Title 5. GROWTH AND CULTIVATION OF VIRUS.pdf ✅

PHARMD GURU Page 3 4) They do not need feeding and caging. 5) They do not have an immune mechanism like animals to counteract virus infection. 6) Chick embryo offers several sites for cultivation of viruses. DISADVANTAGES OF EMBRYONATED EGGS: 1) Some viruses do not show growth on primary inoculation into the eggs. 2) Slight amount of bacterial contamination in the inoculum may kill the embryo. 3) Eggs may be contaminated with mycoplasma and latent fowl viruses which may interfere with the growth of other viruses. 3. TISSUE CULTURE: The first application of tissue culture in virology was by Steinhardt and colleagues (1913) who maintained vaccinia virus in fragments of rabbit cornea. Maitland (1928) used chopped tissues in nutrient media for the cultivation of vaccinia virus. The turning point which made tissue culture, the most important method for the cultivation of viruses was the demonstration by Enders, Weller and Robbins (1949) for poliovirus. Since, then tissue cultures of human or animal cells are frequently used for the cultivation of viruses. There are mainly three types of tissue cultures: (i) Organ culture: Organ cultures are useful for the isolation of some viruses which appear to be highly specialised parasites of certain organs e.g. tracheal ring organ culture is employed for the isolation of coronavirus. (ii) Explant culture: Minced tissue may be grown as explant embedded in plasma clots. This is not useful in virology. In the past, adenoid tissue explant cultures were used for adenovirus. (iii) Cell culture: This is a very popular and useful technique routinely used for cultivation of viruses. Tissues are dissociated into the component cells by the action of proteolytic enzymes such as trypsin and mechanical shaking. The cells are washed, counted and suspended in a growth medium and distributed in Petri plates, test tubes or bottles. The cells adhere to the glass surface and grow out to form a manolayer sheet. On the basis of their origin and characteristics, cell cultures are classified into three types. a) Primary cell cultures: These are normal cells freshly taken from the body and cultured. They are capable of only limited growth in a culture and cannot be
PHARMD GURU Page 4 maintained in serial culture. They are useful for isolation and cultivation of viruses for vaccine production e.g. rhesus monkey kidney cell culture, human embryonic kidney cell culture, human amnion cell, chick embryo fibroblast culture etc. b) Diploid cell strains: Diploid cell strains are derived from primary cell cultures established from a particular type of tissue, such as a lung or kidney, which is embryonic in origin. They are of a single type and can undergo 50 to 100 divisions before dying. They possess the normal diploid karyotype (set of chromosomes). They are useful for the isolation of fastidious pathogens and also for the production of viral vaccines e.g. human embryonic lung cell strain (WI - 38), rhesus embryo cell strain (HL-8). c) Continuous cell lines: They are single type of cells mainly derived from cancer cells. These also can be grown in successive generations by transferring them from one test tube to another test tube without change in character of cells. Standard cell lines derived from human cancers, such as HeLa, Hep-2 and KB cell lines have been used in different laboratories throughout the world. These cell lines may be maintained by serial subcultivation or stored in the cold (– 70oC) e.g. Human carcinoma of nasopharynx line (KB), Human epitheloma of larynx cell line (Hep-2), Human carcinoma of cervix cell line (HeLa), Baby hamster kidney cell line (BAK 21), etc. Virus growth in cell cultures can be detected by cytopathic effect (Fig. 3.7), transformation, metabolic inhibition, interference, hemadsorption and immuno-fluorescence.