Virus-Infected Cells

One of the most frequent requests we get here at the MicrobiologyBytes Video Library is for "videos of viruses". Slight problem - viruses are submicroscopic parasites, too small to video. However, even if we can't video the viruses themselves, we can show you their effects!

Virus infection of cells is complex and results in many changes to the host cell, known collectively as cytopathic effect, or "c.p.e.". Such changes include:

• Altered shape
• Detachment from substrate
• Lysis
• Membrane fusion
• Altered membrane permeability
• Inclusion bodies
• Apoptosis

This video shows the cytopathic effect of human rhinovirus infection on HeLa-ohio cells. At the start of the video, uninfected cells are shown. Since HeLa cells are an adherent cell line, they normally grow flat and stuck down firmly on the tissue culture flask. After infection with rhinovirus, the cells change shape, becoming round and more refractile (brighter) under phase contrast microscopy. Some infected cells detach from the tissue culture flask and float in the medium:

Actually, rhinoviruses are not usually very cytopathic. Other viruses, such as poliovirus or HHV-1 do much more damage to infected cells, causing complete lysis of infected cells. However, some HeLa cells (HeLa-ohio) are highly permissive for rhinovirus, producing virus titres (concentrations) of 1x10⁶-1x10⁷ ml^-1. On other cells types, the effects of rhinovirus infection are less pronounced, e.g. in MRC5 cells:

MRC5 cells are less permissive for rhinovirus infection than HeLa cells, with virus titres of 1x10⁵-1x10⁶ ml^-1 produced (10-fold less). Rhinovirus c.p.e. in MRC5 cells is less pronounced than in HeLa-ohio cells, inducing a typical granular appearance of infected cells, sometimes described as "salt & pepper" - you'll have to look at the video carefully to see this. In cells which are even less permissive, e.g. BEAB-2B epithelial cells, rhinovirus-induced c.p.e. is even less obvious. A titre of only of 1x10²-1x10³ ml^-1 is produced in BEAB-2B cells:

So, c.p.e. is not always a reliable way of detecting virus infection, and we frequently need to use other techniques. One of these is immunofluorescence. In this technique, infected cells are treated with fluorescent antibodies directed against the virus in question. Cells which contain virus antigens are stained and fluoresce under ultraviolet light in a fluorescence microscope. In the experiment shown in this video, HeLa cells were infected with human rhinovirus. Sixteen hours after infection, the cells were stained with fluorescent anti-rhinovirus antibodies:

Virus-infected cells fluoresce brightly. If you look closely, you can see that the fluorescence is localized to the cytoplasm, and the nucleus does not fluoresce. This is typical of all picornaviruses, which replicate in the cytoplasm. Uninfected cells can be seen as pale green ghosts in this video. This faint appearance is due to autofluorescence, a common property of many biological materials. Again, the brightly-stained virus-infected cells are round - quite different in shape from the uninfected cells. By counting the number of brightly-stained cells and the total number of cells (stained + unstained), it is possible to calculate the proportion of cells in the culture - around 80% in this video.

Virus Culture: A Practical Approach
A.J. Cann (Ed).

Viruses have properties that are distinct from other living organisms and so require different methods to culture them. This volume provides a broad treatment of the principles and practice of virus culture and will be of interest to all those involved in virus culture including academic, industrial, and clinical research groups. Contains over 90 tried and tested protocols for virus culture.
(Amazon.co.UK)

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