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Lecture 6

DIAGNOSTIC & CULTIVATION

of VIRUSES1

A. Laboratory viral diagnosis involves one of three

basic approaches:

o antigens in clinical specimens

o serologic testing of viral specific antibodies.

o virus isolation; direct demonstration of virus, viral

nucleic acid,

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Laboratory Diagnostic Methods in Virology

1) Electron microscope

2) Virus isolation (Tissue culture cells, embryonated eggs,

or animal hosts)

3) Direct examination of clinical specimens

4) Antigen / serologic testing of viral specific antibodies

5) PCR (direct demonstration of virus, viral nucleic acid)

Because the clinical symptoms of a virus are often

distinctive, lab diagnosis is frequently not necessary.

If done, it begins with identification of the most likely

viruses based on clinical symptoms and the patient’s

history.

It is often not possible during the first few days after

infection.

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Electronmicrographs

Adenovirus Rotavirus

1- Electron Microscopy

The sensitivity and specificity of EM may be enhanced by immune electron microscopy

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Problems with Electron Microscopy

Expensive equipment & maintenance

Require experienced observer

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Virus isolation This technique identifies virus replication in susceptible cells.

(Tissue culture cells, embryonated eggs, or animal hosts are oftenused).

In live infected tissue culture cells, replication may be detectedby observing a characteristic cytopathogenic effect (CPE) suchas polykaryocyte formation or hemadsorption (adhesion of redblood cells to infected cells).

In fixed infected tissue culture cells, replication may be detectedby observing characteristic inclusion bodies or performingimmunohistochemical staining of viral antigens.

In embryonated egg, replication is detected by pock formation;in animals, by the development of clinical symptoms.

Proper collection and preservation of specimens are necessaryfor virus isolation.

It is best accomplished during the onset and acute phase ofdisease.

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2- Cell culture

Cell Cultures are most widely used for virus isolation,

there are 3 types of cell cultures:

1. Primary cells - Monkey Kidney

2. Semi-continuous cells - Human embryonic kidney and skin, fibroblasts

3. Continuous cells - HeLa, Vero, Hep2, LLC-MK2, MDCK

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Tissue culture: Tissue culture of human or animal cells are frequently used for

the cultivation of viruses. There are mainly three types of

tissue culture:

1. Organ culture, e.g. tracheal ring organ culture is employedfor the isolation of coronavirus.

2. Explant culture: Minced tissue may be grown as explantembedded in plasma clots. This is not useful in virology. In thepast adenoid tissue explant culture were used for adenovirus.

3. Cell culture: This is very popular and useful techniqueroutinely used for cultivation of viruses. From tissue, fragmentscells are dispersed by proteolytic enzymes like trypsin andmechanical shake. After washing the cells, they aresuspended in growth medium and distributed in petridishes,test tubes or bottles.

The cells adhere to glass surface and grow out to form amonolayer sheet and can be seen in situ under low power.

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Cell Cultures

Growing virus may produce

1. Cytopathic Effect (CPE) - such as the ballooning of cells

or syncytia formation, degradation, necrosis, inclusion

bodies (intranuclear or intracytoplasm or both),

transformation (oncogenic).

- may be specific or non-specific.

2. Haemadsorption - cells acquire the ability to stick to

mammalian red blood cells.

Confirmation of the identity of the virus may be carried out using

neutralization, haemadsorption-inhibition or immunofluorescence

tests.

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Cytopathic Effect

Syncytium formation in cell

culture caused by RSV

(Respiratory syncytial virus

Syncytium formation

in cell culture caused

by measles

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Haemadsorption When hemagglutinating viruses grow in cell cultures, their presence

can be indicated by the addition of guinea pig erythrocytes to the

cultures.

If the viruses are multiplying in the cultures, the erythrocytes will

adsorb onto the surface of cells. This is known as ‘hemadsorption’.

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Problems with cell culture

Long period (up to 4 weeks) required for result.

Susceptible to bacterial contamination.

Many viruses will not grow in cell culture e.g.

Hepatitis B, Diarrhoeal viruses, parvovirus,

papillomavirus.

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3. Direct examination of clinical specimens

General characteristics

Specimens may include sections of tissue biopsies,

tissue imprints or smears, blood, cerebrospinal fluid,

urine, throat swabs, feces, or saliva.

Only those specimens likely to contain the virus (e.g.,

throat swabs for respiratory tract infection) should be

examined.

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4- Antigen / Antibody tests

Antibodies

(also known as immunoglobulins abbreviated Ig) are gamma

globulin proteins that are found in blood and are used by the

immune system to identify and neutralize foreign objects, such

as bacteria and viruses.

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Antigen / Antibody tests

Antigens

A substance that when introduced into the body

stimulates the production of an antibody

Immunoassay

A laboratory technique that makes use of the binding

between an antigen and its homologous antibody in

order to identify and quantify the specific antigen or

antibody in a sample

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Serologic tests1. General characteristics

Serologic tests are used to determine the titer of specific antiviral antibodies.

Paired blood samples are taken (one sample at the onset and one sample during the recovery phase of the illness); at least a fourfold increase in titer between the samples must be present to indicate a current infection.

The test may be diagnostic without the use of paired samples if significant levels of IgM antiviral antibodies are obtained.

Techniques include virus neutralization, complement fixation, hemagglutination inhibition tests, and solid-phase immunoassays.

These tests are expensive to perform and must be standardized for each virus.

