Virus
Cultivation and Quantitation
Viruses are obligate intracellular
parasite. They depend totally on their
host cells for existence. They can be cultivated within suitable living hosts/
cell only. Viruses are cultivated using tissue cultures, embryonated eggs,
bacterial cultures, and other living hosts. Thus animal viruses are cultivated
or grown in the laboratory using embryonated eggs, tissue culture or by using
laboratory animals. To cultivate bacteriophages, bacterial culture is used as
the host and for plant viruses, plant tissue culture or whole plants are used.
Virus cultivation is essential to get
sufficient amount of virus particles for the following applications
·
For conducting studies on virus and their
host interactions and diseases
·
For studying the effectiveness of
antiviral drugs
·
For use as gene vectors in gene therapy
·
Viral pesticide production
·
For vaccine production
Since viruses are host dependent, it is
not possible to cultivate them solely in presence of organic or inorganic
nutrient medium. They can be grown only if living cells and tissues are used as
culture medium. These tissues and cells would act as the host for the virus in
laboratory conditions. For this purpose, the relevant cells or tissues must be
cultivated first.
Cultivation
of Bacterial viruses
Bacterial viruses or bacteriophages are cultivated
in either broth or agar cultures of actively growing host bacterial cells.
In broth culture, due to the destruction
of host cells due to viral multiplication the turbid bacterial cultures will
clear rapidly. If bacteriophage is not lytic, bacteria grow luxuriously on
culture medium and there will not be any clearance of turbidity.
Agar cultures are prepared by mixing the
bacteriophage sample with cool, liquid nutrient media and a suitable bacterial
culture and pouring into a sterile petri dish.
After solidification of media, the plates will be incubated. During incubation, bacteria grow and
reproduce to form a continuous, opaque layer or lawn growth. A virus coming in contact with a bacterial
cell infects it and reproduce, the progeny virus infects the adjacent cells and
reproduces and eventually, there will be a zone where bacterial lysis occurred
and a plaque or clearing in the lawn can be observed. The appearance of plaque
is characteristic of the particular phage.
Each plaque is assumed to come from a
single viral particle. The titer of the virus is given in plaque forming units
or PFU. PFU could be measured using the
plaque assay for bacteriophage quantitation.
The
plaque method: Serial dilutions of Virus suspension,
bacteria, and agar mixed, plated and incubated in a suitable nutrient medium
that allows the growth of the bacteria. During incubation, the bacteria
multiply, and during the replication the virus lyses the bacteria, forming
plaques, or clear zones. Each plaque is assumed to come from a single viral
particle. The titer of the virus is given in plaque forming units.
Cultivation
of Plant Viruses: There are several methods of cultivation
of viruses such as plant tissue cultures, cultures of separated cells, or
cultures of protoplasts, etc. Viruses also can be grown in whole plants.
Leaves are mechanically inoculated by
rubbing with a mixture of viruses and an abrasive such as carborundum. 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. Even when
lesions do not arise, the infected plant may show symptoms such as change in
pigmentation or leaf shape. Some plant viruses can be transmitted only if a
diseased part is grafted onto a healthy plant.
Cultivation
of animal viruses
Viruses cannot replicate in synthetic
media and require living cells for their growth.
The living systems that are commonly used
for cultivation of animal viruses are
i) Inoculation into animals, ii)
Embryonated Eggs and iii) Cell Culture
Whatever system is adopted for cultivation
of viruses, it should be free from bacteriological contamination. This can be
achieved by passing the suspension through membrane filters (0.2 µm) or by
treatment with antibiotics e.g. Penicillin,
streptomycin, etc.
The process of viral replication destroys
the infected living cells and may result in formation of disease lesions or
other abnormalities in the tissues.
I)
Inoculation into animals:
The earliest method for cultivation of
viruses causing human diseases was inoculation into human volunteers. Reed and
his colleagues (1900) used human volunteers for their work on yellow fever. Due
to serious risk involved, human volunteers are involved only when no other
method is available and the virus is relatively harmless.
