LABORATORY

DIAGNOSIS OF
VIRAL
INFECTIONS
DIAGNOSTIC METHODS IN VIROLOGY

1. Direct Examination

2. Indirect Examination (Virus Isolation)

3. Serology
1. DIRECT EXAMINATION

1.1. Light microscopy histological appearance - e.g. inclusion

bodies
1.2. Electron Microscopy morphology / immune electron
microscopy
1.3. Antigen detection immunofluorescence, ELISA etc.
1.4. Molecular techniques for the direct detection of viral
genomes
2. INDIRECT EXAMINATION

2.1. Cell Culture - cytopathic effect, haemadsorption,

confirmation by neutralization,
interference, immunofluorescence etc.
2.2. Eggs pocks on CAM - haemagglutination, inclusion bodies
2.3. Animals disease or death confirmation by neutralization
3. SEROLOGY

Detection of rising titres of antibody between acute and

convalescent stages of infection, or the detection of IgM in
primary infection.

Classical Techniques Newer Techniques

1. Complement fixation tests (CFT) 1. Radioimmunoassay (RIA)

2. Haemagglutination inhibition tests 2. Enzyme linked immunosorbent assay (EIA)
3. Immunofluorescence techniques (IF) 3. Particle agglutination
4. Neutralization tests 4. Western Blot (WB)
5. Counter-immunoelectrophoresis 5. RIBA, Line immunoassay
 SPECIMEN COLLECTION

•Most viruses enter the body through the respiratory or GI tract, so the
most obvious & appropriate specimen is respiratory tract secretions or
the GI tract secretions.

•In case of internal organ involvement where direct organ specimen is

difficult to obtain, sampling from multiple sites seems useful. Eg., in
case of measles, Skin is most dramatically involved. Yet measles virus
may be isolated from respiratory tract or from urine.

•Similarly Central Nervous System & Cardio Vascular System are

commonly affected in serious viral infections, but the virus may be
isolated from the upper respiratory tract or Gastro Intestinal Tract.

•The optimal specimens for viral culture are aspirates of fluids, exudates
or secretion, tissues, washings of upper air ways or stool specimens,
swab specimen are acceptable in most situations. Nasopharyngeal
washings are generally sufficient for respiratory viruses.
•Blood may be useful for entero viral infections in young children
& infants.
•To culture vesicular skin lesions the skin should be cleaned with
an alcohol swab and allowed to dry for at least one minute.
•The vesicle should then be unrolled with a sterile scalpel.
 TRANSPORTATION &
STORAGE OF SPECIMEN

Transport medium

•All the samples should be transported to laboratory in a sterile,

leak proof container.
•Interval between collection of the specimen & its inoculation
should be minimal.
•In case of some fragile viruses for eg RSV virus inoculation of cell
cultures at the bed side has been recommended.
•Swabs should be placed in viral transport media swabs, any swabs
are not acceptable.
•Specimens are refrigerated till they are further processed. The
specimens should be maintained at -70°C if they are to be stored for
a very long time (weeks or months). When the delay is short (less
than 24 hours) specimens are stored at 4°C temperature.
LABORATORY DIAGNOSIS
1. Direct Examination
•Direct examination methods are often also called rapid diagnostic
methods because they can usually give a result either within the same
or the next day.
•This is extremely useful in cases when the clinical management of
the patient depends greatly on the rapid availability of laboratory
results e.g. diagnosis of RSV infection in neonates, or severe CMV
infections in immunocompromised patients.
•However, it is important to realize that not all direct examination
methods are rapid, and conversely, virus isolation and serological
methods may sometimes give a rapid result.
•With the advent of effective antiviral chemotherapy, rapid diagnostic
methods are expected to play an increasingly important role in the
diagnosis of viral infections.
1.1. Light Microscopy
•Light Microscopy has been traditionally used in directly
demonstrating viral infections by detecting the viral inclusion body in
smear & tissue.
•Inclusion bodies are dense aggregates of stainable substances,
usually proteins.
•They can be either intra nuclear (present inside the nucleus) of
infected well or intracytoplasmic (present inside the cytoplasm of the
infected cell).
•Viruses that are assembled in the nucleus (usually DNA viruses) like
herpes simplex viruses, Varicella zoster virus, Cytomegalo virus,
adeno virus & papova viruses forms intranucleus inclusions.
•While viruses that are assembled in the cytoplasm (usually RNA
viruses) like Respiratory syncytial virus, rabies virus & viruses of the
pox group forms intra cytoplasmic inclusion bodies.
•The intracytoplasmic lesions of rabies virus are known as Negri
bodies, intracytoplasmic inclusions of the pox viruses known as
Guarneri bodies.
 Inclusion bodies
Negri bodies

