Isolation cultivation purification animal viruses

Isolation and Cultivation of Animal Viruses

1. Dr. R.S. Jadhav Department of Microbiology VNBN Mahavidyalaya , Shirala Isolation and Cultivation of Animal Viruses

2. Introduction There are three methods used for isolation and cultivation of animal viruses. These methods include: I. Embryonated chicken eggs, II. Tissue cultures III. Laboratory Animals Embryonated chicken eggs Tissue cultures and Laboratory Animals

3. I. Embryonated chicken eggs Select Fertile chicken eggs ( 5to 12 days old). Embryonated chicken eggs have several site for the cultivation of viruses. The egg inoculation steps is as follows: Located air sac in the egg by candling. Drill a small hole in the shell aseptically. Inoculate sample through the opening. Close the opening with paraffin wax. Incubate the eggs at 36 0C for 36-72 hrs. Embryonated chicken eggs method has been used for production of vaccines, such as Small pox, Yellow fever, Influenza and other diseases. This is most economical and convenient method for cultivation of animal viruses.

4. Table:1 Embryonated egg inoculation sites Sr. No. Inoculation site Cultivation of viruses 1 Chorioallantoic membrane Vaccinia , Herpes simplex, Pox and Rous sarcoma virus 2 Allontoic cavity Influenza, Mumps, Newcastle disease and Avian adenovirus 3 Amniotic sac Influenza and Mumps 4 Yolk Herpes simplex II. Tissue cultures (Cell culture) Tissue cultures or cell culture are three types based on organ, chromosomal characters and no. of generation they divide. Primary cell culture Diploid cell strains Continuous cell lines

5. 1. Primary cell culture This culture is obtained from normal animal tissues and grow on nutrient surface in glass or plastic for the first time. Mouse, hamster chicken monkey or human embryonic or young tissue cells are usually used for the cell cultures. Embryonic or young tissue cells undergo more division and sensitive to wide variety of viruses. Primary cell culture do not have long life in vitro, only few serial subcultures are possible because limited no. of divisions ( Few to fifty ). Monkey kidney, human amnion and chick embryo used for cell culture Primary cell cultures used for isolation of viruses, cultivation of obligate intracellular parasites and production of viral vaccines.

6. II. Diploid cell strains The cell cultures that contain diploid karyotype are called diploid cell strains. Lung or kidney (embryonic origin) is used for diploid cell strains. Diploid cell strains retain normal diploid chromosome number during subculture. Finite serial subculture are possible due to limited number of divisions (fifty to hundred). Human fibroblast (fetal lung fibroblast- WI38 cell line) are used for cultivation of many human viruses, fastidious pathogens and vaccine production.

7. III. Continuous cell lines The cell line that undergoes infinite number of divisions is called continuous cell line. Such cells may arise by mutation or derived from cancerous tissue. Continuous cell line have abnormal chromosome number. Continuous cell line derived from human cancer include HeLa (Henrietta Lacks carcinoma of cervix) and HEP-2 (Human epithelioma of larynx). These are used only for virus isolation not for vaccine producton . These cell lines are stored in cold at -70 C.

8. III. Animals Now , the use of animals for experiments of virus cultivation is limited for ethical reasons. Some viruses is not used method other than animals such as HIV. Some animals such as Primates, mice, guinea pigs and rabbits are commonly used. The turkey hemorrhagic enteritis virus can either be propagated in live birds or in cell culture for vaccine production. However spleen (live-bird) propagated product is widely used than cell culture propagated.

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