Monkeys were used for the isolation of
Poliovirus by Handsteiner and popper in 1909. Due to their cost, and risk to
handlers, they have limitations. Mice are most widely used animals in virology.
Infant mice are very susceptible to Coxsackie’s and arboviruses. Mice can be
inoculated through several routes i.e. intracerebral, subcutaneous,
intraperitonial, intranasal, etc. Other animals such as guinea rabbits, ferrets,
birds such as chicken etc are also used. They should be germ-free and are
termed as SPF (Specific Pathogen Free) birds and animals. The growth of virus
in inoculated animals is indicated by death, disease or visible lesions.
Experimentally inoculated/infected animals
are examined daily for;
i)
Clinical signs of disease, respiratory distress, CNS involvement, and visible
lesions on skin and membranes.
ii)
Abnormal behaviour of the animal
iii)
Blood samples are taken daily for antibodies titer determination.
iv)
Death of the animal
Biopsy material or tissue specimens should
be examined for;
i.
Microscopically for lesions (Cytopathic
effects)
ii.
Histopathologically for pathological
changes
iii.
Serologically for presence of specific
viral antigens by, e.g. gel diffusion,
CFT etc.
iv.
By electron microscope, for identification
of viral particles
Animal inoculation has a disadvantage that
immunity may interfere with viral growth and that animals often harbor latent
viruses.
II)
Embryonated eggs:
The Embryonated hen’s egg was first used
for cultivation of viruses by Good Pasteur and Burnet (1931). Since the early 1950 the Embryonated hen’s
eggs have been used widely for cultivation of animal viruses. Embryonated egg
does not support the growth of all animal viruses but most of the avian viruses
grow.
The eggs should be free from any kind of
germ and thus SPF-Eggs laid by SPF-birds are used
This is the most suitable means for
primary isolation and identification and production of viral vaccines. The major advantages of embryonated eggs over
other systems are;
i.
Easily available, economical and
convenient to handle.
ii.
Relatively free from bacterial and many
latent viral infections.
iii.
Generally free from immune mechanisms.
The developing chick embryo, 10 to 14 days
after fertilization, provides a variety of differentiated tissues, including
the amnion, allantois, chorion, and yolk sac, which serve as substrates for
growth of a wide variety of viruses, including orthomyxoviruses,
paramyxoviruses, rhabdoviruses, togaviruses, herpesviruses, and poxviruses.
To prepare the egg for virus cultivation,
the shell surface is first disinfected with iodine and
penetrated with a small sterile drill. After inoculation, the drill hole is
sealed with gelatin and the egg incubated. Viruses may be able to reproduce
only in certain parts of the embryo; consequently they must be injected into
the proper region. For example, the myxoma virus grows well on the
chorioallantoic membrane, whereas the mumps virus prefers the allantoic cavity.
The infection may produce a local tissue lesion known as a pock, whose
appearance often is characteristic of the virus.
The sites for the
cultivation of viruses in embryonated egg:
1) Chorioallantoic membrane (CAM): CAM is
inoculated mainly for growing poxvirus and Herpes simplex virus. Virus
replication produces visible lesions, grey white area in transparent CAM. Pocks
produced by different virus have different morphology. Each pock is derived
from a single virion. Pock counting, therefore can be used for the assay of
pock forming virus such as vaccinia.
2) Allantoic cavity: Inoculation into the
allantoic cavity provides a rich yield of influenza and some paramyxoviruses.
Duck eggs are bigger and were used for the preparation of the inactivated
non-neural rabies vaccines.
3) Amniotic cavity: The amniotic sac is
mainly inoculated for primary isolation of influenza a virus and the mumps
virus.
4) Yolk sac: It is inoculated for the
cultivation of some viruses as well as for some bacteria like Chlamydiae and
Rickettsiae.
