Virology Handouts: Detection Methods and Virus Classifications - Prof. Elizabeth Fortunato, Study notes of Virology

Download Virology Handouts: Detection Methods and Virus Classifications - Prof. Elizabeth Fortunato and more Study notes Virology in PDF only on Docsity! MMBB 432/532 Virology Handouts for Topics 2 and 3 Topic 2 - Methods of Detection A. Virus cultivation – the advent of tissue culture (see FIGURE 1) 1. Most tissue culture is performed in either epithelial or endothelial cell monolayers. There are primary tissue cells and then there are cell lines (which are immortal). 2. The cells can be synchronized in different cell cycle phases to see the effects on both the virus and the host. B. The evidence for viral growth in culture (cytopathic effects) (see FIGURE 2) – the virus often causes very distinct phenotypic changes that can easily be viewed under the microscope. C. The need for animal work still exists for many viruses (lack of good model, pathogenesis studies important) D. Detection of viruses – there are many methods used to detect virus, depending upon how it interacts with the host cell 1. Measurement of infectious units – All of these methods involve SERIAL DILUTION of a viral stock and then reinfection onto a new monolayer of cells. a. Plaque Assays (see FIGURE 3) – Here we are looking for PFU (plaque forming units). Virus will lyse the original cell and will spread to surrounding cells in the monolayer, forming a clearly visible plaque. The spread of the virus is inhibited by an agarose overlay. b. There are variations on this theme, depending upon what the virus does to the cell. If it does not LYSE the cell, we can perform infectious center assays (must stain for a viral antigen) and if the virus transforms the cells, we can use focus forming assays. c. There is an important concept here known as PARTICLE TO PFU RATIO (i.e.- of the “stuff” that’s coming out of an infected cell, how much is INFECTIOUS?) 2. Measurement of viral particles and components – this is not so much to specifically quantitate how much virus is in your stock, but to get a feel for relative concentrations (i.e. – to compare different samples to one another). a. Hemagglutination assays – (see FIGURE 4) – these are used to get relative concentrations of viral particles in different samples. Twofold serial dilutions are performed on stocks, which are then mixed with RBCs. We look for lattice formation at the highest dilution. b. We can detect specific viral protein components using specific antibodies that have been raised against these proteins. These antibodies can then be used to track and quantitate proteins via: