Productive infection of field strains of avian coronavirus infectious bronchitis virus in chicken peripheral blood-derived monocyte

Yueting Zhang; Gary Whittaker

IBV strains used in this study were M41, Cal99, Conn46, Iowa97 (all field strains) and Beaudette (a laboratory--adapted version of M41). Virus stocks were produced in embryonated chicken eggs and titered by EID50 or TCID50 assay. Chicken peripheral blood monocytes were prepared as described (20). Briefly, blood was obtained by cardiac puncture from 6--8 week--old specific-pathogen--free White Leghorn chickens. Monocytes were isolated with a Ficoll gradient (GE Healthcare) and were cultured in RPMI 1640 supplemented with 10% heat--inactivated chicken serum and penicillin/streptomycin. 24h after cell seeding, non--adherent were washed away. Monocytes for each independent experiment were obtained from an individual blood donor. All work with chickens was carried out according to the Cornell University Animal Care and Use program and complied with the Public Health Service Policy on Humane Care and Use of Laboratory Animals For immunofluorescence assay, cells were fixed with methanol for detection of IBV viral antigens; otherwise cells were fixed with paraformaldehyde. Anti--S1 monoclonal antibody 15:88 was used for IBV--M41 and Beaudette, and anti--M monoclonal antibody 9:19 was used for IBV--Cal99, IBV--Conn46, and IBV--Iowa97. Monoclonal antibody KUL01 was purchased from Abcam. Secondary antibody was AlexaFluor goat anti--mouse 488 and nuclei were stained with Hoechst 33258 (Molecular Probes). Cells were quantified by scoring the % cells positive for viral antigen with All rights reserved. No reuse allowed without permission.