Metode Yang

Digunakan Dalam
Virologi
Lindi Grahawanti Haritsyah, S.Si., M.Biomed

20 Juni 2019
MATERI PEMBELAJARAN
Metode yang digunakan dalam virologi:
1. Kultivasi virus (dalam kultur sel hewan)
2. Isolasi virus
3. Sentrifugasi (memanen virion)
4. Identifikasi struktur sel & virion
5. Kuantitatif assay
6. Deteksi virion
7. Deteksi virus yang infektif
8. Deteksi Ag virus
9. Deteksi asam nukleat virus

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CAPAIAN PEMBELAJARAN
Mahasiswa mampu memahami berbagai metode yang digunakan
dalam virologi.

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 Cultivation of viruses
 Virus cultivation → virus propagation or growth.
 All viruses are obligate intracellular parasites → need an appropriate
host cell if they are to replicate:
1) Phages are supplied with bacterial cultures.
2) Plant viruses may be supplied with specially cultivated plants or
with cultures of protoplasts (plant cells from which the cell wall has
been removed).
3) Animal viruses are grown
in cultured animal cells or
may be supplied with whole
organisms (such as mice,
eggs containing chick
embryos, insect larvae).
The culture of animal viruses in embryonated eggs
 Cultivation of viruses in eggs containing chick embryos.
Inoculation can be made into the developing embryo itself or into one of the
various membranes and cavities such as the chorioallantoic membrane or the
allantoic cavity. Viral propagation is demonstrated by death of the embryo, or
the appearance of lesions on the membranes.
 Animal cell culture
 Animal cell culture techniques →
used continuous cell lines*
derived from humans and other
animal species.
 Continuous cell lines → HeLa
cells** (taken from cervical
carcinoma), human hepatoma
cells (from hepatic carcinoma),
vero cells (from kidney epithelial
cells African green monkey), etc.

* Consist of cells that have been immortalized, either in the laboratory or in the body
** HeLa cell derived from cervical cancer cells taken on Feb 8, 1951 from Henrietta
Lacks, a patient who died of cancer on Oct 4, 1951
 Animal cell culture

 Cells are cultured in media that provide nutrients,

antibiotics, and high concentration of CO2.
 Most media are supplemented with animal serum (contains
substances that promote the growth of many cell lines).
Other important roles for the medium are the maintenance
of optimum osmotic pressure and pH for the cells.

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Viruses can be cultivated in cells growing on the surface of a variety of
plastic vessels with the cells bathed in the growth medium.
 Animal cell culture

 Most cells grow on a plastic

or glass surface as a single
layer of cells, known as
a monolayer.

 Alternatively the cells can be suspended in the medium,

which is stirred to keep them in suspension.

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 Isolation of viruses
 Viruses can be isolated as a result of their ability to form discrete
visible zones (plaques) in layers of host cells.
 A plaque is the result of the infection of a cell by a single virion.
 There is a possibility that a plaque might be derived from two or
more virions → to increase the probability that a genetically pure
strain of virus has been obtained,
material from a plaque can be
inoculated onto further monolayers
and virus can be derived from
an individual plaque. The virus
is said to have been plaque purified.
 Centrifugation

 After a virus has been propagated it is usually necessary to

remove host cell debris and other contaminants before the
virus particles can be used for laboratory studies, for
incorporation into a vaccine, or for some other purpose.
 Many virus purification procedures involve centrifugation:
1) Partial purification can be achieved by differential
centrifugation.
2) Higher degree of purity can be achieved by some form of
density gradient centrifugation.

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 Partial purification of virions by differential centrifugation.
A crude preparation of virus containing host debris is subjected to low-speed/short-time
centrifugation (e.g. 10.000 g/20 minutes) followed by high-speed/longtime centrifugation
(e.g. 100.000 g/2 hours). This cycle can be repeated to obtain a higher degree of purity.
The final pellet containing partly purified virus is resuspended in a small volume of fluid.
 Purification of virions by density gradient centrifugation.
A partly purified preparation of virus is further purified in a density gradient. Rate zonal
centrifugation involves layering the preparation on top of a pre-formed gradient.
Equilibrium centrifugation can often be done starting with a suspension of the impure
virus in a solution of the gradient material; the gradient is formed during centrifugation.
Structural investigations of cells and virions