The presence of viral growth may be
identified in embryonated egg by;
1.
Death of the embryo (Toga virus)
2.
Deformities such as dwarf growth (IB-virus)
3.
Hemorrhages (ND-virus)
4.
Oedema and pock lesions on CAM (Cow pox, Herpes B-virus)
5.
Intracytoplasmic inclusion bodies (Herpes virus)
III)
Tissue culture:
Cell culture (earlier called tissue
culture) is the most widely used method for cultivation of viruses. Cell
culture allows the primary isolation of viruses, performance of infectivity
assays and biochemical studies and the production of viral vaccines.
The main advantages of cell culture method
over the other two systems are;
1.
Growth of most viruses can be detected easily in cell culture.
2.
Viruses can be grown in bulk.
3.
Cells can be stored for longer period of time.
Disadvantages are
1.
Requirement of good laboratory facility.
2.
More costly as compared to the embryonated eggs.
3.
There are chances for the presence of latent viruses in the cultured cells.
There
are three types of tissue cultures:
1) Organ culture:
Small bits of organs can be maintained in
vitro for days and weeks. Organ culture is useful for the isolation of some
viruses which appear to be highly specialized parasites of certain organs.
Example: Tracheal ring organ culture is
employed for the isolation of corona virus, a respiratory pathogen.
2) Explant culture:
Fragments of minced tissues can be grown as explants embedded in plasma clots.
They may also be cultivated in suspension.
Example: Adenoid tissue explant culture
was used for the isolation of adenovirus.
3) Cell culture: The
cell culture is the method routinely employed nowadays for identification and
cultivation of viruses.
Procedure
·
To obtain a primary cell culture, tissue
or organs preferably from embryonic or infant (e.g. chicken embryo, embryonic
liver) are cut up in small fragments.
·
These fragments are mixed with Trypsin,
which will dissolve the connective tissue and thus cells becomes separated.
This step is called as trypsinization.
·
The washed suspended cells are then
cultivated in a suitable growth medium in a flat bottomed tissue culture flask.
The essential constituents of growth medium are essential amino acids,
vitamins, salts and glucose and a buffering system and about 5% calf or fetal
calf serum. Antibiotics are added to prevent bacterial contaminants and phenol
red as indicator. Such media will allow most cell types to multiply with a
division time of 24-48 hrs in a CO2 incubator at 37oC.
·
After a period of time, the cells attach
to the bottom of the flask and start dividing until a monolayer is formed. This
kind of cell culturing is known primary cell culture.
·
The inoculum suspected to contain a
particular virus type is inoculated and allowed to absorb on the cell
monolayer.
·
Add an adequate amount of maintenance
medium and incubate the flask at 37oC.
Types
of cell cultures:
On the basis of origin, chromosomal
characters, and the number of generations through which they can be maintained,
cell cultures are classified in three types.
1) Primary cell culture:
These are normal cells obtained from fresh
organs of animals and cultured. Once the cells get attached to the vessel
surface, they undergo mitosis until a confluent monolayer of cells covers the
surface. These layers are capable of limited growth in culture and cannot be
maintained in serial culture. They are commonly employed for primary isolation
of viruses and in preparation of vaccine. Primary cell cultures are generally
best for viral isolation.
Examples: Rhesus monkey kidney cell
culture, Human amnion cell culture.
2) Diploid cell culture:
It is also called as semi continuous cell
lines. These are cultures derived from primary cell cultures. These are cells
of single type that retrain the original diploid chromosome number and
karyotype during serial sub cultivation for a limited period of time. There is
rapid growth rate and after 50 serial subcultures, they undergo senescence and
the cell strain is lost. The diploid cell strains are susceptible to a wide
range of human viruses. They are also used for isolation of some fastidious
viruses and production of virus vaccines,
Examples: Human embryonic lung strain
(WI-38) and Rhesus embryo cell strain (HL-8)
3) Continuous cell culture:
These are cells of a single type, usually
derived from the cancer cells that are capable of continuous serial
cultivations indefinitely. These cells grow faster and their chromosomes are
haploid. They are also called as permanent cell lines. Permanent cell lines
derived from a single separated cell are called as clones. One common example
of such clone is HeLa strain derived from cervical cancer of a lady named HeLa.
