Cultivation of viruses

viruses are obligate intracellular parasites and so they cannot be grown on any inanimate culture
medium.

Three methods are employed for the cultivation of viruses:

1. Animal inoculation
2. Embryonated Hen’s egg
3.Tissue cultures

1. Animal inoculation:
• The earliest method for the cultivation of viruses causing human diseases was inoculation
into human volunteers.
• Reed and Colleagues (1900) used human volunteers for their pioneering work on yellow
fever.
• Theiler in 1903 used white mice for the cultivation of viruses.

• Mice are still the most widely used animals in virology

 • Infant mice (suckling mice) are very susceptible to coxsackie and Arbovirus, many of
which do not grow in any other system.
• Mice may be inoculated by several routes: intracerebral, subcutaneous, intraperitoneal or
intranasal.
• Other animals such as guinea pigs, rabbits and ferrets are used in some situations.
• The growth of the virus in inoculated animals may be indicated by death, disease or visible
lesions.
• Animal inoculation is also used for the study of pathogenesis, immune response,
epidemiology and oncogenesis.
Disadvantages:
The immunity may interfere with viral growth and that animals often harbour latent viruses.

2. Embryonated eggs
Embryonated hen’s egg was first used for the cultivation of viruses by Good Pasture in 1931.
The method was further developed by Burnet.
The embryonated egg offers several sites for the cultivation of viruses. -
1. Chorioallantoic membrane(CAM),
2. Allantoic cavity,
3. Amniotic sac and
4. Yolk sac
For cultivation of viruses in Yolk sac, 4-6 days incubated eggs chosen and for CAM, allantoic
cavity and amniotic sac, 10-12 days incubated eggs are selected.

a. Chorioallantoic membrane
CAM is mainly employed in the growth of pox virus. Inoculation on the chorioallantoic membrane
produces visible lesions called pocks.
Different viruses have different pock morphology. Under optimal conditions, each infectious viral
particle can form one pock.

Pock counting, therefore can be used for the assay of pock-forming viruses such as variola or
vaccinia.
Herpes simplex virus is also grown on CAM.
b. Allantoic cavity
Inoculation into allantoic cavity provides a rich yield of Influenza and some Paramyxoviruses.
Allantoic inoculation is employed for growing the Influenza virus for vaccine production.
other chick embryo vaccines in routine use are the yellow fever(17D strain) and Rabies (Flury
strain) vaccines.
c. Amniotic cavity
The amniotic sac is employed for the primary isolation of the Influenza virus and the mumps virus.
d. Yolk sac inoculation is used for the cultivation of some viruses, Chlamydiae and Rickettsiae.
Duck eggs are bigger and have a longer incubation period than hen’s eggs. They therefore provide
a better yield of Rabies virus and were used for the preparation of the inactivated non-neural
vaccine.
Advantages of Inoculation into embryonated egg
• Widely used method for the isolation of virus and growth.
• Ideal substrate for the viral growth and replication.
• Isolation and cultivation of many avian and few mammalian viruses.
• Cost effective and maintenance is much easier.
• Less labor is needed.
• The embryonated eggs are readily available.
• Sterile and wide range of tissues and fluids
• They are free from contaminating bacteria and many latent viruses.
• Specific and non-specific factors of defense are not involved in embryonated eggs.
• Widely used method to grow virus for some vaccine production.

Disadvantages of Inoculation into embryonated egg

The site of inoculation for varies with different virus. That is, each virus has different sites for their
growth and replication.
3. Tissue culture:

• Cell culture is most widely used in diagnostic virology for cultivation and assays of viruses.
• The tissue culture was first applied in diagnostic virology by Steinhardt and colleagues in
1913.
• They maintained the vaccinia virus by culture in tissues of rabbit cornea. Subsequently,
Maitland (1928) used cut tissues in nutrient media for cultivation of vaccinia viruses.
• Enders, Weller, and Robins (1949) were the first to culture poliovirus in tissue cultures of
nonneural origin. Since then, most of the virus had been grown in tissue culture for
diagnosis of viral diseases.
• Different types of tissue cultures are used to grow viruses. Tissue culture can be of three
different types as follows:
Types of tissue culture
1. Organ Culture
In this method, small bits of the organs are maintained in vitro for days and weeks preserving their
original morphology and function.
This was used earlier for the isolation of some viruses, which appear to show affinity for certain
tissue organs. For example, coronavirus, a respiratory pathogen, was isolated in the tracheal ring
organ culture.
Nowadays, organ culture is not used.

2. Explant Culture
In this method, components of minced tissue are grown as explants embedded in plasma clots.
Earlier, adenoid tissue explant cultures were used for isolation of adenoviruses. This method is
now seldom used in virology.

3. Cell Culture:

• Cell culture is now routinely used for growing viruses.

• In this method, tissues are dissociated into component cells by treatment with proteolytic
enzymes such as trypsin or collagenase followed by mechanical shaking.
• The cells are then washed, counted, and suspended in a growth medium containing
essential amino acids and vitamins, salts, glucose, and a buffering system generally
consisting of bicarbonate in equilibrium with 5% Co2.
• This medium is supplemented by up to 5% of fetal calf serum and antibiotics are added to
prevent bacterial contaminants. Phenol red is used as indicator.
• The cell suspension is dispensed in glass or plastic bottles, tubes or Petri dishes.
• On incubation, the cells adhere to the glass surfaces and divide to form a confluent
monolayer sheet of cells covering the surface within a week.
• The cell culture may be incubated in a sloped horizontal position either as a stationery
culture or may be rolled in special roller drums to provide better aeration. Some fastidious
viruses grow only in such roller cultures.
• The cell cultures are classified into three different types based on their origin,
chromosomal characters, and number of generations for which they can be maintained.

