Virus stocks of the IBV strains QX and B1648 were obtained by propagation in specific pathogen free embryonated chicken eggs (VALO SPF, Cuxhaven, Germany). The allantoic fluid was harvested, clarified by low–speed centrifugation and stored at −80 °C. The viral titer was determined by titration in primary chicken embryo kidney cells. The IBV strain QX was kindly provided by Hans Christian Philipp (Lohmann Tierzucht, Cuxhaven, Germany). The IBV strain B1648 was kindly provided by Dave Cavanagh (Institute for Animal Health, Compton, UK). S1 sequences can be found in Additional file 1.
Primary chicken embryo kidney cells were prepared from 20 day old SPF chicken embryos as described previously .
Epithelial cells of the chicken oviduct from 16–19 weeks old chickens were isolated by opening the magnum segment of the oviduct longitudinally and cutting it into small pieces. After incubation with 0.4 mg protease from Streptomyces griseus, Type XIV (Sigma-Aldrich, Steinheim, Germany)/mL medium for 3 h, the remaining tissue was removed and the cells in the supernatant were pelleted by centrifugation. The pellet was resuspended with Dulbecco’s modified Eagle medium and Ham’s F12 1:1, supplemented with 5% fetal bovine serum (Biochrom, Berlin, Germany), 1% chicken serum (Sigma-Aldrich, Steinheim, Germany), 1% nonessential amino acids (PAA, Pasching, Austria), 1% Penicillin/Streptomycin (PAA, Pasching, Austria), 0.01% Gentamycin (PAA, Pasching, Austria), 0.01% Amphothericine B (Sigma-Aldrich, Germany), filtered through 100 μm cell strainer and seeded in tissue culture flasks (75 cm2). After incubation for 2 h the cells were seeded in 6-well-plates and incubated at 37 °C and 5% CO2.
Preparation of oviduct explants
The oviduct was aseptically collected from 16 to 18 weeks old premature SPF-chicken. After washing the oviduct with phosphate-buffered saline (PBS), the different segments of the oviduct (infundibulum, magnum, isthmus, uterus) were cut into approximately 5 mm thick rings and incubated at 37 °C For cultivation we used the same medium mentioned above. The next day the explants were used for infection studies with the strains QX and B1648. All experiments were performed in accordance with German animal welfare regulations.
Viability of oviduct explants
In a live and dead staining (LIVE/DEAD Viability/Cytotoxicity Kit, Invitrogen, Darmstadt, Germany) we tested the oviduct rings for viability. Living cells are stained in green, dead cells are stained in red. Additionally we analyzed the oviduct rings for ciliary activity under the light microscope.
Preparations of cryosections of infected oviduct explants
The explanted oviduct rings were infected 24 h post preparation with the IBV strains IBV QX and B1648 at a titer of 1 × 104 foci-forming units (FFU)/ring in duplicates in each of in total three experiments. The infected rings were incubated for 1 h at 37 °C on a shaker. After 12 h, 24 h and 48 h the rings were mounted on filter papers with tissue freezing medium (Jung, Heidelberg, Germany) and were subsequently frozen in liquid nitrogen and stored at −80 °C until they were sectioned (10 μm) with a cryostat (Reichert-Jung, Nußloch, Germany).
The cryosections were fixed with 3% paraformaldehyde for 20 min and subsequently permeabilized with 0.2% Triton-X-100 followed by three washing steps with PBS. To detect infected cells, a monoclonal antibody Ch/IBV 48.4 (Prionics, Lelystad, the Netherlands) directed against the N protein of IBV in a dilution of 1:100 was used. Bound antibodies were visualized by FITC-labeled anti-mouse antibodies (Sigma-Aldrich, Steinheim, Germany). All antibodies were diluted in 1% bovine serum albumin (Roth, Karlsruhe, Germany) and incubated with the sections for 1 h at room temperature in a humid incubation chamber. Fluorescence microscopy was performed with a Nikon Eclipse Ti Microscope.
