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Infectious bronchitis corona virus establishes productive infection in avian macrophages interfering with selected antimicrobial functions

Fig 4

IBV M41 and Conn A5968 strains replicate in macrophages in vitro and establish productive infection.

1.5x 106 of MQ-NCSU cells were cultured on plastic coverslips in 6-well plates for 24 hours before infected with 0.1 MOI of IBV Conn A5968 and M41 strains. After 1 hour of adsorption period, inoculum was drained and cells were washed 2 times with HBSS and incubated at 40°C for another 24 hours. At the end of 24 hours, cell membranes of macrophages were stained with horseradish wheat agglutinin conjugated with Alexa Flour 594. Then the cells were fixed using 4% paraformaldehyde, stained for IBV N antigen and visualized using Dylight 488® conjugated secondary antibody and nuclear staining was done with mounting media with DAPI at the time of mounting coverslips on the slides. (a) Percentage of infected macrophages (MQ-NCSU cells) is shown for each IBV strain. (b) Representative confocal microscopy images showing M41 and Conn A5968 IBV antigens in macrophages. (c) The MQ-NCSU cells were cultured on 6-well plates for 24 hours before infected with 0.1 MOI of IBV Conn A5968 or M41 strains. After 1 hour of adsorption period, inoculum was drained and cells were washed 2 times with HBSS to preclude subsequent quantification of the residual IBV inoculum and continued to incubate at 40°C before collecting cell culture supernatant at 6, 12, 18, 24 and 48 hpi for IBV viral RNA concentration determination using a qPCR assay. All the treatments were done in triplicate and qPCR reactions were carried out in triplicate for each sample as well. For each time point, average number of IBV copies per 200 ng of starting RNA of both IBV Conn A5968 and M41 strains are shown with SEM. ANOVA test was performed to identify group differences and the differences were considered significant at P<0.05. The results represent pooled data of three independent experiments.

Fig 4

doi: https://doi.org/10.1371/journal.pone.0181801.g004