The animal models used for viral inoculation include experimental animals such as transgenic animals, monkeys, primates, hamsters, guinea pig, and laboratory mice or rabbits. Animal inoculation is generally used for the cultivation of viruses that cannot be cultivated in embryonated eggs or cell culture systems. The newborns of these animals are usually preferred in the cultivation of viruses when viral cultivation using animal models is anticipated. Animal inoculation systems however, where mostly used before the advent of the cell/tissue culture systems and embryonated egg systems. Even though animals still play an essential role in studying viral pathogenesis or virulence; the use of animals for viral cultivation is gradually being replaced by other means of viral cultivation such as the cell/tissue culture systems and the use of embryonated eggs as well. The viruses are usually inoculated subcutaneously or intraperitoneally; and the inoculated experimental animal(s) is observed for the development of the disease caused by the said pathogenic virus. Death usually occurs in most cases. And the virus is then isolated from the tissues or organs of the dead animals and purified for further studies. Nevertheless, animal inoculation systems for viral cultivation has some disadvantages and advantages. The extinction of these animals aside other factors is one of the reasons limiting the use of animals for viral cultivation.
CELL/TISSUE CULTURE SYSTEMS OF VIRAL CULTIVATION
Cell/tissue culture is the in vitro technique by which cells or tissues obtained from an organism are maintained under controlled laboratory conditions outside its natural host. It provides the most widely used and most powerful hosts for cultivation and assay of viruses since the animal inoculation systems are not widely accepted due to some ethical issues surrounding the use of animals in research. Animal viruses can be grown and/or cultivated efficiently in vitro in cell/tissue culture systems due to the availability of animal cells that can be propagated outside their host organisms in an efficient manner over certain period of time. Table 1 shows the advantages and disadvantages of animal inoculation systems used for viral cultivation.
Table 1: Advantages and disadvantages of animal inoculation systems for viral cultivation
|It is the primary method used for the isolation of some viruses.||Animal systems for viral cultivation are very expensive.|
|Animal inoculation systems helps the researcher to determine the pathogenesis and clinical symptoms of the virus being propagated.||They are difficult to maintain; and choosing a unique animal to cultivate a given virus is usually intricate.|
|They provide a reliable method of studying viral replication patterns. Animal inoculation systems helps in the identification of antibodies produced against the virus.||Not all human viruses are cultivated in animals. Ethical issues limit the use of animals for viral cultivation since animal welfare is given top-most priority in some regions.|
|It is the perfect medium for studying immune responses to a particular virus.||Some animals such as mice do not provide the ideal environment for the development of human vaccines.|
Tissue culture has enhanced the process of viral cultivation; and the practice is now widely used for the isolation and identification of viruses. The development of growth media that support the thriving of animal cells outside their normal environment as wells as the development of antibiotics and drugs that inhibit the growth of bacteria and fungi in cell/tissue culture plates during cell culture has also enhanced the use of this technique to cultivate viruses in vitro. The growth or replication of virus in cell culture plates can be deciphered in several ways including the development of cytopathic effects, hemadsorption and the formation of plaques. Cytopathic effects are observable morphological changes that occur in cells because of viral replication. Death of the cells in culture, ballooning and the clustering or binding together of the cells are some examples of cytopathic effects observable in cell/tissue culture plates during viral cultivation.
Hemadsorption is the phenomenon that occur when red blood cells (RBCs) added to a cell culture plate during the incubation of the plate gets attached to the plasma membrane of the infected cultured cells which have been altered by the cultivated virus. Plaque is the area of lysis or hole formed in a lawn of cells in cell/tissue culture due to the infection or replication of a virus. It is the localized areas of cellular destruction and lysis of cells in cell culture due to viral infection. Plaque formation is not usually used to measure or detect viral replication in cell cultures because they do not always form when viruses are cultivated in vitro in tissue culture vessels. Nevertheless, plaque assay is an important viral assay used to quantify the amount of infectious virus in a sample. Plaque assay is a technique that is used to determine the concentration of infective particles in a virus solution or sample; and it is usually expressed as plaque forming units per ml (pfu/ml).
In plaque assay, the number of plaques induced on a lawn of bacteria or eukaryotic cells in cell culture plates is generally used to evaluate the concentration of infective particles of viruses present in a given sample. PFU is defined as the number of plaques formed per unit of volume or weight of a virus suspension or sample. Though the number of plaques formed in the cell culture plates does not necessarily reflect or show the actual number of virus particles present in the samples, it gives a proportionate ratio of the infecting agent present. It is noteworthy that the number of plaques formed is generally expressed as pfu/ml because of the uncertainty that a single plaque arose from a single infectious viral particle. To avoid contamination of the process, viral cultivation is undertaken inside a biosafety laminar flow cabinet or hood. Both cytopathic effects and hemadsorption can be detected microscopically using inverted microscopes meant for this purpose. Cell/tissue culture techniques for viral cultivation are divided into three (3) types.
Acheson N.H (2011). Fundamentals of Molecular Virology. Second edition. John Wiley and Sons Limited, West Sussex, United Kingdom.
Alan J. Cann (2005). Principles of Molecular Virology. 4th edition. Elsevier Academic Press, Burlington, MA, USA.
Alberts B, Bray D, Johnson A, Lewis J, Raff M, Roberts K and Walter P (1998). Essential Cell Biology: An Introduction to the Molecular Biology of the Cell. Third edition. Garland Publishing Inc., New York.
Balows A, Hausler W, Herrmann K.L, Isenberg H.D and Shadomy H.J (1991). Manual of clinical microbiology. 5th ed. American Society of Microbiology Press, USA.
Barrett J.T (1998). Microbiology and Immunology Concepts. Philadelphia, PA: Lippincott-Raven Publishers. USA.