The day after I infected the cells I went to the fluorescence microscope and looked for fluorescence. The uninfected control? Nothing, black. The culture with cells infected with BTV-mCherry had islands of red. It had worked. I had made a bluetongue virus which, when it infects cells, expresses a fluorescent protein, in this case mCherry (one of many options - a girl working with me wanted mPlum).
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| BTV-mCherry replicating in a Drosophila fruit fly. Green fluorescence represents autofluorescence of fly tissues. Red = fluorescence generated upon BTV-mCherry infection. |
But why bother? What use is a virus which makes cells light up?
The reason it came in useful for us is that we wanted to see where in an insect the virus replicated. A more straightforward approach would be to find the virus using antibodies and then reveal the antibodies - immunolabelling. But this resulted in the insect muscles fluorescing. Using the fluorescent virus meant that we didn't need immunolabelling; instead we simply sliced up the insect and looked at which bits lit up when illuminated at the wavelength for mCherry.
Another reason is so that virus infections can be filmed - live cell imaging - to reveal dynamic processes. For example, virus protein 'X' is tagged with a green molecule. Where does it go? Fixing the cells means looking at just a point in time, rather than 'the virus protein travels from here (A) to here (B)'. A fluorescently tagged virus therefore lets you film the virus in real-time. I saw this in action with a fluorescently labelled Rinderpest virus (prior to its eradication). As the virus spread it formed large syncytia, swallowing up cells in an ever growing expanse of green.
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| Cells infected with GFP-expressing Rinderpest virus from Banyard et al., (2010). A; individual cells. B; syncitia. |
The ability to film dynamic processes of an infection has other advantages. Fluorescence can be measured, and can be used to reflect the extent or rate of replication: in addition to its use in virus research, this can obviouly be used as a measure of therapeutic value.
And in the animal?
The simplicity of looking for these sorts of viruses in animal tissue means it's possible to sample from more tissues of the infected animal, increasing our knowledge of the virus in the host. Even during infection, a machine can sort a blood sample so that we can find which blood cells the virus is replicating in. The insect infected with bluetongue is one example, but the most impressive I've seen so far was at the SGM in Dublin (now published in J. Virology). A canine distemper virus expressing GFP (green) or dTomato (red) was used to infect ferrets. This time it wasn't just a case of looking at dissected tissues, the authors looked at entire organs to determine where the virus replicated: virus infection at the macroscopic level - something impossible without such a virus.
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| Ferret tissues infected with green or red expressing Canine distemper virus (from Ludlow et al, 2012); A, infection around the eye and mouth, but not the nose; B, gingiva; C, skin epidermis; D, tongue and tonsils; E, salivary gland and lymph nodes; F, lungs; G, liver; H, spleen; I, stomach/gastrointestinal tract; J, infected B cell follicles in Peyer's patches; K, absence of fluorescence in the leptomeninges (redCDV); L, fluorescence in the leptomeninges (greenCDV). |
So, there you have it, not so useless after all. As freaky as they may be, fluorescently tagged viruses certainly have their place.
Banyard AC, Simpson J, Monaghan P, & Barrett T (2010). Rinderpest virus expressing enhanced green fluorescent protein as a separate transcription unit retains pathogenicity for cattle. The Journal of general virology, 91 (Pt 12), 2918-27 PMID: 20719989
M. Ludlow, D. T. Nguyen, D. Silin, O. Lyubomska, R. D. de Vries, V. von Messling, S. McQuaid, R. L. De Swart and W. P. Duprex (2012). Recombinant Canine Distemper Virus Strain Snyder Hill Expressing Green or Red Fluorescent Proteins Causes Meningoencephalitis in the Ferret Journal of virology, 88 (14) DOI: 10.1128/JVI.06725-11
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