Continuous cell lines are maintained either by serial subculture or by storing
in deep freeze at -70°c.
Examples: Vero i.e. Vervet monkey kidney
cell line, BHK, i.e. Baby Hamster kidney cell line.
Most animal cells are anchorage dependent
and thus surfaces of glass, plastics, natural polymers such as collagen, or
other support materials are used. In lab
scale, T- flasks, spinner bottles, roller bottles and trays containing shallow
liquid cultures are used for cell culture.
Large scale reactors for animal cell
culture includes microcarrier systems, hollow fiber reactors, ceramic matrix
systems, weighted porous beads, etc. for anchorage dependent cells and Stirred
tank reactors and bubble column reactors and perfusion bioreactors for
suspension cultures.
1) Roller bottles: Bottles are rotated
about the long axis with the cells adhered to its sides. They are therefore
dipped in the medium and are aerated alternatively.
2) Microcarriers of DEAE or dextran are
used for anchorage dependent cells. Cells grow on the surface of the
microcarriers, usually in the form of monolayers and sometimes as multilayers. Microporous
microcarriers are also used in which cells grow inside them.
3) Hollow fiber reactors are used to
provide a high growth surface- volume ratio. Cells are immobilized on the
external surfaces of hollow fibres, and nutrients pass through the tubes.
4) Other immobilization based reactors:
Tubular ceramic matrix reactors, microencapsulation in spherical membranes and
gel encapsulation
Detection of virus growth in cell cultures
- Cytopathic
effects (CPE) – morphological changes in cultured cells, seen under
microscope. The degenerative
changes of cells that are linked with the multiplication of certain
viruses are known as the cytopathic effect (CPE). The characteristics of cytopathic effect
produced on different cell culture can be used to identify viral
infection. Common examples are rounding of the infected cell, fusion with
adjacent cells to form a syncytia and the appearance of nuclear or
cytoplasmic inclusion bodies. Inclusion bodies may represent either
altered host cell structures or accumulations of viral components.
Cytopathic
effects (CPE) Formation of syncytia
- Metabolic
Inhibition – no acid production in presence of virus
- Hemadsorption
– influenza & parainfluenza viruses, by adding guinea pig erythrocytes
to the culture
- Interference
– growth of a non cytopathogenic virus can be tested by inoculating a
known cytopathogenic virus: growth of first virus will inhibit the infection
by second
- Transformation
– oncogenic viruses induce transformation & loss of contact inhibition
- microtumors
- Immunofluorescence
– test for viral Ag in cells from viral infected cultures.
Assays
for viral infectivity
This could be done by either of two
methods, by assaying the Infectivity (plaque assay) or by Physical measurement
of virus particles and their components (Hemagglutination, Electron microscopy,
Viral enzymes, Serology, Nucleic acids).
Two types of assays are used to determine
the viral infectivity primarily in cell cultures and occasionally in other
systems.
i)
Quantitative assays
ii) Quantal
assays are used
Quantitative
assays: actual no. of infectious particle in an inoculum
These assays quantify the number of virus
particles in an inoculum. The commonly used assay in cell culture is Plaque
Assay or Pock Assay.
Quantification
of viruses
The quantification of viruses in a
sample/suspension is important for diagnosis and for experimental purposes for
vaccine preparation, virus cultivation etc.
Methods
of Quantification
The methods of quantification are divided
into two categories;
i.
Physical Method
ii.
Biological Method
I.
Physical method
In this method, electron microscope is
used for quantification.
Electron
Microscopy
Through EM, besides the size and shape of
viruses, we can quantify/count the viral particles.
