virology practice
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Virology practice. Cultivation of viruses. Since viruses are obligate intracellular parasites they have to be grown in living cells. There are three systems for their cultivation: 1- tissue culture. 2- embryonated eggs. 3- intact animals. 1- tissue cultures: - PowerPoint PPT PresentationTRANSCRIPT
Virology practice Dr. Y. Hamadt Allah Elhaj
Ass. Prof. medical microbiology
Cultivation of viruses • Since viruses are obligate intracellular
parasites they have to be grown in living cells.• There are three systems for their cultivation:1- tissue culture.2- embryonated eggs.3- intact animals.1- tissue cultures: Pieces of animal or human tissues are
trypsinized to get separate cells. Thses are grown in presentceof growth medium containing serum, on glass or plastic tubes, bottles or plates with a flat side.
A monolayer or sheet of cells is formed on the
flat side of container within few days. Viruses
are inoculated on the monolayer. There are
three types of tissue cultures:
1. Primary cell lines: these are prepared from
organ fragments e.g. monkey kidney.
such cells can only divide for several
passages (4 - 6) and then degenerate.
2. Continuous cell lines: these are derived
from tumour cells and they can divide
indefinitely e.g. hela cells which are derived
from carcinoma of the cervix.
3. Human diploid cell lines: these
are usually fibroblasts derived
from human embryo tissues .
They have diploid number of
chromosomes. They grow rapidly
and can be subcultured up to
about 50 passages in culture e.g.
human embryo lung tissue.
Detection of virus replication in cell
culture:
1- cytopathgenic effects (CPE): these are
changes in cells that can be observed
microscopically:
a. Cell death and detachment from the glass
surface is produced by many viruses e. g.
poliovirus.
b. Rounding and grape like cluster formation
is produced by adenovrus.
c. Cyncytium or multinucleated giant cell
formation are characteristic of measles or
mumps.
d. Cell transformation: the cells lose
the property of contact inhibition
present in normal cells and pile
up to form foci of malignantly
transformed cells e.g. tumour
viruses.
2- Plaque formation: plaques are virally
infected areas in tissue culture monolayer.
They can be seen by the naked eye as
unstained areas when using vital stains.
3- Inclusion bodies: these are intranuclear or
intracytoplasmic structures which may
appear in virus infected cells and can be
seen by the light microscope. They are
often the site of virus replication. Their
presence is of diagnostic value e.g. the
(Negri bodies) in nerve cells of rabid
animals.
4 -Haemadsoption:When RBCs are added to infected cells they will appear as rosettes or clumps on the areas where the virus is growing. This is useful in haemagglutinating viruses such as influenza virus.5- Fluorescent antibody staining: infected cell sheets on cover slips or microtiter plates may be treated with fluorescein labelled specific antibody and examined for positive fluorescence.
6- Interference: in some viruses which
do not produce CPE their growth can be
proved by their ability to interfere with
the growth of another CPE producing
virus.
7- detection of viral antigens by
serology: including complement
fixation test, haemagglutination test ,
…..
8- neutralization tests: neutralization of
effects of virus on tissue culture by
specific antisera can be used to identify
and type the virus isolated.
Plaque
CPE: inclusion bodies
Negribodies
Negribodies
neuron
Immunohistochemical staining of intra-cytoplasmic viral inclusions in the neuron of a human rabies patient. (Fields Vriology (2007) 5th edition, Knipe, DM & Howley, PM, eds, Wolters Kluwer/Lippincott Williams & Wilkins, Philadelphia
Fig. 39.9)
Hemadsorbtion
add red blood cells
BEST WISHESDr. yagoub Hamadt
Allah
2- embryonated eggs:Different viruses can grow in various cavities of embryonated eggs or in the developing embryo itself. The age of the embryo used and the site of inoculation vary according to the virus inoculated.Chorioallantoic membrane inoculation is used in pox and herpes viruses.The influenza virus can readily grow in the amniotic sac and in the respiratory cells of the embryo.
3- intact animal:Animal inoculation was mainly used in the past when tissue culture methods were not known, however, animal inoculation is still used for studying viral oncogenesis, pathogenesis of viral disese, immune response to viruses and for primary isolation of some viruses.The white sucking mouse is the most widely used; it is susceptible to encephalitis viruses by the intracerebral route.
Diagnosis of virus infectionthe laboratory procedure used in diagnosis of viral disease include:a. Direct detection of viruses or their antigens in clinical specimens.b. Isolation of viruses.c. Serologic detection of antiviral antibodies.
a. Direct detection of viruses or their antigens in the
specimens:
Can be achieved by different techniques:
1- Light microscope: this can be used to visualize some
large viruses e.g. poxviruses in which elementary bodies
can be seen in skin lesions (papules and vesicles).
Inclusion bodies can also seen under the light microscope
in several viral infections. In rabies, intracytoplasmic
inclusions called (negri bodies) can be detected in nerve
cells.
