Infectious bronchitis corona virus establishes productive infection in avian macrophages interfering with selected antimicrobial functions
MQ-NCSU cells (0.75 x 106 per well) were seeded on 12-well plates and either infected or treated with 0.1 MOI of IBV Conn A5968 strain, IBV M41 strain, 1μg/ml LPS (positive control for NO production), 50μg/ml dsRNA (positive control for type 1 IFN activity) or RPMI media (negative control). (a) After 24-hour of stimulation period, cell culture supernatants were collected and NO content in a portion was measured using the Griess assay. Average nitrite contents with each treatment and controls (a) are shown. (b) Following the collection of culture supernatants, the MQ-NCSU cells were scraped and trypan blue dye exclusion method was used to count dead and live cells. The percentage of dead cells with each treatment and controls (b) are given. (c) The remaining portions of macrophage cell culture supernatants were transferred (250μl) to DF-1 cell monolayer for the quantification of type 1 IFN activity. Twenty-four hours following the transfer of culture supernatants, the DF-1 cells were infected with VSV-GFP at the rate of MOI = 0.1. At 24 hours post infection of DF-1 cells, the cells expressing GFP signals were observed under epifluorescent microscope after fixation with 4% paraformaldehyde and nuclear staining with Hoechst 33342 (Image-iT™ LIVE Plasma Membrane and Nuclear Labeling Kit (I34406), Invitrogen, Eugene, Oregon, USA). Average fluorescent percentages with each treatment and controls (c) are shown. The results represent pooled data of three independent experiments. * = p< 0.01, ** = p< 0.001, *** = p<0.0001.