(tutorial-voiced)labatory diagnosis of viral infections ii
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By
Dr. Nashwa Abo Khadr
Microlobiogy and Immunology Dep.
Alex. University
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Depend on three principle
techniques
Direct demonstration of
viral Ag or nucleic acid
Isolation and identification ofthe virus.
Serological
demonstration of Ab
to the virus.
EM
Florescent Ab.
Double I.D.
ELISA
POLYMERASE
CHAIN
REACTION
((PCR)
EM
Florescent Ab.
Double I.D.
ELISA
POLYMERASECHAIN
REACTION
((PCR)
T.C.
Chick
embryo
Lab.animal
T.C.
Chick
embryo
Lab.animal
I.F.
ELISA NT
HA
I.F.
ELISA NT
HA
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1-Direct detection
a. Electron microscopy:
It show the detail structure . Of the v. with
diameters of ( 0.o1-0.2nm )
E.M. require high titer of the v.
1-Direct detection
a. Electron microscopy:
It show the detail structure . Of the v. with
diameters of ( 0.o1-0.2nm )
E.M. require high titer of the v.
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B-Fluorescent antibody test
Viral antigens may be detected in
smears of lesions by direct
immunofluorescent ( DIF) techniquedemonstrating specific fluorescence
with standard antiviral serum
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This test
Gives rapid diagnosis as E.M.
Does not require high titer.
Non specific staining may be a problem
careful; control to avoid false +ve
results
This test
Gives rapid diagnosis as E.M.
Does not require high titer.
Non specific staining may be a problem
careful; control to avoid false +ve
results
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C- Double immunodiffusion
Viral antigens may also be detected
by a precipitation reaction against
antiviral serum in agar gel This is a relatively rapid means of
detecting virus in a specimen and
takes from 2-6 hours
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Rapid method and takes 2-6 hours
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D- ELISA
Using the double antibody sandwitch
technique for detection of antigen
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E- Nucleic acid probing
A probe is a unique piece of NA that
can be used to demonstrate the
presence of a complementarysequence of eithr DNA or RNA in
another sample of N.A ( See
practicle )
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F- Polymerase chain reaction ( PCR )
It is a process of amplification of
specific DNA sequences .
The target DNA could be amplified
more than a million fold .
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A quantitative PCR ( Real time PCR ) isnow available to detect the viral load
( no. of viral particles /ml of blood )
This is important in monitoring the
progression of the disease and the
response to treatment .
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Direct cytological examination i.e
cytopathology A rapid presumptive diagnosis of viral
infection could be done by demonstrating
characteristic cytological changes fromsmears of infected tissues
e.g conjunctival scraping , the base of
skin lesion ,desquamated cells found in
urine and in affected neuron
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The most important cytological
changes of viral infection are the
formation of syncytia ormultinucleated giant cells and the
detection of inclusion bodies .
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Inclusion bodies are acidophilic
staining masses seen in the nucleus
or cytoplasm of infected cells ,composed of viral related structure
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Viral disease for which direct
microscopic examination of smears
has been proven to be useful includerabies , HSV infection and v-z skin
infection
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Specimen reach the lab as fast as
possible.Specimen must be kept cold.
Antibiotics and antifungal are added
to the specimen in the lab, avoid Bact.
Contamination.
Specimen is centrifuged to depositthe bacteria
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Laboratory animals
Three main systems are used for virus
isolation
Tissue culture Chick embryo
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1-Tissue culture: Cells from man or
animal are grown on a single layer (mono-
layer) on the wall of the tubes or on one
side of flat bottle. Cells are incubation at
36.5C.Tissue culture media consist of: balanced
salt sol., enriched with amino acids,
vitamins, buffer.
1-Tissue culture: Cells from man or
animal are grown on a single layer (mono-
layer) on the wall of the tubes or on one
side of flat bottle. Cells are incubation at
36.5C.Tissue culture media consist of: balanced
salt sol., enriched with amino acids,
vitamins, buffer.
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Three main types of tissue culture media
1-Primary culture:
Derived from the initial growth of cells
Cells are dispersed with trypsin. Little cell division during growth.
It form mono layer on the surface of the glass
tube.
Three main types of tissue culture media
1-Primary culture:
Derived from the initial growth of cells
Cells are dispersed with trypsin. Little cell division during growth.
It form mono layer on the surface of the glass
tube.
