LABORATORY DIAGNOSIS OF VIRUS

INFECTION
Laboratory technology is changing and
automated techniques are now replacing
traditional, but often labour intensive, methods.
Rapid methods: based on detection of virus
antigen or of virus IgM are now widely used to
give a faster turn-round of report to clinicians.
Virus diseases are diagnosed in the laboratory
by :
1. Serology, i.e. demonstration of virus antibody
(or antigen)
2. Isolation of virus
More rarely, virus particles can be demonstrated
in a specimen by electron microscopy.

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SEROLOGY
Now the principal diagnostic technique in virology.
DIAGNOSIS
Antibody is detected in patients serum by reaction with known virus
preparation: now increasingly used for antigen detection using
known antibody.
Assays
The reaction is detected by:
1. LABELLING one of the detecting reagents with:
i. Enzyme (enzyme-linked immunoassay or ELISA)
when tested with chromogenic substrate a
colour develops.
ii. Radioactive isotope (radioimmunoassay or RIA)
the labelled reagent is demonstrated in a radioactivity
counter or by exposure on X-ray film.
iii. Fluorescein : fluorescence is detected under UV light
microscopically.

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2. COMPLEMENT FIXATION
When antigen and antibody react
together, complement is fixed or
used up. Fixation is detected by
addition of red cells with anti-red cell
antibody: these lyse in the presence
of free or unfixed complement, i.e. a
negative test.

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ELISA
Now the most widely used serological test.
Used mainly for testing for antibodyIgM or IgG.
With some viruses, used for testing for antigen directly in
patiens sample.
The principles of an ELISA are illustrated diagrammatically in
under it.
ELISAs can be varied by :
i. Use of monoclonal virus antibodyto increase specificity.
ii. Use of class-specific antibody (e.g. anti IgG, anti IgM or anti
IgA) to detect one class of antibody in patiens serum.
iii. Competitive assay in which antibody is detected by
competing with known (labelled) antibody for combining sites
on the known virus preparation.

Diagram for ELISA test for virus IgM antibody

Virus

Patients serum

Add enzyme-labelled
anti-human IgM antiserum
Incubate
stop reaction

add substrate
Measure reaction by colour intensity
in optical density reader
Calculate as positive or negative
reaction by comparison with controls

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Enzyme substrates used in ELISAs

include:
i. Horseradish peroxidase and orthophenyldiamine.
ii. Alkaline phosphatase and pnitrophenyl phosphate.

Note: conjugation of antibody with

biotin and substrate with avidin or
streptavidin greatly increases
binding and so the sensitivity of the
assay.

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Quantitation:
One disadvantage is that antibody
cannot be titrated by ELISA, so the
traditional diagnostic criterion of fourfold rise in antibody titre (see below)
cannot be used: this is not necessary
in the case of IgM detection, whith by
itself proves recent infection

RADIOIMMUNOASSAY

Based on similar principles to ELISA, but

seldom used because of difficulty in handling
radioisotopes in diagnostic laboratories.
IMMUNOFLUORESCENCE
Antibody detection
Dilutions of patiens serum are added to spots
of virus infected cells on microscope slide.
Positive reaction is detected by addition of
fluorescein-labelled anti-human IgG (or antiIgM) with fluorescence seen under UV light.

Complement Fixation Test

Still used in must virus laboratories,
but being phased out and replaced
by more sensitive techniques like
ELISA that can be automated.
Note: positive reaction is indicated by
lack of lysis of the indicator system
of sheep red cells sensitized with
anti red cell antibody

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Other rarer tests for virus antibody include:

i.
Haemagglutination-inhibition test: Many viruses
haemagglutinate erythrocytes, but virus antibody
blocks this. Antibody can be detected in a patients
serum by inhibition of virus haemagglutination.
ii.
Radial immune haemolysis: A useful qualitative test for
antibody detectionbut not titrationwith
haemagglutinating viruses. Widely used as a screen
test for immunity to rubella. Virus and erythrocytes are
mixed in an agar gel in plate with added complement.
Patients sera are added to wells cut in the agar: if
antibody is present, zones of haemolysis appear round
the wells on incubation.
iii. Neutralization: Antibody prevents virus infection of
cells. Antibody can be detected by neutralization of
virus cytopathic effect (CPE) tissue culture. Timeconsuming and labour-intensive, so not widely used.
iv. Western blot: Patients serum is tested for reaction with
individual virus proteins separated by gel
electrophoresis on a strip of membrane.

Virus Isolation
Virus isolation requires the use of
cells since viruses cannot grow on
inanimate media. Now used for
diagnosis less than formerly. There
are three main systems:
1. Tissue culture
2. Chick embryo*
3. Laboratory animals*
Note : * now rarely used.

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1.

Tissue culture
Tissue culture is really cell culture in vitro and consists
of a single layer (monolayer) of actively metabolizing
cells adherentto glass or plastic surface in a test tube,
petri plate or on one side bottle.

2.

Chick embryo
Rarely used now for virus diagnosis. Useful for
preparation of bulk virus, e.g. for antigen or vaccine
production.

3.

Laboratory animals
Some viruses can only be isolated by inoculation of
laboratory animals, usually mice. After inoculation the
animals are observed for signs of disease or death. The
viruses are identified by testing for neutralization of
their pathogenicity for animals by standard antiviral
sera

Direct Demonstration Of Virus

A rapid method of virus diagnosis. Virus or virus antigen is
detected in lesions, fluids, tissues, or excretions from
the patient and a result can be obtained within an hour
or two of receipt of the specimen.
The main techniques are:
1. Serological: preferably with monoclonal antiviral
antibody. The most populer method is
immunofluorescence, but ELISA now also used for this,
especially useful for rapid diagnosis of respiratory virus
infection.
1.

Electron microscopy: virus particles are detected and

provisionally identified (but not serologically typed) on
the basis of morphology. Mainly used for detection of
the faecal viruses that cause gastroenteritis.

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Probes: Radiolabelled virus DNA sequences can

be used to detect virus genome or mRNA in
tissues and fluids by molecular hybridization.
4. Polymerase chain reaction (PCR) technique
provides a powerful tool for amplification of
virus nucliec acid in tissues, cells, body fluids:
an extremely sensitive technique with great
promise, although not yet much used in
diagnosis.
3.

Inclution bodies: are virus-induced masses seen in

the nucleus or cytoplasm of infected cells. With
a few exeptions, e.g. rabies too non-specific to
be useful in diagnosis.