Virus Culture Techniques (PDF)

CULTIVATION OF VIRUS  Isolation of Viruses Unlike bacteria many of which can be grown on an artificial nutrient medium, viruses require a living host cell for replication Infected host cells (eukaryotic or prokaryotic) can be cultured and grown, and then the growth medium...

CULTIVATION OF VIRUS  Isolation of Viruses Unlike bacteria many of which can be grown on an artificial nutrient medium, viruses require a living host cell for replication Infected host cells (eukaryotic or prokaryotic) can be cultured and grown, and then the growth medium can be harvested as a source of virus Virions in the liquid medium can be separated from the host cells by either centrifugation or filtration. Filters can physically remove anything present in the solution that is larger than the virions; the viruses can then be collected in the filtrate  Viruses can be grown in vivo (within a whole living organism, plant, or animal) or in vitro (outside a living organism in cells in an artificial environment, such as a test tube, cell culture flask, or agar plate). Bacteriophages can be grown in the presence of a dense layer of bacteria (also called a bacterial lawn) grown in a 0.7 % soft agar in a Petri dish or flat (horizontal) flask. For lytic bacteriophages, lysing of the bacterial hosts can then be readily observed when a clear zone called a plaque is detected As the phage kills the bacteria, many plaques are observed among the cloudy bacterial lawn  Animal viruses require cells within a host animal or tissue-culture cells derived from an animal. Animal virus cultivation is important for 1) identification and diagnosis of pathogenic viruses in clinical specimens, 2) production of vaccines, and 3) basic research studies. In vivo host sources can be a developing embryo in an embryonated bird’s egg (e.g., chicken, turkey) or a whole animal. For example, most of the influenza vaccine manufactured for annual flu vaccination programs is cultured in hens’ eggs. The embryo or host animal serves as an incubator for viral replication Location within the embryo or host animal is important. Many viruses have a tissue tropism and must therefore be introduced into a specific site for growth.  Within an embryo, target sites include the amniotic cavity, the chorioallantoic membrane, or the yolk sac. Viral infection may damage tissue membranes, producing lesions called pox; disrupt embryonic development; or cause the death of the embryo. For in vitro studies, various types of cells can be used to support the growth of viruses. A primary cell culture is freshly prepared from animal organs or tissues. Cells are extracted from tissues by mechanical scraping or mincing to release cells or by an enzymatic method using trypsin or collagenase to break up tissue and release single cells into suspension. Because of anchorage-dependence requirements, primary cell cultures require a liquid culture medium in a Petri dish or tissue-culture flask so cells have a solid surface such as glass or plastic for attachment and growth.  Primary cultures usually have a limited life span. When cells in a primary culture undergo mitosis and a sufficient density of cells is produced, cells come in contact. This is called a secondary cell culture. Periodically, cell density must be reduced by pouring off some cells and adding fresh medium to provide space and nutrients to maintain cell growth. In contrast to primary cell cultures, continuous cell lines, usually derived from transformed cells or tumors, are often able to be subcultured many times or even grown indefinitely (in which case they are called immortal). An example of an immortal cell line is the HeLa cell line, which was originally cultivated from tumor cells obtained from Henrietta Lacks, a patient who died of cervical cancer (a) Primary cell cultures grow attached to the surface of the culture container. Contact inhibition slows the growth of the cells once they become too dense and begin touching each other. At this point, growth can only be sustained by making a secondary culture. (b) Continuous cell cultures are not affected by contact inhibition. They continue to grow regardless of cell density. Assays To identify a specific pathogenic virus using hemagglutination we must use an indirect approach. Proteins called antibodies, generated by the patient’s immune system to fight a specific virus, can be used to bind to components such as hemagglutinins that are uniquely associated with specific types of viruses. The binding of the antibodies with the hemagglutinins found on the virus subsequently prevent erythrocytes from directly interacting with the virus So when erythrocytes are added to the antibody-coated viruses, there is no appearance of agglutination agglutination has been inhibited. We call these types of indirect assays for virus- specific antibodies hemagglutination inhibition (HAI) assays.