Figure 3.

Necrosis in CuniNPV-infected C. quinquefasciatus larvae. (a) Cells infected with CuniNPV at 48 h p.i. were identified with a pool of fluorescein-labeled cRNA probes against CuniNPV genes, cun24, cun75, and cun85. Virus-infected cells at this time demonstrated hypertrophied nuclei (arrow) compared with uninfected cells (arrow head). Note that there is clear separation between cells that were positive for viral gene expression versus those that were negative (dashed line). More than 100 midguts were examined in several independent experiments; all cells positive for viral gene expression at this time point have hypertrophied nuclei (additional images in Supplementary Figure S2). (b) Cells with hypertrophied nuclei have compromised cell membrane integrity. Live larvae at 48 h p.i. were exposed to 1 μg/ml PI in culture media for 10 min before the midguts were dissected out, fixed with paraformaldehyde, and counterstained using DAPI. All the cells with hypertrophied nuclei (arrow) are also permeable to PI, indicating necrotic cell death. In contrast, cells in the anterior midgut (arrow head) have normal nuclei and intact membrane. More than 40 midguts were observed in three independent experiments, and essentially all of them showed the described association between nuclei morphology and permeability to PI