Techniques of isolation and cultivation of Animal and Plant viruses

1. Dr. R.S. Jadhav
Department of Microbiology
VNBN Mahavidyalaya, Shirala
Isolation and Cultivation of Animal
and plant 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
 Good pasture in 1931 first used
embryonated chicken eggs for the
cultivation of viruses.
 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

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.
1. Primary cell culture
2. Diploid cell strains
3. 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,

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),
HEP-2 (Human epithelioma of larynx) ,Vero (Vervet
monkey) kidney cell lines and BHK-21 (Baby Hamster
Kidney cell line)..
 These are used only for virus isolation not for vaccine
producton.
 These cell lines are stored in cold at -70 0C.

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.

9. Plant Viruses
 There are some methods of Cultivation of plant viruses
such as plant tissue cultures, cultures of separated cells,
or cultures of protoplasts, etc. viruses can be grown in
whole plants.
 1968, Takebe et al., developed a technique for the
production of large amounts of active protoplasts from
mesophyll cells of Nicotiana tabacum that became a
standard.
 Leaves are mechanically inoculated by rubbing with a
mixture of viruses and an abrasive. When the cell wall is
broken by the abrasive, the viruses directly contact the
plasma membrane and infect the exposed host cells.
 A localized necrotic lesion often develops due to the
rapid death of cells in the infected area. Some plant

10.  The insect cell cultures have been used for the
cultivation of plant viruses. Many plant viruses use
insects as their vectors. Thus it is possible to cultivate
them in insect cell cultures.
 Rhabdoviruses are cultivated in grasshopper cell
culture.

11. Thanking You