Viruses are cultured in embryonated egg, cell line culture, and animal inoculation because they can only replicate inside host cells, so viruses cannot be cultured in non-living medium as bacteria and fungi (Bacteria and fungi are living creatures). The main purposes of virus cultivation are the identification and isolation of viruses in clinical samples, research on viral structure, replication, genetics, and their effects on host cells, and production of viral vaccines. Hence, cultivation of viruses is essential which is carried out by three methods viz. Animal inoculation, Embryonated egg culture, and Cell culture.
Flowchart: Methods of cultivation of viruses
1. Animal inoculation: Animal inoculation is one of
the primary methods for the isolation of certain viruses and for the study of the
pathogenesis of certain viral diseases. This method is carried out for those
viruses that are not cultivated in embryonated egg and tissue culture methods.
In this method, healthy and disease-free laboratory mice (white mice) are
particularly used for virus cultivation. Suckling mice of age less than 48
hours are used for culture of Toga virus and Coxsackie virus. The inoculation
of viruses is carried out by intracerebral and intranasal routes. The
inoculation is also carried out by intraperitoneal and subcutaneous routes.
Other healthy and disease-free animals such as hamsters, guinea pigs, rabbits, chimpanzees,
and primates are also used as alternatives for virus culture. After inoculation
of the virus sample into the host cell, the animals are observed for symptoms
of disease till death and then the virus is isolated from the tissue of an animal.
Live inoculation was first used to study of yellow fever virus on human
volunteers.
Advantages of Animal inoculation:
• Diagnosis, Pathogenesis, and clinical symptoms are
determined.
• Antibodies productions are identified.
• Mice are the reliable model for virus replication.
• This method helps to study immune responses, epidemiology,
and oncogenesis.
Disadvantages of Animal inoculation:
• Animal experiments are costly and difficult to maintain.
• Animal selection for specific viruses is difficult.
• Some human viruses are not grown in animals.
• Vaccine production is not possible with mice models.
2. Embryonated egg culture: For virus cultivation, an
egg embryo of 7-12 days is used. At first, the egg is kept in an incubator for
embryo development for up to 7-12 days and then the virus sample is inoculated
into the egg. For inoculation, eggs are first prepared for cultivation, and the
shell surface is first disinfected with iodine and penetrated with a small
sterile drill. After inoculation, the opening is sealed with paraffin and
incubated at 36°C for 2-3 days. The egg is broken after incubation and the virus
is isolated from the tissue of the egg. The virus can be cultured in different
parts of the embryonated egg, such as the chorioallantoic membrane (Pox virus
are cultured), amniotic sac (Influenza virus, Mumps virus, Yellow fever virus,
and Rabies virus are cultivated), amniotic cavity (Influenza virus is cultured)
or yolk sac (Herpes virus is cultured) depending upon types of virus. In 1931,
Scientist Pasture first used the embryonated hen’s egg for the cultivation of the
virus (Fig).
Fig: Technique of embryonated egg culture
Advantages of Embryonated egg culture:
• This method is a widely used method for the isolation of
viruses.
• It is an ideal substrate for the viral growth and
replication.
• This method is cost effective and maintenance is much
easier.
• Less labor is needed.
• The embryonated eggs are readily available.
• They are free from contaminating bacteria and many latent
viruses.
Disadvantages of Embryonated egg culture:
• Each virus has different sites for their growth and
replication hence the site of inoculation varies with the viruses.
3. Cell culture (tissue culture) technique: This
technique is the most commonly used technique for the cultivation of viruses.
There are three types of cell culture techniques viz. organ culture, explant
culture, and cell culture.
(i) Organ culture: This culture method is mainly done
for highly specialized parasites of certain organs. Like, tracheal ring culture
is done for isolation of coronavirus.
(ii) Explant culture: In this method, a small
fragment of tissue is extracted from a human or animal and used for virus
culture but this technique is very rarely used.
(iii) Cell line culture: This is the most commonly
used technique. Cell line culture is routinely used in the lab for virus
culture, isolation, and identification. In this method, the growth media is
prepared by maintaining a balanced salt concentration, all essential amino acids,
glucose, buffering agents, some antibiotics, serum, etc. Thereafter some tissue
fragment is obtained which is trypsionized to dissociate cells. These cells are
washed and suspended in culture media in Petri plates and incubated for
sufficient time. On incubation, the cell divides and spreads out on the glass
surface to form a confluent monolayer of cells that is used for virus culture.
Based on origin, chromosomal characteristics, and number of
generations through which cell culture can be maintained, cell line culture is
classified into three types viz. Primary cell line, Semi-continuous cell line,
and Continuous cell line.
I. Primary cell line: These are normal cells,
obtained from fresh organs of animals or humans and cultured. These primary
cell line cultures are used for the isolation of viruses and for the preparation
of vaccines. These cells are capable of limited growth for limited generations.
They cannot be maintained in a serial sub-culture. Examples: Monkey kidney cell
line, Human amnion cell line, etc.
II. Semi-continuous cell line: These cells are
fibroblastic cells also known as diploid cells because they contain the same
number of chromosomes as the parent cell. These diploid cells are sub-cultured
for a limited generation. They are susceptible to a wide range of Human virus
cultures and are also used for vaccine production. Examples: Rhesus embryo
cell, human embryonic lung strain, etc.
III. Continuous cell line: These are cells of a single
type capable of infinite growth in vitro. These are usually cancer cells
derived from cancerous tissue. These cells grow faster and they are haploid
cells. These cells are maintained by serial sub-culture or by deep freezing at
−7°C so that these cells are reused when necessary. This cell line is used for
virus culture but not for vaccine preparation. Examples: HeLa cell is obtained
from cervical cancer, 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:
• The method is easy, broad-spectrum, cheaper, and good
sensitivity.
Disadvantages of Cell Culture:
• The process requires a skilled person.
• Tissue or serum is sent to central laboratories for
analysis to identify the virus.