Cultivation of Viruses Lecture 3

Lecture (2) Cultivation of virus  CULTIVATION OF VIRUSES The cultivation of virus is an essential prerequisite for their isolation Viruses are obligatory intracelluler parasite, virus can only replicate in cells. The primary purpose of virus cultivation is: 1. To isolate...

Lecture (2) Cultivation of virus  CULTIVATION OF VIRUSES The cultivation of virus is an essential prerequisite for their isolation Viruses are obligatory intracelluler parasite, virus can only replicate in cells. The primary purpose of virus cultivation is: 1. To isolate and identify viruses in clinical samples. 2. To do research on viral structure, replication, genetics and effects on host cell. 3. To prepare viruses for vaccine production.  Cultivation of plant virus Plant cells don’t possess receptors to allow entrance of the virus inside its cell but have cell wall consist of cellulose and hemicellulose. Cultivation can be provided by artificially wounding the cells of plant epidermis. Wound have to be very minute to avoid death of the wounded plant cells. using minute fine powders of sharp particles e.g. fine sand, carborundum powder or fine glass powder by which the epidermis have to be scratched, then the viral suspension (sap containing virus particles) will be sprayed on the wounded cells and scratched again by a cotton or rubber piece. Infected plants have to be grown in glass houses away from insects and other healthy or other infected plants to get pure virus suspension. Notice that: subculture the viruses, in young plants of the same host species. Three basic laboratory substrate for this purpose these are ; 1. Embryonated Eggs 2. Tissue culture 3. Animal inoculation  Viruses are obligate intracellular parasites so they depend on host for their survival. They cannot be grown in non-living culture media or on agar plates alone, they must require living cells to support their replication. There are three methods of Viruses Cultivation: 1. Animal Inoculation 2. Inoculation into embryonated egg 3. Cell Culture  Inoculation into embryonated egg Pasture in 1931 first used the embryonated hen’s egg for the cultivation of virus. The process of cultivation of viruses in embryonated eggs depends on the type of egg which is used. Viruses are inoculated into chick embryo of 7-12 days old. For inoculation, eggs are first prepared for cultivation, the shell surface is first disinfected with iodine and penetrated with a small sterile drill. 1. Embryonated Eggs Although not all virus can be grown in embryonated egg, many viruses are cultivate in this host. Eggs are now used for rapid isolation of a few viruses and also used for vaccine or antigen production This method is more economical and convenient than animal inoculation. Embryonated eggs may be infected by various route ; amniotic, allantoic, chorio allantoic, yolk sac, in special cases intravenous and intra embryonal  The choice of an particular route is again depend upon the ability of tissue to support virus replication.  Before inoculation eggs are candled to check that the embryo is alive.  The presence of virus in inoculated eggs can be detected by mortality, deformities and haemorrhagic of the embryo, lesion in the form of pock, oedema of the developing membrane, the presence of specific antigen in the fluid as shown by HI test, SN test, FAT and other serological test.  Eggs provide a suitable means for: the primary isolation and identification of viruses, the maintenance of stock culture and, the production of vaccines. Advantages An embryo is an early developmental stage of animals marked by rapid differentiation of cells Birds undergo their embryonic period within the closed protective case of an egg, which makes an incubating bird egg a nearly perfect system for viral propagation It is an intact and self-supporting unit, complete with its own sterile environment and nourishment It furnishes several embryonic tissues that readily support viral multiplication Defence mechanisms are not involved in embryonated eggs Cost- much less, Maintenance-easier, 2- Animal Inoculation Viruses which are not cultivated in embryonated egg and tissue culture are cultivated in laboratory animals such as mice, guinea pig, hamster and rabbits. The selected animals should be healthy and free from any communicable diseases. Viruses can be inoculated by intraperitoneal and subcutaneous route. After inoculation, virus multiply in host and develops disease. Note: The animals are observed for symptoms of disease and death. After the animal is inoculated with the virus suspension, the animal is: observed for signs of disease visible lesions or is killed so that infected tissues can be examined for virus Then the virus is isolated and purified from the tissue of these animals. Live inoculation was first used on human volunteers for the study of yellow fever virus. o Advantages of Animal Inoculation: 1. Diagnosis, Pathogenesis and clinical symptoms are determined. 2. Production of antibodies can be identified. 3. Primary isolation of certain viruses. 4. Mice provide a reliable model for studying viral replication. 5. Used for the study of immune responses, epidemiology and oncogenesis. o Disadvantages of Animal Inoculation: 1. Expensive and difficulties in maintenance of animals. 2. Difficulty in choosing of animals for particular virus 3. Some human viruses cannot be grown in animals, or can be grown but do not cause disease. 4. Mice do not provide models for vaccine development. 5. It will lead to generation of escape mutants 6. Issues related to animal welfare systems. 3- Cell Cultures: Prior to the advent of cell culture, animal viruses could be propagated only on whole animals or embryonated chicken eggs Cell cultures have replaced embryonated eggs as the preferred type of growth medium for many viruses Cell culture consists of cells grown in culture media in the laboratory. These cultures can be propagated and handled like bacterial cultures; they are more convenient to work with than whole animals or embryonated eggs  There are three types of tissue culture; organ culture, explant culture and cell culture. Organ cultures are mainly done for highly specialized parasites of certain organs e.g., tracheal ring culture is done for isolation of coronavirus. Explant culture is rarely done. Cell culture is mostly used for identification and cultivation of viruses. Cell culture is the process by which cells are grown under controlled conditions. Cells are grown in vitro on glass or a treated plastic surface in a suitable growth medium.  Types of cell culture 1. Primary cell culture: These are normal cells derived from animal or human cells. They are able to grow only for limited time and cannot be maintained in serial culture. They are used for the primary isolation of viruses and production of vaccine. Examples: Monkey kidney cell culture, Human amnion cell culture 2. Diploid cell culture (Semi-continuous cell lines): They are diploid and contain the same number of chromosomes as the parent cells. They are used for the isolation of some fastidious viruses and production of viral vaccines. Examples: Human embryonic lung strain, Rhesus embryo cell strain 3. Heteroploid cultures (Continuous cell lines): They are derived from cancer cells. They can be maintained either by serial subculture or by storing in deep freeze at -70°c. Due to derivation from cancer cells, they are not useful for vaccine production. Examples: HeLa (Human Carcinoma of cervix cell line), HEP-2 (Human Epithelioma of larynx cell line), Vero (Vervet monkey) kidney cell lines, BHK-21 (Baby Hamster Kidney cell line).  Advantages of cell culture 1. Relative ease, broad spectrum, cheaper and sensitivity Disadvantage of cell culture 1. The process requires trained technicians with experience in working on a full-time basis. 2. State health laboratories and hospital laboratories do not isolate and identify viruses in clinical work. 3. Tissue or serum for analysis is sent to central laboratories to identify virus.