1. Primary cell culture:

 2.
• Primary cell cultures are normal cells freshly taken from the body and cultured.
• They are capable of only limited growth in culture and cannot be maintained in serial
culture.
• These cells have the normal diploid chromosomal number and are capable of only limited
growth (5–10 divisions) in culture.
• Monkey kidney cell culture, human embryonic kidney cell culture, Human amnion and
Chick embryo fibroblast cell culture are the common examples of Primary cell culture.
• Primary cell cultures are highly useful for the primary isolation of Myxovirus,
Paramyxovirus, many Enteroviruses, and some Adenoviruses.

2. Diploid cell strains:

• Diploid cell strains are of a single cell type that retains the original diploid chromosome
number and karyotype during serial subcultivation.
• After about 50 serial passages before they undergo senescence (die off) or undergo a
significant change in their characteristics.
• Diploid cells derived from human fibroblasts are useful for isolation of some fastidious
viruses and wide range of human viruses.
• They are also used for production of vaccines; for example, WI-38(Human embryonic lung
cell strain) is used for the cultivation of fixed rabies virus, and human fetal diploid cells for
isolation of adenovirus, picornaviruses, HSV, CMV, and VZV.
 • HL-8(Rhesus embryo cell strain)

3. Continuous cell lines:

• Continuous cell lines are cells of a single type, which are derived from cancerous cells that
are capable of continuous serial cultivation indefinitely without senescing.
• Hep-2(Human epithelial type 2) is derived from human laryngeal carcinoma used to
cultivate RSV (Respiratory Syncytial virus), Adenovirus and HSV.
• HeLa is derived from human cervical cancer cells. HeLa cells have been infected with
various types of viruses including HIV, Zika, herpes, and mumps to test and develop new
vaccines and drugs and also used in the development of Human Papilloma viral vaccines.
• Vero cell line is derived from African green monkey kidney cell line.
• These cell lines have been used extensively for the growth of a number of viruses. These
cell lines are usually stored at -70°C for use when necessary or are maintained by serial
subculture.

Detection of viruses in cell lines:

Growth of viruses in cell cultures can be detected by the following methods:

1.Cytopathic effect:

Crenation of cells Cell rounding or ballooning by Herpes virus

 Syncytium formation
• Many viruses cause morphological changes in cultured cells in which they grow.
• These changes can be observed by microscopic examination of cultures.
• These changes are known as cytopathic effect (CPE) or cytopathogenic effect.
• Viruses causing CPE are called cytopathogenic viruses
• The CPE produced by different types of viruses are characteristic and help in the initial
identification of virus isolates.
• For example,
• Herpes virus produces discrete focal degeneration.
• Focal degeneration causes a localized attack of the host cell monolayer. Although this type
of CPE may eventually affect the entire tissue, the initial stages and spreading occur at
localized viral centers known as foci. Focal degeneration is due to direct cell-to-cell
transfer of the virus rather than diffusion through the extracellular medium.
• Initially, host cells become enlarged, rounded, and refractile. Eventually, the host cells
detach from the surface. The spreading of the virus occurs concentrically, so that the cells
lifting off are surrounded by enlarged, rounded cells that are surrounded by healthy tissue.
This type of CPE is characteristic of herpesviruses and poxviruses
• Enteroviruses cause crenation of cells and degeneration of the entire cell sheet.
• Measles virus produces syncytium formation.
• Adenoviruses produce large granular changes resembling bunches of grapes.
 • Syncytium formation by Measles virus and large granular clumps by Adenovirus in Rhesus
monkey kidney cell line
• Crenation of cells and cell rounding or ballooning by Herpes virus
2. Metabolic inhibition
• In normal cell cultures, the medium turns acid due to cellular metabolism.
• When viruses grow in cell cultures, cell metabolism is inhibited and there is no acid
production.
• This is detected by the colour of the indicator (phenol red) incorporated in the medium.
3. Hemadsorption:
• Hemadsorption is the process of adsorption of erythrocytes to the surfaces of infected cells.
• Viruses, such as influenza virus, parainfluenza virus, mumps virus, and togavirus, express
hemagglutinins on the infected cell membrane.
• These hemagglutinins bind some erythrocytes to the infected cell surface.
4. Heterologous interference:
• This property is used to detect viruses that do not produce classic CPEs in the cell lines.
• In this method, the growth of non-CPE-producing virus in cell culture can be tested by
subsequent challenge with a virus known to produce CPEs.
• The growth of the first virus will inhibit infection by the cytopathic challenge virus by
interference.
• For example, rubella virus usually does not produce any CPE, but prevents the replication
of picornaviruses, which is inoculated as a cytopathic challenge virus.
5. Transformation:
 • Oncogenic viruses that are associated with formation of tumors induce cell
transformation and loss of contact inhibition in the infected cell lines.
• This leads to surface growth that appears in a piled-up fashion producing microtumors.
• Examples of such oncogenic viruses that produce transformation in cell lines are some
Herpes viruses, adenoviruses, hepadna viruses (Hepatitis B virus), Papovavirus, and
retroviruses.
6. Immunofluorescence:
• Direct immunofluorescence using specific antibodies is frequently used to detect viral
antigens in inoculated cell lines for identification of viruses.
7. Electron microscopy:
The viruses can also be demonstrated in infected cell lines by EM.
Influenza virus by TEM