Neuraminidase treatment of oviduct explants
Explants of the oviduct had been prepared as described above and 24 h post preparation the explants were treated with each 300 mU neuraminidase from Clostridium perfringens (Sigma-Aldrich, Steinheim, Germany) for 1 h at 37 °C. After careful washing of the explants they were infected with 1 × 104 FFU/explant of IBV QX. 24 hours post infection (hpi) the explants were snap frozen in liquid nitrogen and were used for cryosection preparation.
Lectin staining of oviduct epithelial cells
To detect alpha 2,3-linked sialic acids we used the lectins Maackia amurensis agglutinin (MAA) in its two isoforms MAA I and MAA II (Vector Laboratories, USA). The MAA I lectin was FITC labeled. To detect alpha 2,6-linked sialic acids we used the lectin Sambucus nigra agglutinin (SNA) labeled with FITC (Vector Laboratories, USA). Cryosections of the different oviduct parts were incubated with the indicated lectins for 1 h at room temperature. MAA II was used after preincubation of sections with an Avidin/Biotin Blocking kit (from Vector Laboratories, USA). Detection of bound MAA II lectins was carried out with streptavidin labeled with Cy3 (Sigma-Aldrich).
Preparation of soluble spike proteins
cDNA of the S1 subunits was generated by RT-PCR of the indicated strains and cloned into the vector pCG1_Fc to obtain fusion proteins consisting of the S1 gene part (AA 1–536 for B1648 and AA 1–524 for QX) fused to the human IgG Fc domain. For the production of soluble spike proteins, BHK 21 cells were transfected using polyethylenimine (Polysciences, Eppelheim, Germany) with the plasmids following the instructions of the manufacturer. Supernatants containing the soluble proteins were harvested 24 h and 48 h post transfection.
To concentrate the proteins in the supernatant, Amicon Ultra-15 Centrifuge Filter devices were used. Aliquots of the soluble proteins were stored at −20 °C.
Binding assay on cryosections of the oviduct
To analyze binding of the soluble spike proteins of the strains QX and B1648, we incubated the soluble proteins on cryosections of the oviduct at 4 °C for 1 h. To test if binding is sialic acid dependent we incubated the cryosections with 300 mU neuraminidase from Clostridium perfringens (Sigma-Aldrich, Steinheim, Germany) for 1 h at 37 °C before the incubation with the soluble proteins. Binding was detected with a secondary anti-human-Fc Cy-3 labeled antibody in a concentration of 1:750 (Sigma-Aldrich, Steinheim, Germany).
Binding of virus particles
Allantoic fluid containing 3 × 105 FFU of either QX or B1648 and 3 mL allantoic fluid of uninfected SPF chicken eggs was ultracentrifuged through a 25% sucrose cushion at 100 000 × g for 2 h at 4 °C. The pelleted virus particles were resuspended in PBS and applied to cryosections of the isthmus part of the chicken oviduct for 1 h at room temperature. After three washing steps a monoclonal antibody directed against the spike protein (Prionics, Lelystad, the Netherlands) and followed by a FITC labeled secondary antibody was used to detect bound virus particles.
Flow cytometric analysis of isolated oviduct epithelial cells with bound spike proteins
To quantify the binding of soluble S1 proteins to isolated chicken oviduct epithelial cells, the isolated cells were seeded in 6-well plates (Greiner, Frickenhausen, Germany). The next day the cells were washed with PBS and detached using 400 μL trypsin and the cells were subsequently resuspended in PBS containing 1.0% bovine serum albumin. After pelleting by centrifugation at 1000 rpm for 5 min, the cells were incubated with the soluble S1 proteins for 1 h at 4 °C in an overhead shaker. After 3 washing steps with PBS containing 1.0% BSA and pelleting of the cells by centrifugation at 1000 rpm for 5 min after every washing step, the cells were incubated with an anti-human PE-conjugated secondary antibody in a concentration of 1:200 (Beckman-Coulter, Marseille, France) for 1 h at 4 °C in an overhead shaker. After three more washing steps with PBS containing 1.0% BSA, the cells were directly subjected to flow cytometric analysis on a Beckman Coulter Epics XL flow cytometer and analyzed using EXPO32 analysis software.