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After 2-3 weeks the cells degenerate
and are discarded
.e.g primary rabbit kidney ,
1ry chick emryo fibroblast
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2-Semi continuous cell lines Cells are usually fibroblasts derived from
embryonic tissue
e.g human diploid cell line they have diploid no. of
chromosomes.
There is rapid growth rate.
Cells can be subcultured (50 pass ages)
2-Semi continuous cell lines Cells are usually fibroblasts derived from
embryonic tissue
e.g human diploid cell line they have diploid no. of
chromosomes.
There is rapid growth rate.
Cells can be subcultured (50 pass ages)
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3-Contineous cell line Usually derived from malignant cells
e.g Hela cells cervical cancer. Cells are often epithelial cells.
Chromosomes are hetero ploid.
Rapid growth rate
Cells sub cultured indefinitely
3-Contineous cell line Usually derived from malignant cells
e.g Hela cells cervical cancer. Cells are often epithelial cells.
Chromosomes are heteroploid.
Rapid growth rate
Cells sub cultured indefinitely
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Virus growth is recognized by:
1) Cytopathic effect: CPE i.e. cell degeneration or
death which is recognized by
Rounding shrinkage ballooning
As the cell die, it is detached from the glass
Some virus produce (multi nucleated giant cell).
Virus growth is recognized by:
1) Cytopathic effect: CPE i.e. cell degeneration or
death which is recognized by
Rounding shrinkage ballooning
As the cell die, it is detached from the glass
Some virus produce (multi nucleated giant cell).
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2) Haemadsorption
Seen with haemagglutinating viruses which
mature at the cell surface
Virus infected cell + Erythrocyteserythrocytes. adherent to the infected cell
Virus infected cell
+
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3) HaemagglutinationAppearance of haemagglutinin in the cell cult.
haemagglutination test is done on the cellfluid to detect the presence of
haemagglutinating virus.
3) HaemagglutinationAppearance of haemagglutinin in the cell cult.
haemagglutination test is done on the cellfluid to detect the presence of
haemagglutinating virus.
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Haemagglutination pattern.
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4) Interference: In case of Vs which do not
produce CPE even up to one week cells
appear normal but unable to be infected with
CPE producing V.
Virus with known CPE is added to a cell linepreviously infected No CPE will beproduced.
4) Interference: In case of Vs which do not
produce CPE even up to one week cells
appear normal but unable to be infected withCPE producing V.
Virus with known CPE is added to a cell linepreviously infected No CPE will beproduced.
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5) FluorescenceVs antigen is detected in infected cell by
florescence due to Ag-Ab reaction between:
Ag virus + antiviral serum (labeled with flu.)
5) FluorescenceVs antigen is detected in infected cell by
florescence due to Ag-Ab reaction between:
Ag virus + antiviral serum (labeled with flu.)
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Ag Ab if +ve
O + if +ve green
Ag Ab if -ve
O + No colour
No attachmentO
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Virus identification:
The isolated v. is identified by testing for inhibition of
these effects in tissue culture with standard antiviral
serum except in case of fluorescence which it self is an
immunologically specific technique.
For cytopathic viruses; the most widely used test for
identifying virus isolated is the neutralization test
Virus identification:
The isolated v. is identified by testing for inhibition of
these effects in tissue culture with standard antiviral
serum except in case of fluorescence which it self is an
immunologically specific technique.
For cytopathic viruses; the most widely used test for
identifying virus isolated is the neutralization test
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a) Neutralization tests: Can be done with
any virus which produce CPE in tissue
culture.
Known antisera + unknown virus itneutralize infectivity.
prevent the usual CPE produced by thevirus
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b) Haemagglutinating viruses are typed or identified
by testing for neutralization by standard antiserum
which will inhibit heamadsorption HI.
b) Haemagglutinating viruses are typed or identified
by testing for neutralization by standard antiserum
which will inhibit heamadsorption HI.
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2. Chick embryo
Fertile hens egg were widely used before the
advent of the tissue culture techniques but
now been largely replaced by tissue culture
for virus isolation chick embryos are
susceptible to far fewer viruses than tissue
culture
2. Chick embryo
Fertile hens egg were widely used before the
advent of the tissue culture techniques but
now been largely replaced by tissue culture
for virus isolation chick embryos are
susceptible to far fewer viruses than tissue
culture
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C- laboratory animals
Animals are recognized for the
isolation of some viruses
Animals are observed for signs ofdisease or death after inoculation
Identification of the virus is done by
neutralization with standardantiserum
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3- Serological diagnosis
Infection is diagnosed by the
demonstration of the development of
virus antibody . Virus antibodies are common in
healthy populations and can remain
at high level for many years afterinfection .