·
Mix known no. of latex beads with the
diluted purified virus suspension.
·
Put this suspension on the copper
grid/mesh of the microscope.
·
Examine it under microscope and count the
particles in a specific area.
·
Find a ratio between the latex beads and
virus particles being seen under microscope.
If we know the number of latex beads per
ml of the suspension then the number of virus particles can be calculated. The counted virus particles can be expressed
as No. of virus particles/ml.
Electron
Microscope - Immune Electron Microscopy.
Light
microscope – Inclusion bodies. eg Negri Body in Rabies
Fluorescent
Microscope -Fluorescent antibody technique.
II.
Biological method
This method includes a number of important
techniques used for quantification of viruses.
Plaque
Assay: Purified virus suspension is inoculated on the
monolayer cell culture in vitro. For
bacteriophages, bacterial colonies are used for culturing process.
·
Make serially diluted suspension of
bacteriophage or virus
·
Make a lawn culture of bacteria or a
monolayer cell culture
·
Add the serially diluted virus suspension
into it and incubate
·
Examine for plaque formation and count the
plaques formed.
Virus particles = Plaque number X reciprocal of
dilution or dilution factor X reciprocal of volume in ml.
Plaques are clear zones that develop on
lawns of host cells.
This method is also now used for animal
virus quantitation by a modification (In 1952 by Renato Dulbecco, Nobel Prize, 1975),
where monolayer cultures of cells were used instead of bacterial lawn. After addition of virus suspension, an agar
overlay is done. The plaques could be
observed after staining the cell monolayer after incubation for appropriate
time.
Pock
Assay: In this technique, pock lesions formed on the chorio
allantoic membrane (CAM) of chicken embryos are counted. The counted pocks can
be expressed as Pock FU/ml. (Pock FU stands for Pock Forming Units).
There are several serological and
immunological methods
Haemagglutination
Assay
Many viruses have the property to bind to
erythrocytes (RBCs) of different species through complementary receptor sites
on the erythrocyte surface. A quantitation of viruses based on this is known as
haemagglutination assay (HA). Haemagglutination is a particular form a
agglutination which involves the participation of red blood cells (RBC).
A type of lattice will be formed by red
blood cells when virus with surface or enveloped proteins stick to human or animal
red blood cells and bind to its N-acetylneuraminic acid. The clump
formation/haemagglutination ability of a virus is known as HA titre.
Greater the ability of clump formation
---- Stronger will be the virus.
Lower the ability of clump formation
------ Weaker will be the virus.
This method is relatively fast and easy
and large amounts of samples could analyzed.
Virus
neutralization assay
This is a method in which antibodies are
added to a virus preparation, and the infectivity of this preparation is measured
using cells. Antibodies produced against any virus will have the ability to
interfere with the interaction between the virus and its host cell receptor. Such antibodies will have the ability to
neutralize the infectivity of the virus.
Immunostaining
This is a method in which antibodies are
used to detect viral proteins in infected tissues or cells.
Immunoblotting
and immunoprecipitation
These methods allow detection of specific
viral proteins in lysates from infected tissues or cells.
Enzyme-linked
immunosorbent assay (ELISA)
ELISA allow the detection of viral antigen
using a specific antibody
Nucleic
acid detection
Methods for the detection of viral nucleic
acids in clinical and laboratory specimens include Southern blot analysis, in
situ hybridization, polymerase chain reaction (PCR) and the use of gene arrays.
Quantal
assays - presence or absence of infectious viruses
These assays do not count the number of
infectious virus particles present in an inoculum.
Serial dilution of a virus inoculum is
made and is inoculated into tubes containing cell monolayer. After incubation,
the incubated tubes are examined for virus infection i.e. by looking to the
changes (e.g. CPE) in the inoculated cells, the titre of the inoculum is
determined.
The infectivity of virus is expressed as
the 50% lethal dose, the dose required to infect and kill 50% inoculated cells.
