1.2 DEVELOPMENT OF VIRUS ASSAYS

 

 Much of the early analytical virus work was carried out with bacterial viruses. Virologists of the time would much rather have worked with agents that caused disease in humans, animals, or crop plants, but the technology was not sufficiently advanced. It is simply not possible to analyze the details of virus growth in whole animals or plants, although viruses could be assayed in whole organisms (see below). Animal cell culture was not a practicable proposition until the 1950s when antibiotics became available for inhibiting bacterial contamination; plant cell culture is still technically difficult. This left bacterial viruses which infect cells that grow easily, in suspension culture, and quickly – experiments with bacterial viruses are measured in minutes, rather than the hours or days needed for animal viruses. The observations of d’Hérelle in the early part of the twentieth century led to the introduction of two important techniques. The first of these was the preparation of stocks of bacterial viruses by lysis of bacteria in liquid cultures. This has proved invaluable in modern virus research, since bacteria can be grown in defined media to which radioactive precursors can be added to “label” selected viral components. Many animal viruses can be similarly grown in cultures of the appropriate animal cell. Secondly, d’Hérelle’s observations provided the means of assaying these invisible agents. One method is to grow a large number of identical cultures of a susceptible bacterium species and to inoculate these with dilutions of the virus-containing sample. With more concentrated samples all the cultures lyse, but if the sample is diluted too far, none of the cultures lyse. However, in the intermediate range of dilutions not all of the cultures lyse, since not all receive a virus particle, and quantitation of virus is based on this. For example, in 10 test cultures inoculated with a dilution of virus corresponding to 10−11 ml, only three lyse. Thus, three cultures receive one or more viable phage particles while the remaining seven receive none, and it can be concluded that the sample contained between 1010 and 1011 viable phages per ml. It is possible to apply statistical methods to end-point dilution assays of this sort and obtain more precise estimates of virus concentration, normally termed the virus titer. The other method suggested was the plaque assay, which is now the more widely used and more useful. d’Hérelle observed that the number of clear spots or plaques formed on a lawn of bacteria (Fig. 1.1a) was inversely proportional to the dilution of bacteriophage lysate added. Thus the titer of a viruscontaining solution can be readily determined in terms of plaque-forming units (PFU) per ml. If each virus particle in the preparation gives rise to a plaque, then the efficiency of plating is unity, however for many viruses preparations have particle to PFU ratios considerably greater than 1. Both these methods were later applied to the more difficult task of assaying plant and animal viruses. However, because of the labor, time, cost, and ethical considerations, end-point dilution assays using animals are avoided where possible. For the assay of plant viruses, a variation of the plaque assay, the local lesion assay was developed by Holmes in 1929. He observed that countable necrotic lesions were produced on leaves of the tobacco plant, particularly Nicotiana glutinosa, inoculated with tobacco mosaic virus and that the number of local lesions depended on the amount of virus in the inoculum. Unfortunately, individual plants, and even individual leaves of the same plant, produce different numbers of lesions with the same inoculum. However, the opposite halves of the same leaf give almost identical numbers of lesions so two virus-containing samples can be compared by inoculating them on the opposite halves of the same leaf (Fig. 1.1b). A major advance in animal virology came in 1952, when Dulbecco devised a plaque assay for animal viruses. In this case a suspension of susceptible cells, prepared by trypsinization of a suitable tissue, is placed in Petri dishes or other culture vessel. The cells attach to the surface and divide until a monolayer of cells (one cell in depth) is formed. The nutrient medium bathing the cells is then removed and a suitable dilution of the virus added. After a short period of incubation to allow virus particles to attach to the cells, nutrient agar is placed over the cells. After a further period of incubation of usually around 3 days, (but ranging from 24 hours to 24 days depending on the type of virus), a dye is added to differentiate living cells from the unstained circular areas that form the plaques (Fig. 1.1c). These days plaque assays are conducted using cell lines that can be maintained for many generations in the laboratory, rather than generating them from fresh tissue every time. Some viruses are not cytopathic (i.e. do not kill cells), but infected cells can always be recognized by the presence of virus protein or nucleic acids that they produce, providing that the appropriate specific detection reagents are available. An alternative for those tumor viruses that cause morphological transformation of cells (Chapter 20), is a focus-forming assay in which a single infectious particle leads to the formation of a discrete colony of cells; colonies can be counted as a measure of the input virus.