 Light microscopy has useful applications in detecting virus-infected cells, for

example by observing cytopathic effects or by detecting a fluorescent dye
linked to antibody molecules that have bound to a virus antigen
(fluorescence microscopy).
 Large magnifications are achievable with a transmission electron
microscope but the specimen, whether it is a suspension of virions or an
ultrathin section of a virus-infected cell, must be treated so that details can
be visualized.
 X-ray crystallography revealing detailed information about the 3D structures
of virions (and DNA, proteins & DNA–protein complexes). This technique
requires the production of a crystal of the virions or molecules under study.
 Other techniques that are providing useful information about the structure
of viruses are nuclear magnetic resonance and atomic force microscopy.
 Quantitative assays
 A plaque assay can be carried out with any virus that can form plaques,
giving an estimate of the concentration of infective virus in plaque-
forming units (pfu).
 The assay is done using cells grown in Petri dishes, or other appropriate
containers, under conditions that will allow the formation of plaques.
 Cells are inoculated with standard volumes of virus dilutions. After
incubation, dishes that received high dilutions of virus may have no
plaques or very few plaques, while dishes that received low dilutions of
virus may have very large numbers of plaques or all the cells may have
lysed.
 Dishes are selected that have plaque numbers within a certain range,
e.g. 30–300. The plaques are counted and the concentration of virus in
the sample (pfu/ml) is calculated.

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Detection of viruses and virus components

 A wide range of techniques has been developed for the

detection of viruses and virus components & many of them
are used in laboratories involved with diagnosis of virus
diseases.
 The techniques can be arranged in 4 categories:
1) Detection of virions.
2) Detection of infectivity using cell cultures.
3) Detection of virus antigens.
4) Detection of virus nucleic acids.

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 1) Detection of virions

 Specimens can be negatively stained & examined in an

electron microscope for the presence of virions.
 Limitations → high costs of the equipment & limited
sensitivity; the minimum detectable concentration of virions
is about 106/ml.
 Application → examination of faeces from a patient with
gastroenteritis for the presence of rotavirus particles.

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2) Detection of infectivity using cell cultures

 Sample/specimen can be inoculated into a culture of cells or a

host organism, known to support the replication of the virus
suspected of being present.
 After incubation of an inoculated cell culture at an appropriate
temperature it can be examined by light microscopy for
characteristic changes in the appearance of the cells resulting
from virus-induced damage. A change of this type is known as a
cytopathic effect (CPE).
 Examples of CPEs induced by poliovirus and HSV. The poliovirus-
infected cells have shrunk and become rounded, while a multi-
nucleated giant cell known as a syncytium (plural syncytia) has
been formed by the fusion of membranes of HSV-infected cells.

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Cytopathic effects caused by replication of poliovirus & HSV in cultures of Vero (monkey
kidney) cells. (a) Uninfected Vero cells. (b) Infected with poliovirus. (c), (d) Infected with
HSV. The cells were stained with haematoxylin and eosin.
 3) Detection of virus antigens
 Virus antigens can be detected using virus-specific antisera or
monoclonal antibodies.
 Positive results are indicated by detecting the presence of a label,
which may be attached either to the antivirus Ab (direct tests) or to a
2nd Ab (indirect tests).

 The anti-virus Ab is produced by injecting virus Ag into one animal

species and the 2nd Ab is produced by injecting Ig from the first
animal species into a second animal species.
Antibodies can have many types of label attached & the labels can be
detected using a range of methods. Some types of label and some
methods for detecting them are listed in Table 2.1.
 4) Detection of virus nucleic acids

Hybridization
 Virus genomes or virus messenger RNAs (mRNAs) may be detected
using sequence-specific DNA probes carrying appropriate labels.
Some of the labels that are used for Ab detection can be used to
label the probes (Table 2.1).

Polymerase chain reaction (PCR)

 When a sample is likely to contain a low number of copies of a virus
nucleic acid the probability of detection can be increased by
amplifying virus DNA using a PCR.
 RNA can be copied to DNA and amplified using a RT (reverse
transcriptase)-PCR.

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 DAFTAR PUSTAKA

1. Carter J & Saunders V. Virology: Principles And Applications.

England: John Wiley & Sons Ltd.; 2007.
2. Hogg S. Essential Microbiology. England: John Wiley & Sons
Ltd.; 2005.
3. Slonczewski JL, Foster JW. Microbiology – An Evolving
Science. New York: W. W. Norton & Company, Inc.; 2009.

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Figure 10.17. The plaque assay for
bacteriophages. The number of
particles in a phage preparation can
be estimated by means of a plaque
assay. Phages and bacteria are mixed
in a soft agar then poured onto the
surface of an agar plate. Bacteria grow
to develop a confluent lawn, and the
presence of phage is indicated by
areas of clearing (plaques) where
bacteria have been lysed. From Reece,
RJ: Analysis of Genes and Genomes,
John Wiley & Sons, 2003. Reproduced
by permission of the publishers
• In the 1950s, cell culture techniques advanced, thanks
in part to the widespread availability of antibiotics,
making the control of bacterial contamination much
more readily achieved.
• Cells are usually grown as monolayers in tissue culture
flasks containing a suitable liquid growth medium.
Treatment with the protease trypsin dissolves the
connective tissue matrix between the cells, allowing
them to be harvested, and used to seed new cultures.
• Changes in cell morphology, known generically as
cytopathic effects, are indicators of viral infection, and
may be used diagnostically in the identification of
specific viral types.