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Diagnosis of recent infection depends
on the following criteria
1- detection of IgM the earliest
antibody to appear
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2- Rising titer :
Increase in the level of antibody at least 4
fold over the course of infection from acuteto convalescence
( Titer is the highest dilution of antiserum
at which activity is demonstrated
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3- High stationary titer :
If the titer of antibody is
considerably higher than that found
in the general population recent
infection with the virus can beassumed
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Some tests used in
serology
1- Immunofluorescence
Virus specific antibody is detected
usually by indirect technique
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2- Enzyme linked immunosorbent
assay (EIISA ) The indirect technique for detection
of antibodies is used
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The ELISA test in a microplate.
Microtitre plates of special quality and with flat bottomed wells are used.
The colour produced is the basis for interpretation of the reaction, which is
read by a photometer. The four rows on the left are the controls.
The ELISA test in a microplate.
Microtitre plates of special quality and with flat bottomed wells are used.
The colour produced is the basis for interpretation of the reaction, which is
read by a photometer. The four rows on the left are the controls.
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3-Heamagglutination inhibition test :
Many viruses haeagglutinate
erythrocytes but virus antibody
blocks this Antibodies can be detected in
patients serum by inhibition of virus
haemagglutination
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++
++
Haemagglutination testHaemagglutination test
Antigen
(virus)
Antigen
(virus)RBCsRBCs
HaemagglutinationHaemagglutination
Haemagglutination inhibition testHaemagglutination inhibition test
AntigenAntigen antibodyantibody
No Ag-Ab
complex
No Ag-Ab
complex
+ RBCs+ RBCs
+ve
Haemagglutination
+ve
Haemagglutination
No specific Abs
Negative test
No specific Abs
Negative test
Ag-AbcomplexAg-Abcomplex
+RBCs+RBCsNo Haemagglutination
specific Abs positive
test
No Haemagglutination
specific Abs positive
test
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4- Neutralization test
Antibody prevents virus infection of
cells , antibody can be detected by
neutralization of the biologic effect ofthe virus e.g CPE
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Immunoblotting
techniques These techniques recognize the
component of the antibody
response ;e.g western blot ( fordetection of HIV antibodies )
RIBA ( recombinant immunoblotassay ) for detection of HCV
antibodies )
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1- Separation of the viral lysate
antigens by electrophoresis through a
polyacrylamide gel (PAGE )
2-Transfer ( blotting ) of the
separated antigens from within thegel onto nitrocellulose paper that is
subsequently cut into strips
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3- Strips are used as solid phases
and incubated with the patient serum
i.e patient serum is allowed to react
with the nitrocellulose bound and
separated viral antigen
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GEL ELECTROPHORESIS
http://images.google.com/imgres?imgurl=http://oceanexplorer.noaa.gov/explorations/03bio/logs/sept10/media/lasonolide3_600.jpg&imgrefurl=http://oceanexplorer.noaa.gov/explorations/03bio/logs/sept10/media/lasonolide3.html&usg=__w2QroaydWhIo3FxAELYfa2lzf4Y=&h=450&w=600&sz=35&hl=en&start=9&tbnid=BAwN9-A1polORM:&tbnh=101&tbnw=135&prev=/images%3Fq%3DGEL%2BELECTROPHORESIS%26gbv%3D2%26hl%3Denhttp://images.google.com/imgres?imgurl=http://www.moleculardetective.org/TutorialProteomics/GelElectrophoresis02.JPG&imgrefurl=http://www.moleculardetective.org/TutorialProteomics/TutorialProteomicsPage6.html&usg=__eI7tHXmtMcAZQsKyGqR-2bCtWh8=&h=400&w=327&sz=31&hl=en&start=15&tbnid=p_Z8jTJ02K8woM:&tbnh=124&tbnw=101&prev=/images%3Fq%3DGEL%2BELECTROPHORESIS%26gbv%3D2%26hl%3Den -
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4- The indirect ELISA is performed
on the strips
These techniques owe their
specificity to component separation
and component concentration
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