Techniques that identify specific viral proteins or genomes provide ways to rapidly identify viruses

Techniques that identify specific viral proteins or genomes provide ways to rapidly identify viruses. virtually any viral protein. Powerful genetic techniques can also be used to generate designer cells or organisms. There are a variety of methods for quantitating viruses. Infectivity assays measure the ability of a virus to productively infect a cell. Techniques that identify specific viral proteins or genomes provide ways to rapidly identify viruses. Some of these assays can be used at the bedside, or in the field. Powerful and inexpensive DNA sequencing technologies are being used to identify new viruses, many of which could not be found by other methods. The challenge is to understand how or if these viruses impact their hosts. Keywords: Electron microscopy, fluorescence microscopy, hemagglutination, immunoassay, plaque assay, infectivity assay, polymerase chain reaction, cell culture, centrifugation This chapter describes methods for growing, purifying, counting, and characterizing viruses. It also presents general principles of diagnostic virology. After studying this chapter, you should be able to: ? Describe general requirements for culturing cells and tissues. ? Describe differences between cultures of primary and transformed cells. ? Describe how centrifugation is used to purify viruses. ? Understand the types of information provided by negative staining electron microscopy (EM), thin sectioning EM, cryo-EM, and confocal microscopy. ? Understand what is being measured by each of the following techniques: plaque assays, PCR, ELISA, hemagglutination, and hemagglutination inhibition assays. Growing Viruses Viruses replicate only within living cells, thus many early studies of viruses were done in bacteria or plants. Tobacco mosaic virus (TMV) was an early model virus as it replicates in a variety of plants, at levels sufficient for biochemical analysis and imaging. Growing TMV is as simple as applying virus to abraded leaves of a susceptible plant. The earliest studies of animal viruses were Sapacitabine (CYC682) limited to using whole animals. When possible animal pathogens were adapted to small animals such as mice, rats, and rabbits. These small animal models provided a means to study viral pathogenesis and vaccine efficacy. Fertile chicken and duck eggs were, and continue to be, widely used for propagating viruses. In the 1940s and 1950s development of robust cell culture techniques revolutionized the study of animal viruses. Today, most animal viruses are grown in cultured cells. Generating Cell Cultures The following steps describe an overall strategy for generating primary cell cultures. It is of utmost importance that all work is done under sterile conditions (Fig. 4.1 ): 1. The desired tissue is removed from the animal and IL17B antibody is chopped or minced. 2. Tissue fragments are treated with enzymes such as collagenase to degrade the extracellular matrix and release single cells and small aggregates of cells. 3. Cells are pelleted by centrifugation and are resuspended in buffered saline or cell culture media. 4. Additional centrifugation steps may be performed to separate single Sapacitabine (CYC682) cells from cell aggregates. 5. Cells and growth media are added to culture dishes and are maintained in a humidified incubator (37C, 5% CO2). 6. Cells attach to the bottom of the dish where they grow and divide to form a monolayer. 7. The cells can be removed with trypsin, washed, and divided among new culture plates or dishes. This is called a passage, and is done to increase cell number. Open in a separate window Figure 4.1 Generating cell cultures begins with removing tissues (normal or tumor) from an animal. Tissues are minced and treated with enzymes to degrade the extracellular matrix. Centrifugation is used to pellet the cells. Cells are resuspended in media and placed in culture vessels. Primary cells can be propagated for only a limited number of passages before the cells undergo a crisis and the culture dies. Embryonic cells can be passaged many more times than cells taken from adults. Some types of cells (for example, fibroblasts) divide more readily than do cells that are normally nondividing in the adult animal (for example, neurons). Tumors provide another source of cells for virus culture. Tumor-derived cells can often be passaged indefinitely. These immortalized cells are excellent tools for the virologist. They are relatively easy to culture, many types are commercially available and they can be Sapacitabine (CYC682) genetically modified. Multiple genes can be introduced, mutated, or deleted to generate an unlimited.

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