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Improved membrane preservation of flavivirus-infected cells with cryosectioning
J M Mackenzie1, M K Jones, P R Young
1Sir Albert Sakzewski Virus Research Centre, Royal Childrens' Hospital, Herston, Queensland, Australia. mackenzi@biosci.uq.oz.au
Abstract:
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.
Insights
Ultra-cryomicrotomy revealed novel membrane structures in dengue virus-infected cells. This electron microscopy technique offers superior visualization of virus-induced membrane rearrangements.
Area of Science:
- Cell Biology
- Virology
- Microscopy
Background:
- Dengue virus infection induces significant cellular membrane rearrangements.
- Previous studies utilized resin embedding for ultrastructural analysis.
- Understanding these changes is crucial for comprehending viral replication.
Purpose of the Study:
- To investigate membranous structures in dengue virus-infected cells using ultra-cryomicrotomy.
- To compare cryo-sectioning with traditional resin-embedding methods.
- To identify novel virus-induced membrane structures.
Main Methods:
- Ultra-cryomicrotomy and electron microscopy were employed.
- Mammalian and insect cells infected with dengue virus were analyzed.
- Modified staining techniques and a diamond knife were utilized.
Main Results:
- Cryo-sectioned samples showed ultrastructure comparable to resin-embedded samples.
- Previously unidentified membrane-bound packets of vesicles (100-200 nm) were observed.
- Virus-induced modifications of Golgi-like and ER membranes were noted.
Conclusions:
- Ultra-cryomicrotomy is the preferred method for studying virus-induced membrane rearrangements.
- The technique enhances morphology and section preservation.
- Novel vesicle structures and membrane modifications provide insights into dengue virus replication.