Utilization of cellulose microcapillary tubes as a model system for culturing and viral infection of mammalian cells

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 2 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.

Comparative ultrastructural characterization of African horse sickness virus-infected mammalian and insect cells reveals a novel potential virus release mechanism from insect cells

“…Cells were cultured either on glass coverslips until ∼75 % confluent for AFM, SEM and confocal imaging or as a monolayer in culture flasks for TEM visualizations as described previously (Venter et al , 2012). KC and C6/36 cells were infected with AHSV-3 at an m.o.i.…”

“…In an attempt to observe this release, we meticulously screened more than 300 cells but were never able to capture any budding-type event similar to that observed in mammalian cells (see below). We even cultured and processed the cells directly in 200 mm diameter cellulose microcapillary tubes, as we recently reported that this reduces mechanical disruption and increases the chance of observing virus release (Venter et al, 2012), but with no success. However, we visualized extracellular viruses, present either as individual particles or as clusters of aggregated viruses, between neighbouring infected cells with intact plasma membranes (Fig.…”

Investigating the Role of African Horse Sickness Virus VP7 Protein Crystalline Particles on Virus Replication and Release

“…To date, AHSV VP7 crystalline particles have been observed in cells infected with AHSV serotypes 3, 4 and 9 [ 20 , 22 , 23 ]. These crystals have been observed in both mammalian cells and a Culicoides -derived insect cell line (KC cells) infected with AHSV [ 22 , 24 ]. The fine structure of these flat crystals of AHSV VP7 were revealed to be highly ordered hexagonal lattices [ 20 ].…”