Inclusion bodies
 1.2. ELECTRON MICROSCOPY
•Viruses are very small and most of them can be seen only by TEM
(transmission electron microscopy).
•TEM has therefore made a major contribution to virology, including
the discovery of many viruses, the diagnosis of various viral infections
and fundamental investigations of virus-host cell interactions.
• However, TEM has gradually been replaced by more sensitive
methods, such as the PCR.
•In research, new imaging techniques for fluorescence light microscopy
have supplanted TEM, making it possible to study live cells and
dynamic interactions between viruses and the cellular machinery.
• Nevertheless, TEM remains essential for certain aspects of virology.

• It is very useful for the initial identification of unknown viral agents in

particular outbreaks, and is recommended by regulatory agencies for
investigation of the viral safety of biological products and/or the cells
used to produce them.
•In research, only TEM has a resolution sufficiently high for
discrimination between aggregated viral proteins and structured viral
particles.
•EM is now mainly used for the diagnosis of viral gastroenteritis by
detecting viruses in faeces e.g. rotavirus, adenovirus, astrovirus,
calicivirus and Norwalk-like viruses.
•Occasionally it may be used for the detection of viruses in vesicles
and other skin lesions, such as herpesviruses and papillomaviruses.
•The sensitivity and specificity of EM may be enhanced by immune
electron microscopy, whereby virus specific antibody is used to
agglutinate virus particles together and thus making them easier to
recognize, or to capture virus particles onto the EM grid.
•The main problem with EM is the expense involved in purchasing
and maintaining the facility.
•In addition, the sensitivity of EM is often poor, with at least 105 to
106 virus particles per ml in the sample required for visualisation.
•Therefore the observer must be highly skilled. With the availability of
reliable antigen detection and molecular methods for the detection of
viruses associated with viral gastroenteritis, EM is becoming less and
less widely used.
•106 virus particles per ml required for visualization.

Faeces Rotavirus, Adenovirus

Norwalk like viruses
Astrovirus, Calicivirus

Vesicle Fluid Herpes Simplex Virus

Varicella Zooster Virus

Skin scrapings papillomavirus, ORF (a type of Pox virus,

sore mouth infection)
molluscum contagiosum
 Adenovirus Rotavirus
(courtesy of Linda Stannard, University of Cape Town, S.A.)

The Semliki forest virus, induces the formation of a

Budding of HIV cytopathic vacuole (CPV), surrounded by the ER
 PROBLEMS WITH ELECTRON MICROSCOPY

• Expensive equipment

• Expensive maintenance

• Require experienced observer

• Sensitivity often low

 IMMUNE ELECTRON MICROSCOPY
•IEM is used to localize molecules at the ultrastructural level by
labeling them with specific antibodies, which are visualized by
electron-opaque markers (colloidal gold particles) attached to them.
• The effect is to produce an electron-dense label at the site of the
antigen-antibody reaction.
•When the antigen is located inside of the cell, then transmission
electron microscopy is required to see it.
•The labeling can be done pre-embedding or post-embedding.
•When the antigen in question is located on the surface of the
specimen, scanning electron microscopy can be used to see it.
•The electron dense label can then be viewed using the back scatter
image on an appropriately equipped microscope.
1.3. Antigen Detection
•Examples of antigen detection include immunofluorescence testing
of nasopharyngeal aspirates for respiratory viruses e.g.. RSV, flu A, flu
B, and adenoviruses, detection of rotavirus antigen in faeces, the
pp65 CMV antigenaemia test, the detection of HSV and VZV in skin
scrappings, and the detection of HBsAg in serum.
•(However, the latter is usually considered as a serological test).
•The main advantage of these assays is that they are rapid to perform
with the result being available within a few hours.
•However, the technique is often tedious and time consuming, the
result difficult to read and interpret, and the sensitivity and
specificity poor.
•The quality of the specimen obtained is of utmost importance in
order for the test to work properly.
Immunofluorescense Assay
An-Ab rx. on glass slide (Indirect Fluorescence Antibody Test)

