Foot-and-mouth disease virus (FMDV) is the type species of the Aphthovirus genus of the Picornaviridae. Infection by picornaviruses results in a major rearrangement of the host cell membranes to create vesicular structures where virus genome replication takes place. In this report, using fluorescence and electron microscopy, membrane rearrangements in the cytoplasm of FMDV-infected BHK-38 cells are documented. At 1?5-2?0 h post-infection, free ribosomes, fragmented rough endoplasmic reticulum, Golgi and smooth membrane-bound vesicles accumulated on one side of the nucleus. Newly synthesized viral RNA was localized to this region of the cell. The changes seen in FMDV-infected cells distinguish this virus from other members of the Picornaviridae, such as poliovirus. Firstly, the collapse of cellular organelles to one side of the cell has not previously been observed for other picornaviruses. Secondly, the membrane vesicles, induced by FMDV, appear distinct from those induced by other picornaviruses such as poliovirus and echovirus 11 since they are relatively few in number and do not aggregate into densely packed clusters. Additionally, the proportion of vesicles with double membranes is considerably lower in FMDV-infected cells. These differences did not result from the use of BHK-38 cells in this study, as infection of these cells by another picornavirus, bovine enterovirus (a close relative of poliovirus), resulted in morphological changes similar to those reported for poliovirus-infected cells. With conventional fixation, FMDV particles were not seen; however, following high-pressure freezing and freeze-substitution, many clusters of virus-like particles were seen.

SummaryHigh-pressure freezing is applicable to both morphological and immunocytochemical studies. We are investigating the morphogenesis of foot-and-mouth disease virus and African swine fever virus by the use of high-pressure freezing of infected cells. Foot-and-mouth disease virus particles are not detected in sections of conventionally immersion-fixed infected cells, but when the cells are prepared by high-pressure freezing, newly formed virions are readily seen throughout the cell. We report two methods for high-pressure freezing of virally infected cells: first, two sapphire discs frozen 'face to face' with a narrow spacer to prevent cell damage and, second, a fibrous filter substrate that can be easily cut into discs to fit into the freezing planchettes. Cells readily adhere to the fibres in vitro , and the complete disc can be rapidly transferred to the planchettes for freezing. Immunolabelling studies of the microneme proteins of the parasite Eimeria tenella indicate that high-pressure freezing followed by freeze-substitution in acetone with uranyl acetate allows high-sensitivity immunolabelling for these proteins.

Detection of rare events within solid tissues by immunocytochemistry is aided by imaging thick sections. Sections of 40–100 µm thickness of paraformaldehyde‐fixed solid tissue can be prepared by use of a vibrating microtome and when immunolabelled these sections can be imaged in a confocal microscope. This approach provides excellent preservation of the structure of the sample and imposes minimal antigenic damage. In studies of the invasion of the bovine intestinal epithelium by Salmonella, this method has allowed detection of individual invading bacteria within large samples. The thick vibrating microtome sections were also used for the detection of rare apoptotic cell nuclei identified by TUNEL staining.