briefly, the HisTrap-column (1 mL, prepacked with Ni Sepharose High Performance) was equilibrated with ten column volumes (CV) of binding buffer (8 mol/L urea, 25 mmol/L imidazole in PBS, pH 7.4) before the protein solution was loaded. Non-binding components were washed out with binding buffer (10× CV) and the protein was eluted with a linear 20 min gradient to 100% elution buffer (8 mol/L urea, 0.5 mol/L imidazole in PBS, pH 7.4). The eluate fraction containing the protein was dialyzed against urea in PBS (4 mol/L, pH 7.4), and stored at -80 °C. Protein concentrations were determined on a NanoDrop 2000c spectrophotometer (Thermo Fisher Scientific, USA) with dialysis buffer as blank.
SDS-PAGE
Protein samples and fractions were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) on a Mini-Protean 3 cell (Bio-Rad Laboratories) using stacking (T = 4%, C = 2.67%) and separation gels (T = 12%, C = 2.67%; 6 cm long) that were 1 mm thick. Samples were diluted (1:4, v/v) with sample buffer (62.5 mmol/L Tris/HCl, pH 6.8, 20% (v/v) glycerol, 2% (w/v) SDS, 5% (v/v) β-mercaptoethanol, 0.5% (w/v) bromophenol blue) to a total volume of 20 μL, heated (5 min, 95 °C), and loaded on the stacking gel. Separation was performed by applying voltages of 100 V for 10 min and 200 V for 45 min using a PowerPac 300 (Bio-Rad Laboratories). Proteins were visualized by colloidal Coomassie Brilliant Blue (CBB) G-250 [32], or Oriole™ fluorescent stain (λexc = 270 nm, λem = 604 nm) according to the manufactures protocol. Images were taken on a ChemiDoc MP CCD camera system (Bio-Rad Laboratories).
Immunoblot
Proteins were electroblotted onto a PVDF membrane using a Trans-Blot Turbo transfer cell (Bio-Rad Laboratories) for 10 min (25 V, 1.3 A, RT). Membranes were blocked with Advan Blocking solution (30 min, RT) and incubated with serum samples (3 μL serum in 10 mL Advan Blocking solution) overnight at 4 °C. Afterwards, membranes were washed two times with Advan Washing solution (5 min), incubated with peroxidase-conjugated goat anti-mouse IgG + IgM (0.5 μL in 10 mL Advan blocking solution plus 0.5% BSA (w/v), 90 min, RT), and washed again with Advan Washing solution (3×, 5 min each). The membrane was incubated with Immuno Blue HRP-Substrate (NH Dyeagnostics, Halle, Germany) for 10 min, washed in Advan washing solution and the fluorescence was recorded (ChemiDoc MP CCD camera system, Bio-Rad Laboratories).
Enzyme-linked immunosorbent assay (ELISA)
Sera were tested with the Mouse Reovirus type 3 ELISA Kit (XPressBio-ELISA, BioCat, Heidelberg, Germany) according to the manufacturer’s instructions. Briefly, sera were diluted in sample diluent (1:50, v/v) and 0.1 mL were transferred to the wells of a microtiter plate. The plate was covered and incubated (45 min, 37 °C). Wells were washed five times with wash solution (0.3 mL per well). Peroxidase conjugate was added (0.1 mL per well), incubated (45 min, 37 °C), and the wells were washed five times with wash solution (0.3 mL per well). Peroxidase substrate solution containing 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) was added (0.1 mL per well) and incubated (30 min, RT). The absorbance was recorded at 405 nm (Infinite F50 absorbance microplate reader, Tecan, Männedorf, Switzerland). Difference absorbance values were calculated by subtracting the absorbance recorded for a control well.
Alternatively, Medium Bind microplates (Brand, Wertheim, Germany; 96-well, U-shape) were coated with Strep-rσ-1-His (0.1 μg) in carbonate-bicarbonate buffer (50 mmol/L, pH 9.6) overnight at 4 °C. Wells were washed three times (PBS, 300 μL) using a Columbus Pro ELISA washer (Tecan, Männedorf, Switzerland), blocked with Superblock (300 μL, 30 min, RT), and stored at 4 °C. During all incubations, plates were covered with an adhesive foil (SealPlate, Excel Scientific, CA, USA). Wells were incubated with diluted murine serum (1:50 in Assay Diluent; 100 μL, RT) for 45 min. The wells were washed three times with PBS-T (300 μL per well) and conjugate solution was added (100 μL per well, goat anti-mouse-HRP, 1:30,000 in Stabilzyme Select), incubated (30 min, RT), and washed three times, before TMB was added (100 μL per well). After 15 min at RT the reaction was stopped with sulfuric acid (0.5 mol/L, 100 μL per well) and the absorbance recorded at 450 nm.
Indirect immunofluorescence assay (IFA)
The immunofluorescence assay used acetone-fixed mammalian orthoreovirus type-3-, type-2-, and mock-infected (negative control) MA-104 cells. Mouse serum samples were diluted (1:75) with blocking buffer (5% bovine serum albumin in PBS) and incubated on the cells for 1 h at 37 °C. Cells were washed thoroughly and incubated with Alexa Fluor 488-conjugated goat anti-mouse IgG secondary antibody (1 μg/mL; Thermo Fisher Scientific, MA, USA) and DAPI (final concentration of 0.5 μg/mL) for 1 h at 37 °C. Cells were then washed twice with PBS and images were taken using a fluorescence microscope (IX70, Olympus, Japan).