Fluorescence microscope
The working model of florescence microscope
 Positive immunofluorescence
test for rabies virus antigen.
(Source: CDC)
CMV pp65 antigens
detected in nuclei of
peripheral blood
neutrophils

HSV-infected epithelial cell

from skin lesion (DFA)
 1.4. Molecular techniques
•Methods based on the detection of viral genome are also commonly
known as molecular methods.
•However in practice, although the use of these methods is indeed
increasing, the role played by molecular methods in a routine diagnostic
virus laboratory is still small compared to conventional methods.
•Classical molecular techniques such as dot-blot and Southern-blot
depend on the use of specific DNA/RNA probes for hybridization. The
specificity of the reaction depends on the conditions used for
hybridization.
•These techniques may allow for the quantification of DNA/RNA present
in the specimen.
•However, it is often found that the sensitivity of these techniques is not
better than conventional viral diagnostic methods.
•Newer molecular techniques such as the polymerase chain reaction
(PCR), ligase chain reaction (LCR), nucleic acid based amplification
(NASBA), and branched DNA (bDNA) depend on some form of
amplification, either the target nucleic acid, or the signal itself.
Southern Blotting
New molecular methods
1. Polymerase chain reaction
2. Real time polymerase chain reaction
3. Nucleic acid sequence based amplification
(NASBA)
4. Transcription mediated amplification
5. Genome sequencing (2 techniques)
Polymerase Chain Reaction (PCR)
Real-Time PCR

• Also called quantitative polymerase chain reaction (qPCR), is one of the

most powerful and sensitive gene analysis techniques available and is
used for a broad range of applications including quantitative gene
expression analysis, genotyping, SNP analysis, pathogen detection, drug
target validation and for measuring RNA interference.
• In a Real–Time PCR, a basic PCR mix is first made and then fluorescent
labels are added.
•A light source in the Real-Time PCR instrument excites the fluorescence.
•A camera captures the fluorescent signals.
•As amplification proceeds, the fluorescence accumulation is captured by
the instrument after every cycle, and is translated into a Real-Time PCR
graph .
•SYBR green is a dye that fluoresces only when bound to double stranded
DNA (i.e the PCR product)
•Taqman probes are made of a gene-specific nucleic acid probe, joined to
reporter and quencher molecules.
•Taqman probes are made of a gene-specific nucleic acid probe, joined
to reporter and quencher molecules.
•The probe binds to the DNA between the forward and reverse primer.
While the reporter and quencher are bound to the probe, the quencher
absorbs the fluorescence emitted by the reporter.
• During the extension phase of the PCR reaction the probe is degraded,
releasing the reporter and allowing its fluorescence to be detected.
•The advantage of the Taqman method is that probes with different
coloured reporters can be combined in multiplex assays.
•Several different fluorophores (e.g. 6-carboxyfluorescein,
acronym: FAM, or tetrachlorofluorescein, acronym: TET)
and quenchers (e.g. tetramethylrhodamine, acronym: TAMRA) are
available.
TaqMan assay SYBR green assay
 2. INDIRECT EXAMINATION (VIRUS ISOLATION)
The method used for isolation of virus depend upon the virus sought. In
general they consist of innoculation into animals, eggs or tissue culture.
After that isolates are identified by neutralization or other suitable
serological process.

