Fig. 2.

No mature virus in the feather follicle epithelium (FFE) at 5 dpi. Chicken embryonated cells were inoculated with viruses isolated from the FFE at 5, 6, and 7 dpi, and the cells were incubated for 7 days, followed by passaging three times. After the 3rd passage, the cells were observed via inverted microscopy with a 10× objective lens (A). For immunofluorescence assay, the cells were fixed and incubated with α-HVT serum (green signals) to stain the HVT viral proteins. The stained cells were observed with an immunofluorescence microscope (B). For qPCR, viral DNAs were extracted from the cells after the 3rd passage and subjected to the qPCR using the gene-specific primers for SORF1 gene of HVT and chicken α2 (VI)-collagen gene for internal control. The relative expression levels of viral DNA were calculated as described in the legend to Fig. 1A. No HVT DNA was detected in the FFE at 5 dpi after passaging twice.