Trypsin digest
Gel bands in CBB G-250-stained gels were cut manually or automatically using an EXQuest™ Spot Cutter (Bio-Rad Laboratories), transferred to 0.5 mL polypropylene tubes (Eppendorf, Hamburg, Germany), and destained with aqueous acetonitrile (30%, v/v) containing ammonium bicarbonate (100 μL, 50 mmol/L, w/v) at RT for 5 min. The solution was discarded and the destaining procedure repeated twice before acetonitrile was added. After 5 min, supernatants were discarded and gel pieces were dried on air. A solution of trypsin (Serva Electrophoresis GmbH, 5 μL, 25 ng/μL in 3 mmol/L aqueous ammonium bicarbonate) was added. After incubation (4 h, 37 °C), each solution was transferred to a 0.5 mL polypropylene tube (Eppendorf). Acetonitrile (50 μL) was added and incubated in an ultrasonic bath (Bandelin, Berlin, Germany) for 5 min at RT. The supernatant was transferred to the tube containing the first supernatant and dried in a vacuum concentrator 5301 (Eppendorf) for 1 h at 60 °C. Dried peptides were stored at -20 °C.
NanoRP-HPLC-ESI-QTOF-MS/MS
Extracted peptides were dissolved in aqueous acetonitrile (20 μL; 3%, v/v) containing formic acid (0.1%, v/v) and analyzed on a Waters nano ACQUITY Ultra Performance Liquid Chromatography (nano UPLC) system coupled online to an electrospray ionization quadrupole time-of-flight mass spectrometer (ESI-QTOF-MS, Synapt G2Si MS, Waters, MS Technologies, Manchester, UK). Samples were loaded on a C18-trap column (2G-V/M Trap Symmetry, Waters, 180 μm internal diameter (ID), 2 cm length, and 5 μm particle size) and separated on a C18-column (nanoACQUITY UPLC Peptide BEH, 75 μm ID, 10 cm length, and 1.7 μm particle size) at a column temperature of 35 °C using a linear gradient from 3 to 40% aqueous acetonitrile (0.1% formic acid) in 18.5 min and to 85% aqueous acetonitrile (0.1% formic acid) in 3 min. Peptides were identified by data independent acquisition (DIA) in positive ion MSE mode using the following settings: m/z 50 to 2000, sampling cone of 40 V, source offset of 80 V, source temperature of 80 °C, cone gas flow of 30 L/h, purge gas flow of 150 mL/h, nanoflow gas pressure of 0.3 bar, and a scan time of 0.5 s. Fragmentation was triggered in the trap before the IMS cell using a collision energy ramp from 18 to 40 V. The doubly protonated signal of Glu-1-Fibrinopeptide B at m/z 785.8426 was acquired as lock mass. Acquired data were processed with the Progenesis QI for proteomics software 4.0 (Nonlinear Dynamics), which included Apex3D (version 3.0.14.11) and Peptide3D (version 2.120.0.0) as processing tools, using a lock mass tolerance of 0.254 m/z units. Low, elevated and high energy scan counts were individually determined for every sample by the automatic search function of the software. Data were analyzed with the Progenesis QI search engine using the following workflow parameters: database uniprot_sprot (552,259 sequences, downloaded 26.10.2016), precursor and product MHP window (singly protonated peptide mass of the theoretical sequence) was − 1, “number by match for peptide minimum value” was 3, “number peptide for protein minimum value” was 1, “number by match for protein minimum value” was 7, “protein mass maximum atomic mass unit value” was 250,000, false positive rate value was 4, one missed cleavage site, trypsin as “digester reagent”, and methionine oxidation and cysteine carbamidomethylation as variable modifications. The final fragment peptide table generated by the ion accounting output was filtered for proteins from mammalian orthoreovirus 3 type Dearing. Proteins represented by at least three different peptides and identified by “AutoCurate” (> 95% probability) were considered confident.
ELISA validation and statistical analysis
Repeatability of the indirect ELISA was tested by 36 replicates of positive (PC, anti-His antibody, 1:15,000 in StabilZyme Select®) and negative controls each (NC, Reo-3 [-] serum pool, 1:50 in Assay Diluent). Intermediate precision was tested by five replicates of PC and NC each on five consecutive days. Calculations followed ISO 5725–2 [33, 34]. Assay stability was tested with freshly coated ELISA plates covered with an adhesive foil and stored at 4 °C or 37 °C. All materials needed for testing (controls, buffers, conjugate, TMB, and sulfuric acid) were stored under the same conditions for the same time periods. Three replicates of PC and NC each were tested after storing them at 4 °C or 37 °C for nine time periods up to 200 days.
The limit of detection (LOD) was calculated from the mean absorbance value of sera from mock-infected mice or mice infected with other pathogens (LODref) plus three times its standard deviation (SD), respectively. D-SN, D-SP and the cut-off values were determined by receiver operating characteristic (ROC) analyses using Graph Pad Prism 7.0 (Graph Pad Software, La Jolla, CA, USA).
Ethical approval
Animal experiments were approved by the Animal Care and Usage Committee of the Landesdirektion Sachsen, permission no. TVV 04/14.