Diagnostic Virology
Precise diagnosis requires the assistance of the
laboratory.
Animal host systems still have their uses in virology:
To produce viruses which cannot be propagated in
vitro (HBV).
To study the pathogenesis of and immunity to viral
infections.
To test vaccine safety.
Nevertheless, they are increasingly being discarded
because:
Breeding & maintenance of animals infected with viruses
is expensive
Whole animals are complex systems, in which it is
sometimes difficult to interpret
Results obtained are not always reproducible, due to
host variation
Unnecessary or wasteful use of experimental animals is
morally repugnant
They are rapidly being overtaken by cell culture &
molecular biology
Diagnostic Methods in Virology
1. Direct Examination
2. Indirect Examination (Virus Isolation)
3. Serology
Direct Examination
1. Antigen Detection: - immunofluorescence, ELISA.
2. Electron Microscopy: - morphology of virus particles
- immune electron microscopy
3. Light Microscopy: - histological appearance
- inclusion bodies
4. Viral Genome Detection: - hybridization specific nucleic
acid probes
- polymerase chain reaction
(PCR)
Indirect Examination
1. Cell Culture: - cytopathic effect (CPE)
- haemadsorption
- immunofluorescence
2. Eggs - pocks on CAM
- haemagglutination
- inclusion bodies
3. Animals - disease or death
Embryonated eggs were utilized and in the
1930’s the technique was adopted to isolate
conventional viruses.
Although use of cell culture in the diagnosis of
viral infections started in 1920, it was not until
1949 that this technology was established and
became routinely used for virus isolation.
The use of serologic techniques for virology
began somewhat later to be popular and widely
utilized.
Obtaining specimens for viral Diagnosis
Collecting and submitting proper clinical
specimens is critical for laboratory diagnosis.
Unless the clinician pays close attention to the
details of choice, timing, collection, and
handling, the laboratory can be of little help.
Timing of specimen collection is critical in acute
viral infections while the choice of specimens is
critical in both acute and subacute or chronic
viral infections.
The clinician should indicate to the laboratory which
particular virus or viruses are sought, and with what
urgency, since rapid methods usually require special
attention.
Selection of specimens requires knowledge of the
pathogenesis of the disease.
If a delay is expected, the specimen should be kept at
4◦c in a neutral pH unless a delay of more than 4 days
is anticipated.
If specimens are frozen, they should be held at - 70 ◦c.
Inoculation of Embryonated eggs
Prior to 1950, the standard host for virus isolation was
the embryonated hen’s egg.
Fertilized eggs are incubated for 5-14 days before
inoculation
After an additional 2-5 days of incubation, viruses are
recognized by certain criteria as
death of yolk sac
pocks (chorion)
hemagglutination and hemagglutination inhibition.
Cell culture Methods
Began with whole organs then progressed to methods
involving cells.
It is only since the advent of antibiotics that cell culture
became a matter of simple routine.
In 1949, Enders, Weller, and Rubin propagated
polioviruses in primary human cell cultures marking
the beginning of the “golden age of virology”.
Widespread virus isolation led to the realization that
subclinical viral infections were very common
Types of Cell Cultures
Primary cell cultures
HEK, GPE, CE, PMK.
Epithelial cells in origin.
Fresh tissues from fetuses.
Few (2-3) generations (passages).
Secondary (Diploid) cell cultures
HDF, HNF, MRC-5, WI –38.
Fibroblasts in origin.
40-50 passages
They retain the original diploid chromosome
number throughout.
Continuous cell lines
Cells of a single type capable of indefinite
propagation in vitro (immortal).
Decreased ability to support viral replication
after 100-120 passages.
Hep-2, A - 549, HeLa ( human), Vero
(monkey), MDCk (dogs), L929, 3T3 (mice),
BHK-21 (hamster), and RK –13 (rabbit).
Techniques
Flasks, roller tubes, microtiter trays, and shell vials
utilizing centrifugation techniques.
Preparation
Identification
CPE
EM.
Immunologic methods ( IF, HA, HA
inhibition, hemadsorption and its inhibition,
CF, and neutralization).
