Abstract:
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 neighbouring 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
visualisation of membrane release events. African horse sickness virus (AHSV) is a
non-enveloped virus that employs two different release mechanisms from mammalian cells, i.e. lytic release through a disrupted plasma membrane and a non-lytic buddingtype
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 visualisation, which allowed detection of both types of virus release at the
plasma membrane at a significantly higher frequency than when utilising 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 titres in tissue culture.
