(Tutorial-Voiced)Labatory Diagnosis of Viral Infections II

8/3/2019 (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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(Tutorial-Voiced)Labatory Diagnosis of Viral Infections II

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