2.1. Cell Culture - cytopathic effect, haemadsorption,

confirmation by neutralization,
interference, immunofluorescence etc.
2.2. Eggs pocks on CAM - haemagglutination, inclusion bodies
2.3. Animals disease or death confirmation by neutralization
2.1.Egg inoculation
•Embryonated eggs are among the most useful form of living animal
tissue for the isolation of viruses, for titrating viruses & for large
quantity cultivation in the production of viral vaccines.
•The embryonated egg has various sites namely chorioallantois
membrance, Allantoic membrane, Amniotic sac, Yolk sac & the
embryo proper.
Animal Inoculation

•It is the earliest method for the cultivation of viruses causing human
diseases.
•Reed & colleagues F in 1900 used human volunteers for their work
on yellow fever. In 1909, Landsteiner & Popper used monkeys to
isolate Polio virus.
•Rabbit, Guinea pig, mouse, rat are now commonly used in virus
cultivation.
•It needs to be noted that different animals are used for different
viruses & also there exists different routes to inoculation.
•The growth of virus in inoculated animals may be indicated by death,
disease or visible lesions.
•Disadvantages of animal inoculation are that they may interfere with
viral growth and that animals often harbour latent viruses.
Tissue culture
Tissue culture is often a generic term. Three types of tissue cultures
are available:
a. Organ culture
Small bits of organs are maintained in vitro for days & weeks. They
are used for cultivation of viruses which can be grown only in
specific organ.
b. Explants culture
Fragments of minced tissues is grown as explants embedded in
plasma clot. Eg. Adeno tissue explants for adenovirus.
c. Cell culture
It is routinely used based on their origin. Chromosomal characters &
the number of generation through which they can be maintained,
cell cultures are classified into 3 types
1. Primary cell culture
It consists of normal cells freshly taken from body & cultured. They are
capable of limited growth in culture & cannot be maintained in serial
culture. Eg. Monkey, kidney, human embryonic kidney, human amnion
& chick embryo cell culture. They are useful for isolation of viruses &
for vaccine production.

2. Diploid cell culture

These are used to ensure a continuous supply of cell line. After 50 serial
passages, they undergo senescence. They are useful in viral vaccine
production. Eg. Human fibroblasts

3. Continuous cell culture

These are single type usually derived from cancer cells that are capable
of continous serial cultivation indefinitely. These cells lines many be
maintained by serial sub-cultivation or stored in the cold (-70o C) for
use when necessary. They are useful for vaccine production. Eg. Verocell
for rabies vaccine, Hela, HEp-2, KB
Identification of growing virus
The growth of virus in the cell culture can be detected by
following methods
i. Cytopathic effect
ii. Metabolic inhibitors
iii. Hemadsorption
iv. Interference
v. Transformation
vi. Immunofluorescence
CYTOPATHIC EFFECT

Syncytium formation in cell

culture caused by RSV (top), and
measles virus (bottom).
(courtesy of Linda Stannard, University of Cape
Town, S.A.)
 HAEMADSORPTION

Syncytial formation caused by mumps virus and haemadsorption

of erythrocytes onto the surface of the cell sheet.
(courtesy of Linda Stannard, University of Cape Town, S.A.)
 3. SEROLOGICAL DIAGNOSIS
•It refers to diagnosis of disease based on reactions in the blood
serum.
•The mere presence of an antibody to a virus is not sufficient to
diagnose a viral infection.
•It only denotes that the person’s immune system has been exposed
to the viral antigen, besides a current infection by the virus.
•Variety of methods is available to diagnose viral infection by
antibody detection the choice of the method depends upon the
sensitive of the test and intensity of immune response.
•Various tests available are ELISA, Complement fixation test,
neutralization, hemagglutination tests.
 TYPES OF ELISA
 Direct ELISA
 Indirect ELISA

 Sandwich ELISA
INSTRUMENTS REQUIRED

ELISA READER
 DIRECT ELISA

INDIRECT ELISA
SANDWICH ELISA
Hemagglutination Inhibition test
The HI test involves three main components: antibodies, influenza
virus, and red blood cells that are mixed together in the wells (i.e.,
cups) of a microtiter plate