By Rusul Anwar

Cultivation, Detection and

Quantitation of viruses

CULTIVATION OF VIRUSES
Many viruses can be grown in cell cultures or in fertile eggs under
strictly controlled conditions.
Growth of virus in animals is still used for the primary isolation of
certain viruses and for studies of pathogenesis of viral diseases
and of viral oncogenesis.
 Viruses are extremely small infectious agent that invade
cells of all types
Viruses are obligate intercellular parasites so they depend on
host for their survival
They cannot be growth in non-living culture media or agar
plate alone , they must require living cell to support their
replication .

Viruses are causes many human diseases, including smallpox, flu,

AIDS, and etc……
Techniques used in cultivating and identifying viruses:
Viruses require living cell as their medium
 In vivo = laboratory-bred animals & embryonic bird tissues
 In vitro = cells or tissue culture methods

The primary purposes of virus cultivation are:

 To isolate & identify viruses in clinical samples
 To do research on viral structure , replication , genetic &
effect on host cell
 To prepare viruses for vaccine production

Methods used for cultivation of virus

1- Lab. Animal Inoculation
- Viruses which are not cultivate in empryonated egg & tissue
culture are cultivated in laboratory animals such as ( mice ,
guinea pig , hamster & rabbits ) are used
- The selected animals should be healthy & free from any
communicable diseases
- Mice (less than 48 hours old ) are commonly used
- Mice are susceptible to Togavirus & Coxakie virus which are
inoculated by intracerebral & intranasal routes
Inoculation of virus in lab. animals

Advantages of Animal Inoculation

- Diagnosis , pathogenesis & clinical symptoms are determined
- Production of antibodies can identified
- Primary isolation of certain viruses
- Mice provide a reliable model for studding viral replication
- Use for study of immune response , epidemiology &
oncogenensis

Disadvantages of Animal Inoculation

- Expensive & difficulties in maintenance of animals

- Difficulties in choosing of animals for particular virus
- Some humane viruses cannot be grown in animals or can be
grown but do not cause diseases
 - Mice do not provide models for vaccine development
- Issues related to animals welfare system

2- Inoculation into embryonated egg

- Good Pasture in 1931 first used the embryonated hen’s egg
for the cultivation of virus.
- The process of cultivation of viruses in embryonated eggs
depends on the type of egg which is used
- Viruses are inoculated into chick embryo of 7-12 days old .
- For inoculation, eggs are first prepared for cultivation , the
shell surface is first disinfected with iodine and penetrated
with a small sterile drill
- After inoculation ,the opening is sealed with gelatin or
paraffin & incubate at 36 c for 2-3 days
- After incubation , the egg is broken and virus is isolated from
tissue of egg .
- Viral growth and multiplication in the egg embryo is indicated
by :
 The death of the embryo
 Embryo cell damage
 Formation of typical pocks or lesions on the egg
membranes
- Viruses can be cultivated in various parts of egg like ;
 chorioallantoic membrane
 allantonic cavity
 amniotic cavity
 yolk sac

Chorioallantoic Membrane (CAM):

- Inoculation is mainly for growing poxvirus.

- After incubation , visible lesions called pocks are observed,
which is grey white area in transparent CAM.
- Herpes simplex virus is also grown.
- Single virus gives single pocks
- This method is suitable for plaque studies
Allantoic Cavity
- Inoculation is mainly done for production of vaccine of
 influenza virus,
 yellow fever,
 rabies.
- Most of avian viruses can be isolated using this method.

Amniotic Sac:
- inoculation is mainly done for primary isolation of influenza
virus and the mumps virus.
- Growth and replication of virus in egg embryo can be
detected by haemagglutination assay

Yolk sac inoculation

- It is also a simplest method for growth and multiplication of

virus.
- It is inoculated for cultivation of some viruses and some
bacteria (Chlamydia, Rickettsiae)
- Immune interference mechanism can be detected in most
of avian viruses.
 Advantages of Inoculation into embryonated egg
1. Widely used method for the isolation of virus and growth.
2. Ideal substrate for the viral growth and replication.
3. Isolation and cultivation of many avian and few mammalian
virus
4. Cost effective and maintenance is much easier.
5. Less labor is needed.
6. The embryonated eggs are readily available.
7. They are free from contaminating bacteria and many latent
viruses.
8. 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 have different sites for their growth and
replication.

3- Cell Culture
Cell culture is mostly used for identification and cultivation of
viruses.

- Cell culture is the process by which cells are grown under

controlled conditions.
- Cells are grown in vitro on glass or a treated plastic surface in a
suitable growth medium.
- At first growth medium, usually balanced salt solution
containing 13 amino acids, sugar, proteins, salts, calf serum,
buffer, antibiotics & phenol red are taken and the host tissue
or cell is inoculated.
- On incubation the cell divide and spread out on the glass
surface to form a confluent monolayer

Types of cell culture

( Based on the origin & the chromosome property )

1- Primary cell culture:

- These are normal cells derived from animal or human cells.