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Antigen / Antibody tests

Viral Serology

Criteria for diagnosing Primary Infection

Presence of IgM

Criteria for diagnosing Reinfection

4 fold or more increase in titer of IgG or total antibody between

acute and convalescent sera

Absence or slight increase in IgM

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Western Blot

Western blotting, also known as immunoblotting or

protein blotting, is a technique used to detect the

presence of a specific protein in a complex protein

mixture

It is a core technique in cell biology, molecular biology,

virology and others

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Western Blot

HIV-1 Western Blot

Lane1: Positive Control

Lane 2: Negative Control

Sample A: Negative

Sample B: Indeterminate

Sample C: Positive

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ELISA

Is a biochemical technique used mainly in immunology

to detect the presence of an antibody or an antigen in a

sample.

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Direct ELISA 23

Sandwich ELISA

The ELISA plate is coated with Antibody to detect specific antigen

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Sandwich ELISA25

Indirect ELISA 26

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ELISA :SUMMARY27

Immunofluorescense

Immunofluorescence is a serological test where the labeling of antibodies or antigens is done with fluorescent dyes (fluorochromes).

Fluorochromes are dyes which have the ability to absorb the short wavelength UV radiation and emit light of longer wavelength fluorescence (visible green light).

Examples : FITC, Rhodamine , Acridine orange

There are two ways of doing IF staining

Direct immunofluorescence

Indirect immunofluorescence

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Direct immunofluorescenceUse: Direct detection of Pathogens or their Ag’s in tissues or in pathological

samples

Ag is fixed on the slide

Fluorescein labeled Ab’s are layered over it

Slide is washed to remove unattached Ab’s

Examined under UV light in an fluorescent microscope

The site where the Ab attaches to its specific Ag will show apple green fluorescence

Indirect immunofluorescence Use: Indirect detection of antibodies against viruses in serum

Indirect test is a double-layer technique

The unlabelled antibody is applied directly to the tissue substrate

Treated with a fluorochrome-conjugated anti-immunoglobulin serum

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5- Polymerase Chain Reaction

PCR allows the in vitro amplification of specific target DNA

sequences by a factor of 106 and is thus an extremely sensitive

technique.

Detection and identification of the PCR product is usually carried

out by agarose gel electrophoresis, hybridization with a specific

oligonucleotide probe, restriction enzyme analysis, or DNA

sequencing.

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What is qPCR

“quantitative Polymerase Chain Reaction”

A method that allows to follow in real time (that is why is also called

Real-Time PCR) the amplification of a target.

The target can be nucleic acids (RNA or DNA).

Taq polymerase can only synthesize DNA, so how do we study RNA

using qPCR?

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Reverse Transcription

mRNA can be copied to

complementary DNA sequence

(cDNA) using reverse

transcriptase—a DNA polymerase

that uses ssRNA as template.

Processed mRNA will match

protein coding sequence while

unprocessed (nuclear) mRNA will

contain intron sequences.

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Uses of qPCR

Precise quantitation of DNA or RNA in samples

Estimation of gene number

Gene expression studies by quantification of messenger RNA

Mnay technqiues are used for qPCR :

1- SYBRGreen

2- Taqman

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SYBRGreen36

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TaqMan (quencher & reporter)37

CULTIVATION of VIRUSES

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Cultivation of Virus

Since they are obligate intracellular parasites

and cannot grow on inanimate culture

medium, 3 methods are used for their

cultivation:

a. Animals inoculation.

b. Chick embryo.

c. Tissue culture.

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Animal inoculation: It is one of the oldest methods for the cultivation of viruses.

The poliomyelitis virus after intraspinal or intracerebralinoculation in monkeys causes typical paralytic disease andso isolation of viruses.

Suckling mice is susceptible to Cox-sackie viruses withmanifestation of severe myositis and paralysis.

Smallpox virus may be inoculated in the scarfied skin orcornea of rabbit. Brain tissue of rabied dog when inoculatedintracerebrally in mice or rabbit develop encephalitis.

Growth of virus in animals may be known by the disease,visible classical lesions or death.

Sometimes immunity in experimental animal may interferewith the growth of viruses in that animal.

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Chick embryo: They are better than animal inoculation because of following

reasons:

I. They are clean and bacteriologically sterile.

II. They do not have immune mechanism like animals to counteractvirus infection.

III. They do not need feeding and caging.

IV. Chick embryo offers several sites for cultivation of viruses, i.e.

- chorioallantoic membrane (CAM) for variola or vaccinia andherpes viruses,

- allantoic cavity provides rich yield of influenza and someparamyxoviruses, also Allantoic inoculation may be used forgrowing influenza virus for vaccine purposes. Yellow fever

- amniotic sac may be used for the isolation of influenza virus andyolk sac for the cultivation of chlamydiae, rickettsiae and someviruses. and rabies are other vaccines produced from chickembryo.

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Fig. 45.3: Structure of chick embryo

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Disadvantages of Egg Inoculation

1. Eggs may be contaminated with mycoplasma

and latent fowl viruses which may interfere with the

growth of other viruses.

2. The susceptibility of chick embryo is limited to a

few viruses only.

3. Even slight amount of bacterial contamination in

the inoculum may kill the embryo.

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Tissue culture: Tissue culture of human or animal cells are frequently used

for the cultivation of viruses. There are mainly three types of

tissue culture:

1. Organ culture, e.g. tracheal ring organ culture isemployed for the isolation of coronavirus.

2. Explant culture: Minced tissue may be grown as explantembedded in plasma clots. This is not useful in virology. Inthe past adenoid tissue explant culture were used foradenovirus.

3. Cell culture: This is very popular and useful techniqueroutinely used for cultivation of viruses. From tissue,fragments cells are dispersed by proteolytic enzymes liketrypsin and mechanical shake. After washing the cells,they are suspended in growth medium and distributed inpetridishes, test tubes or bottles.

The cells adhere to glass surface and grow out to form amonolayer sheet and can be seen in situ under low power.

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