ANIMAL INOCULATION AND CELL/TISSUE CULTURE SYSTEMS OF VIRUS CULTIVATION

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The animal models used for viral inoculation include experimental animals such as transgenic animals, monkeys, primates or non-human primates (NHPs), 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, were 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. And the reason for this is usually to prevent the extinction of some animals. The viruses are usually inoculated subcutaneously or intraperitoneally; and the inoculated experimental animal(s) is observed for the development of the disease or clinical signs and symptoms caused by the said pathogenic virus being studied.

Death usually occurs in most cases. In some experiments using animals or laboratory mice, the animal is usually euthanized or sacrificed at certain stage of the investigation in order to obtain specific organs, tissues or cells from the sacrificed animal for further viral studies. 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 (Table 1). The extinction of these animals aside other factors as aforesaid is one of the reasons limiting the use of animals for viral cultivation. 

Table 1. Advantages and disadvantages of animal inoculation systems for viral cultivation

ADVANTAGESDISADVANTAGES
Animal inoculation system 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 and disease pathogenesis.   Animal inoculation systems helps in the identification of antibodies produced against the infecting 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 of the world.  
It is the perfect medium for studying immune responses to a particular pathogenic virus.Some animals such as mice do not provide the ideal environment for the development of human vaccines.

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 host cells 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.

Tissue culture has enhanced the process of viral cultivation; and the practice is now widely used for the isolation and identification of viruses including those pathogenic in man, animals and plants. 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 or flasks 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. These features (i.e., cytopathic effects) are usually observable under the inverted microscope when cell culture plates are viewed using certain magnifications of the microscope. 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 plate 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 cell/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 (virus) 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 as aforesaid.

PRIMARY CELL CULTURES OF VIRAL CULTIVATION

Primary cells are cells obtained directly from freshly killed animals; and they can only be passaged or subcultured once or twice. Primary cell culture supports the cultivation of many viruses. The cells used for primary cell culture can also be obtained from humans. Primary cells are heterogeneous at the stage of collection. But they are homogenized with trypsin, a proteolytic enzyme prior to their usage. Chelating agents like ethylene diamine tetra-acetic acid (EDTA) can also be used for the dispersal of cells in tissues prior to primary cell culture. The homogenization of these tissues helps in the release of single cells and small aggregates of cells capable of initiating optimal growth of the cultivated virus(s). Kidney cells and lung tissues from animals are typical examples of sources of cells for primary cell culture. Primary cell culture though best, is expensive; and it is usually difficult to obtain a reliable supply of normal cells from freshly killed animals to undertake primary cell culture during viral cultivation. Primary cell lines are generally used for viral isolation and for vaccine preparation. Other forms of cell culture techniques include continuous cell culture and semi-continuous cell culture.

Continuous cell culture: Continuous cell lines are cell lines that are capable of more prolonged and indefinite growth. They are immortalized cell lines that can be passaged or subcultured several times unlike the primary cell lines that can only be passaged once or twice. Primary cell lines may soon lose viability after several passaging. HeLa cells (human carcinoma of cervix cell line) are typical examples of continuous cell lines capable of indefinite growth. Continuous cell lines are usually derived from tumour or cancerous cells. These cells are not used for vaccine preparation since they are derived from malignant tumours or cancer cells. Continuous cell culture can only handle a limited number of viruses unlike the primary cell culture that handles a wide variety of viruses even though they are the easiest type of cell culture technique to undertake for viral cultivation. 

Semi-continuous cell culture: Semi-continuous cell lines which can otherwise be known as diploid cell lines are cell lines that contain the same number of chromosomes as the parent cells from which they are derived. Though they can be passaged several times, semi-continuous cell lines cannot be subcultured indefinitely like the continuous cell lines used in continuous cell culture. However, semi-continuous cell lines can be passaged up to 50 times unlike the primary cell lines that can only be passaged once or twice. Examples of semi-continuous cell lines include cells from the rhesus monkeys and human embryonic lungs. They can be used for vaccine preparation and for the isolation of fastidious viruses.     

One of the disadvantages of using cell culture techniques in carrying out viral cultivation is that cell culture requires specialized and trained personnel with experience to do it. It is not a routine practice in some hospital laboratories – since they rarely isolate and identify pathogenic viruses from clinical samples. Most clinical samples collected for viral examinations are usually sent to reference laboratories where trained personnel and equipment are available for their processing. Cell/tissue culture technique does not support the cultivation of some animal viruses. Nevertheless, cell culture technique has a high sensitivity in identifying a virus and the technique is relatively cheap and easy to perform by experienced laboratory personnel.   

Further reading

Acheson N.H (2011). Fundamentals of Molecular Virology. Second edition. John Wiley and Sons Limited, West Sussex, United Kingdom.

Brian W.J Mahy (2001). A Dictionary of Virology. Third edition. Academic Press, California, USA.

Cann A.J (2011). Principles of Molecular Virology. Fifth edition. Academic Press, San Diego, United States.

Carter J and Saunders V (2013). Virology: Principles and Applications. Second edition. Wiley-Blackwell, New Jersey, United States.

Dimmock N (2015). Introduction to Modern Virology. Seventh edition. Wiley-Blackwell, New Jersey, United States.

Kudesia G and Wreghitt T (2009). Clinical and Diagnostic Virology. Cambridge University Press, New York, USA. 

Marty A.M, Jahrling P.B and Geisbert T.W (2006). Viral hemorrhagic fevers. Clin Lab Med, 26(2):345–386.

Strauss J.H and Straus E.G (2008). Viruses and Human Diseases. 2nd edition. Elsevier Academic Press Publications, Oxford, UK.

Zuckerman A.J, Banatvala J.E, Schoub B.D, Grifiths P.D and Mortimer P (2009). Principles and Practice of Clinical Virology. Sixth edition. John Wiley and Sons Ltd Publication, UK.

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