Isolation Methods

Viruses are obligate intracellular parasites that require living cells in order to replicate.
Generally cell culture, embryonated eggs and small laboratory animals are used for the isolation of viruses.
Embryonated eggs are very useful for the isolation of influenza and paramyxoviruses. Although laboratory animals
are useful in isolating different kind of viruses, cell culture is still a preferred way for virus isolation in many of the
laboratories.

VIRUS CULTIVATION
 Also known as viral propagation or growth.
 Necessary to supply the virus with appropriate cells in which it can replicate.
 Phages are supplied with bacterial cultures. Plant viruses may be supplied with specially cultivated plants or with
cultures of protoplasts (plant cells from which the cell wall has been removed).
 Animal viruses may be supplied with whole organisms, such as mice, eggs containing chick embryos, insect larvae
or animal cells.
PURPOSE OF VIRUS CULTIVATION
1. To isolate and identify viruses in clinical specimens
2. To prepare viruses for vaccines
3. To do detailed research on viral structure, multiplication cycles, genetics, and effects on host cells.
Bacteriophages — cultivation and identification is simple and easy, due to the simplicity of the host cells.
Animal viruses — difficult, due to the properties of the animal host. Systems of cultivation with broader applications
were developed, including in vitro* cell (or tissue) culture methods and in vivo* inoculation of laboratory-bred
animals and embryonic bird tissues.
VIRUS CULTIVATION SYSTEMS
 Tissue culture system
 Embryonated eggs system
 Whole animal systems a) Natural host b) Experimental animals c) Transgenic animals
Cell/Tissue cultures system
They are generally of 3 types:-
1. Primary culture –Primary culture refers to the stage of the culture after the cells are isolated from the tissue and
proliferated under the appropriate conditions until they occupy all of the available substrate (i.e., reach confluence)
(e.g. Primary monkey kidney, mice fibroblasts)
2. Diploid cell culture – They are derived from neonatal tissues and can be subcultured 5-10 times. e.g. human
diploid fibroblasts cells.
 3. Continuous cells – They are derived from tumor tissues and can be subcultured more than 10 times. e.g. Vero,
Hep2, Hela. When a finite cell line undergoes transformation and acquires the ability to divide indefinitely, it
becomes a continuous cell line. Become immortal through a process called transformation.
Disadvantages
Long period (up to 4 weeks) required for result.
Often very poor sensitivity, sensitivity depends on a large extent on the condition of the specimen.
Susceptible to bacterial contamination.
Susceptible to toxic substances which may be present in the specimen.
Many viruses will not grow in cell culture e.g. Hepatitis B, Diarrhoeal viruses, parvovirus, papillomavirus.
Embryonated eggs system
 The process of cultivation of viruses in embryonated eggs depend on the type of egg which is used.
 The egg used for cultivation must be sterile and the shell should be intact and healthy.
 Use embryonated chicken, duck or turkey for inoculation of viral suspension
 This system is used especially for the influenza viruses isolation.
 7 - 10 days old embryonated eggs are used.
 The egg must be cleaned, the shell decontaminated with a disinfectant
Advantage
 Isolation and cultivation of many avian and few mammalian viruses
 Ideal receptacle for virus to grow
 Sterile & wide range of tissues and fluids
 Cost- much less
 Maintenance-easier
 Less labour
 Readily available
Whole animal systems
 Using live animal eg.mice, rats, rabbits, guinea pigs, hamster, chickens, and monkey.
 The animal is exposed to the virus by injection of a viral preparation or specimen into the brain, blood,
 muscle, body cavity, skin, or footpads.
 Use in example research to study the immune system’s response to viral infections.
 HIV: immunodeficient mice grafted to produce human T cells and human gamma globulin.
 Only system for studying pathogenesis & immune responses
 Used if it’s the only method through which the virus can be isolated.
Mouse model
Advantages
• In-Breed Strains Reduce Genetic Variability
• Genetics Are Well Understood
• Introduce, mutate or inactivation specific genes thought to control the immune response.
Disadvantages
• Sometimes not infected-therefore virus has to be adapted or use a closely related surrogate virus
• Does not always cause same disease state
• Mice are not humans
VIRAL QUANTIFICATION
Virus quantification involves counting the number of viruses in a specific volume to determine the virus concentration.
The methods used include :
i) Hemagglutination assay
ii) Plaque assay
iii)TCID₅₀
Hemagglutination assay
A direct method to titre virus. Based on the ability of some viruses to agglutinate RBCs Virus is tittered by making serial
two fold dilutions of the virus and determining the highest dilution of virus that causes agglutination of RBCs.

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Plaque assay
 When cells grow as monolayers, they can be used to quantify the number of viruses using plaque assay.
 The virus is serially diluted in a liquid medium.
 For each dilution a set amount is added to separate plate containing monolayer of tissue culture cells and the
viruses in that solution are allowed to attach to the tissue culture cells.
 After attachment has been allowed to occur, a semi solid medium is added to restrict the movement of new viruses
produced so that only adjacent cells will be infected.
 Where virus has infected the tissue culture cells, the infected cells will die causing the formation of a clear zone
amongst the otherwise intact monolayer of cells
 This clear zone is called a plaque and it theoretically represents an area where one virus has infected a single tissue
culture cell, has multiplied and been released, and has gone on to infect adjacent cells.
 The number of plaque forming units (pfu)/ml can be calculated based on the dilution of the original viral solution.
 The term pfu/ml is used rather than the number of viruses/ml because it is possible that occasionally more than
one virus infects a single cell.
 Often the cells or plaques are stained to help in visualization of the plaques.
TCID₅₀
 TCID50 is the measure of infectious virus titer.
 This endpoint dilution assay quantifies the amount of virus required to kill 50% of infected hosts or to produce a
cytopathic effect in 50% of inoculated tissue culture cells