Department of Genetics, University of Pretoria, Pretoria 0002, South Africa.
Microsc Res Tech. 2012 Oct;75(10):1452-9. doi: 10.1002/jemt.22111. Epub 2012 Aug 3.
Cryofixation by high-pressure freezing (HPF) and freeze substitution (FS) gives excellent preservation of intracellular membranous structures, ideal for ultrastructural investigations of virus infected cells. Conventional sample preparation methods of tissue cultured cells can however disrupt the association between neighboring cells or of viruses with the plasma membrane, which impacts upon the effectiveness whereby virus release from cells can be studied. We established a system for virus infection and transmission electron microscopy preparation of mammalian cells that allowed optimal visualization of membrane release events. African horse sickness virus (AHSV) is a nonenveloped virus that employs two different release mechanisms from mammalian cells, i.e., lytic release through a disrupted plasma membrane and a nonlytic budding-type release. Cellulose microcapillary tubes were used as support layer for culturing Vero cells. The cells grew to a confluent monolayer along the inside of the tubes and could readily be infected with AHSV. Sections of the microcapillary tubes proved easy to manipulate during the HPF procedure, showed no distortion or compression, and yielded well preserved cells in their native state. There was ample cell surface area available for visualization, which allowed detection of both types of virus release at the plasma membrane at a significantly higher frequency than when utilizing other methods. The consecutive culturing, virus infection and processing of cells within microcapillary tubes therefore represent a novel model system for monitoring intracellular virus life cycle and membrane release events, specifically suited to viruses that do not grow to high titers in tissue culture.
高压冷冻（HPF）和冷冻替代（FS）的冷冻固定可极好地保存细胞内的膜结构，非常适合用于感染病毒的细胞的超微结构研究。然而，组织培养细胞的常规样品制备方法可能会破坏相邻细胞之间或病毒与质膜之间的联系，从而影响研究病毒从细胞中释放的效果。我们建立了一种用于病毒感染和透射电子显微镜制备哺乳动物细胞的系统，该系统允许最佳地观察膜释放事件。非洲马瘟病毒（AHSV）是非包膜病毒，它采用两种不同的从哺乳动物细胞释放的机制，即通过破坏的质膜的裂解释放和非裂解出芽型释放。纤维素微毛细管管用作培养vero 细胞的支撑层。细胞沿着管的内部生长成致密的单层，并且可以容易地被 AHSV 感染。微毛细管管的切片在 HPF 过程中易于操作，没有变形或压缩，并且以其天然状态很好地保存了细胞。有足够的细胞表面积可用于可视化，这使得可以以比使用其他方法更高的频率检测到质膜上的两种类型的病毒释放。因此，在微毛细管管内连续进行细胞培养、病毒感染和处理代表了一种用于监测细胞内病毒生命周期和膜释放事件的新型模型系统，特别适合于在组织培养中不能达到高滴度的病毒。