- They are able to grow only for limited time and cannot be
maintained in serial culture.
- They are used for the primary isolation of viruses and
production of vaccine.
- Examples:
 Monkey kidney cell culture ,
 Human amnion cell culture ,
 chick embryo cell culture
2- Diploid cell culture (Semi-continuous cell lines)

- They are diploid and contain the same number of

chromosomes as the parent cells.
- They can be sub-cultured up to 50 times by serial transfer
following senescence and the cell strain is lost.
- They are used for the isolation of some fastidious viruses
and production of viral vaccines.
- Examples:
 Human embryonic lung strain ,
 Rhesus embryo cell strain

Rhesus embryo cell strain

3- Continuous cell lines

- They are derived from cancer cells.

- They can be serially cultured indefinitely so named as continuous cell
lines o They can be maintained either by serial subculture or by
storing in deep freeze at -70°c.
- Due to derivation from cancer cells they are not useful for vaccine
production.
- Examples:
 HeLa (Human Carcinoma of cervix cell line),
 HEP-2 (Human Epithelioma of larynx cell line),
 Vero (Vervet monkey) kidney cell lines,
 BHK-21 (Baby Hamster Kidney cell line).
They invariably have altered and irregular numbers of chromosome

Advantages of cell culture

- Relative ease ,
- broad spectrum ,
- cheaper
- sensitivity

Disdvantages of cell culture

- The process requires trained technicians with experience in
working on a full time basis.
- State health laboratories and hospital laboratories do not
isolate and identify viruses in clinical work.
 - Tissue or serum for analysis is sent to central laboratories to
identify virus

Detection of Virus-Infected Cells

Multiplication of a virus can be monitored in a variety of ways:
1. Development of cytopathic effects, include cell lysis or
necrosis, inclusion bodies formation, giant cell formation, and
cytoplasmic vacuolization.
2. Appearance of a virus-encoded protein, such as the
hemagglutinin of influenza virus.
3. Adsorption of erythrocytes to infected cells, called
hemadsorption, due to the presence of virus-encoded
hemagglutinin (parainfluenza, influenza) in cellular membranes.
4. Detection of virus-specific nucleic acid by molecular-based
assays such as PCR .
5. Viral growth in an embryonated chick egg may result in :
 death of the embryo (ex: encephalitis viruses),
 Production of pocks or plaques on the chorioallantoic
membrane (ex: herpes, smallpox, vaccinia),
 Development of hemagglutinins in the embryonic fluids
or tissues (ex: influenza)
uninfected early infection late infection
 Inclusion Body Formation
- In the course of viral multiplication within cells, virus-specific
structures called inclusion bodies may be produced.
- They become far larger than the individual virus particle and
often have an affinity for acid dyes (e.g., eosin).
- They may be situated in the nucleus or in the cytoplasm
(poxvirus), or in both.
- In many viral infections, the inclusion bodies are the site of
development of the virions (the viral factories).
- Variations in appearance of inclusion material depend largely
upon the tissue fixative used.
- The presence of inclusion bodies may be of considerable
diagnostic aid.
- The intracytoplasmic inclusion in nerve cells Negri body is
pathognomonic for rabies.

Cytoplasmic inclusion bodies in
cells infected with sheep pox
virus
Intranuclear inclusion bodies in
cells infected with CMV
(Owel’s eye)
Cytoplasmic inclusion bodies (Negri
bodies)
in nerve cells infected with Rabies
virus
Cowdry bodies are eosinophilic or
basophilic nuclear inclusions
composed of nucleic acid and
protein seen in cells infected with
Herpes simplex virus,
Varicella-zoster virus, and
Cytomegalovirus.
Stained by : hematoxylin-eosin
stain
 Quantitation of Viruses
1. Physical Methods
- Quantitative nucleic acid-based assays such as:
- The polymerase chain reaction (PCR) can determine the
number of viral genome (infectious and noninfectious) copies in
a sample.
- Serologic tests such as radio-immunoassays (RIA) and enzyme-
linked immuno-sorbent assays (ELISA) can be standardized to
quantitate the amount of virus in a sample.
- Certain viruses contain a protein (hemagglutinin) that has the
ability to agglutinate red blood cells of humans or some
animals.
- Virus particles can be counted directly in the electron
microscope by comparison with a standard suspension of latex
particles of similar small size.

2. Biologic Methods
- End point biologic assays depend on the measurement of
animal death, animal infection, or cytopathic effects in tissue
culture at a series of dilutions of the virus being tested.
- The titer is expressed as the 50% infectious dose (ID50), which is
the reciprocal of the dilution of virus that produces the effect in
50% of the cells or animals inoculated.
- The plaque assay is the most widely used assay for infectious
virus.
- Monolayers of host cells are inoculated with suitable dilutions
of virus and after adsorption are overlaid with medium
containing agar or carboxymethylcellulose to prevent virus
spreading throughout the culture.
- After several days, the cells initially infected have produced
virus that spreads only to surrounding cells produce a small
area of infection, or plaque.
- Under controlled conditions, a single plaque can arise from a
single infectious virus particle, termed a plaque-forming unit
(PFU) be counted macroscopically.
 Diluted 10 fold Diluted 100 fold Diluted 1000 fold

 Pocks formation: certain viruses, eg, herpes and vaccinia,

when inoculated onto the chorio-allantoic membrane of an
embryonated egg can be quantitated by relating the number of
pocks counted to the viral dilution inoculated.