FIG 4.

Induction of DNA damage in PBMCs of chickens infected with MDV. Specific-pathogen-free (SPF) susceptible White Leghorn chicks (B13/B13 haplotype) were inoculated intramuscularly with 1,000 PFU of the very virulent MDV strain vRB-1B. DNA damage onset in PBMCs from 10 noninfected chickens (circles) and 13 birds infected with vRB-1B (triangles) was assessed. Blood was collected from all birds at the indicated time points. (A) Analysis of DNA damage in PBMCs isolated from mock- and vRB-1B-infected chickens by alkaline comet assays. Two slides per comet assay were prepared for each animal at each time point. A minimum of 50 comets were observed and further analyzed on each replicate slide using CometScore software. Results are presented as a dot plot, with each dot representing an animal and the mean OTM for each group being indicated as a bar. ***, P < 0.001. (B) Viral load estimated after extraction of DNA from whole blood and quantification of MDV genome copies using qPCR. For each group, the number of ICP4 copies in the MDV genome was normalized to 10⁶ copies of the cellular genome, estimated by the detection of iNOS copies. The median copy numbers are indicated as bars. (C) Meq mRNA expression upon MDV infection. Total RNA was extracted from PBMCs isolated from the blood of birds infected with vRB-1B. Quantitative RT-PCRs were performed in order to detect the expression of Meq mRNA. The level of gene expression was normalized to that of GAPDH expression, and fold changes are presented as box plots (minimum and maximum).