Improved membrane preservation of flavivirus-infected cells with cryosectioning.

Ultra-cryomicrotomy and electron microscopy were used to investigate membranous structures in dengue virus-infected mammalian and insect cells. The cryo-sectioned samples displayed ultrastructure comparable to their resin-embedded counterparts with all previously identified virus-induced structures being observed. Structures not previously identified were also found. In particular, membrane-bound packets of vesicles, 100-200 nm in diameter were seen distributed throughout areas of virus-induced membrane proliferation. These packets were clearly distinct from virion arrays. Small smooth membrane vesicles, previously found to contain thread-like enclosures (M.L. Ng, J. Gen. Virol. 68 (1987) 577-582), were frequently observed to contain dense staining material, however the exact nature of this material remains unclear. Virus-induced modification of golgi-like and/or ER membranes was also observed and may represent early events in the generation of the smooth membrane vesicles seen during infection. We suggest that cryosectioning is the method of choice to investigate membrane rearrangement induced by this family of viruses and that a diamond knife and modified staining techniques, as utilised in this report, be employed to enhance morphology and section preservation.