2- Electron microscope: is used to demonstrate virus
particles in vesicular fluid or tissue extracts treated with
special stains. It is only successful if large numbers of
particles (109/ml) are present.
3- Immunoelectron microscope (IEM): addition of
specific antisera to the clinical material leads to
aggregation of virus so it can be detected more readily
than separate virus particles e.g. diagnosis of HAV and
rota virus in stool.
4- Immunofluorescence microscopy: detection of virus in
smears from lesions using fluorescein labeled specific
antisera. E.g diagnosis of rabies virus in brain smears.
5- Solide – phase immunoassay: both radioimmunoassay
(RIA) and enzyme linked immunosorbent assay (ELISA)
can be used for detection of viral antigens in different
clinical specimens such as detect of HCV, HBV,
rotaviruses,….
6- Nucleic acid hybridization: using DNA probes, it is possible to
detect virus nucleic acid in pathologic specimens or in tissue
samples. The probe which is a single strand of the nucleic acid of
the virus in question will hybridize with its complementary strand in
the specimen. Probes are labeled and can be easily detected.
7- Polymerase chain reaction (PCR): is a recent technique which
involves amplification of a short sequence of a target DNA or RNA
( which may be in low concentration e.g one copy). Leading to
accumulation of large amount of that sequence, so it can be easily
detected.
b. Isolation of viruses:
Isolation of viruses from clinical
specimens by inoculation on
tissue culture, chick embryo or
laboratory animals according to
the virus in question.
c. Serologic detection of antiviral antibodies:
Serologic diagnosis of virus infections can be
established by detecting a rising antibody titre to
the virus. the first sample should be collected
early after onset, the second 1 -2 weeks later.If paired sera are not available or rapid diagnosis is needed, as in diagnosis of rubella in early pregnancy; detection of IgM antibodies to the virus is resorted to. The detection of IgM in a single serum sample, indicates recent infection.
The tests used for serologic diagnosis of
viral infection include:
1- Virus neutralization
2- Complement fixation
3- Haemagglutination inhibition.
4- Immunofluorescence.
5- RIA 6- ELISA
Neutralization test
This is based on
the neutralization of the
virus infectivity by mixing it
with specific antibody before
inoculation into
cultures.
1 2 3
B
Following virus isolation:
1.Divide culture yield into small volume in a set of test tubes
2. Prepare the panel of antisera against which the virus isolate is to be challenged
2. To each test tube add one antisera and leave one as a virus control and one as serum control
Incubate for one hour then inoculate each into cell culture tubes, incubate and observe daily.
2 31
Principle of Neutralization test
Viral Hemagglutination Inhibition Test
• Useful for viruses that aren’t cytopathic• Test based on viral hemagglutination,
the ability of some viral surface proteins to clump red blood cells
• Serum from an individual will stop viral hemagglutination if the serum contains antibodies against the specific virus
• Commonly used to detect antibodies against influenza, measles, and mumps
Complement Fixation Test
• Based on the generation of membrane attack complexes during complement activation that disrupt cytoplasmic membranes
• Used to detect the presence of specific antibodies in an individual’s serum
• Can detect antibody amounts too small to be detected by agglutination
Fluorescent Antibody Test
• Uses fluorescent dyes as labels • Fluorescein is the most important dye used
in these test– Chemically linked to an antibody without
affecting antibody’s ability to bind antigen– Glows bright green when exposed to fluorescent
light
• Fluorescein-labeled antibodies used in two types of tests– Direct fluorescent antibody test– Indirect fluorescent antibody tests
Direct Fluorescent Antibody Tests
• Identifies the presence of antigen in tissue– Tissue sample flooded with labeled antibody– Antibody and antigen are allowed to bind for a
short period– Unbound antibody washed from the preparation– Results observed under a fluorescent microscope
• Used to identify small numbers of bacteria in patient tissues
• Not a quantitative test – the amount of fluorescence observed is not directly related to the amount of antigen present
Indirect Fluorescent Antibody Tests
• Can be used to detect antigens in cells or patient tissues
• Also used to detect specific antibodies in serum via a two-step process
Indirect Fluorescent Antibody Test
Figure 17.14
ELISA
• Stands for enzyme-linked immunosorbent assay
• Uses an enzyme as the label– Reaction of the enzyme with its
substrate produces a colored product indicative of a positive test
• Most common form of ELISA is used to detect the presence of antibodies in serum
ELISA
Antibody Sandwich ELISA
• Modification of the ELISA technique• Commonly used to detect antigen• Antigen being tested for is
“sandwiched” between two antibody molecules
Antibody Sandwich ELISA
Advantages of the ELISA
• Can detect either antibody or antigen• Can quantify amounts of antigen or
antibody• Easy to perform, inexpensive, and
can test many samples quickly• Plates coated with antigen and
gelatin can be stored for later testing