Cytopathic Effect (1)
Cytopathic Effect (2)
Syncytium formation in cell culture caused by RSV (left), and
measles virus (right).
Haemadsorption
Syncytia formation caused by mumps virus and haem
-adsorption of erythrocytes onto the surface of the cell sheet.
Problems with cell culture
Long period (up to 4 weeks) required for result.
Often very poor sensitivity, sensitivity depends to a large
extent on the condition of the specimen.
Susceptible to bacterial contamination.
Susceptible to toxic substances which may be present in
the specimen.
Many viruses will not grow in cell culture e.g. Hepatitis
B, Diarrhoeal viruses, parvovirus, papillomavirus.
Rapid Culture Techniques
Rapid culture techniques are available whereby viral antigens
are detected 2 to 4 days after inoculation. The CMV IF test is
the best example, whereby
The cell sheet is grown on individual cover slips in a
plastic bottle.
Following inoculation, the bottle then is spun at a low
speed for one hour (to speed up the adsorption of the virus)
and then incubated for 2 to 4 days.
The cover slip is then taken out and examined for the
presence of CMV early antigens by immunofluorescence.
Immunofluorescense
Rapid Methods
Microscopy (Light, EM, IEM).
Antigen detection (IF, Agglutination, EIA).
Serological methods (CF, IF, RIA, EIA, etc).
Molecular methods
In situ hybridization
PCR
Electron Microscopy
106 virus particles per ml required for visualization, 50,000 -
60,000 magnification normally used. Viruses may be detected
in the following specimens.
Feces Rotavirus, Adenovirus
Noroviruses
Astrovirus, Calicivirus
Vesicle Fluid HSV
VZV
Skin scrapings papillomavirus, orf
molluscum contagiosum
Electronmicrographs
Adenovirus Rotavirus
Molecular Methods
Methods based on the detection of viral genome. It is
often said that molecular methods is the future direction
of viral diagnosis.
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.
It is certain though that the role of molecular methods
will increase rapidly in the near future.
Schematic of PCR
Serology
Criteria for diagnosing -Primary Infection
4 fold or more increase in titer of IgG or total antibody
between acute and convalescent sera
Presence of IgM
Seroconversion
A single high titer of IgG (or total antibody) - very
unreliable
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
Typical Serological Profile After Acute Infection
Usefulness of Serological Results
How useful a serological result is dependent on the
individual virus.
For viruses such as rubella and hepatitis A, the
onset of clinical symptoms coincide with the
development of antibodies.
The detection of IgM or rising titers of IgG in the
serum of the patient would indicate active disease.
Many viruses often produce clinical disease before
the appearance of antibodies such as respiratory and
diarrheal viruses.
So in this case, any serological diagnosis would be
retrospective and therefore will not be that useful.
There are also viruses which produce clinical
disease months or years after seroconversion e.g.
HIV and rabies.
In the case of these viruses, the mere presence of
antibody is sufficient to make a definitive diagnosis.
Problems with Serology
Long period of time required for diagnosis for paired
acute and convalescent sera.
Mild local infections such as HSV genitalis may not
produce a detectable humoral immune response.
Extensive antigenic cross-reactivity between related
viruses e.g. HSV and VZV, Japanese B encephalitis
and Dengue, may lead to false positive results.
immunocompromised patients often give a reduced
or absent humoral immune response.
Patients with infectious mononucleosis and those
with connective tissue diseases such as SLE may
react non-specifically giving a false positive result.
Patients given blood or blood products may give a
false positive result due to the transfer of antibody.
CSF antibodies
Used mainly for the diagnosis of herpes simplex and VZV
encephalitis
CSF normally contain little or no antibodies
Presence of antibodies suggests meningitis or
meningoencephalitis
CSF antibody titer > _1_ is indicative of meningitis
Serum antibody titer 100
Diagnosis depends on the presence of an intact
blood-brain barrier
The diagnosis of viral infections: A summary
visualize the virus
insensitive and laborious
culture the virus
viruses need living cells (animal, egg, culture)
time consuming
a large proportion of clinically relevant viruses
cannot be grown in vitro
detect antibodies against the virus
indirect approach
dependent on host immunity
find the viral nucleic acid
universally applicable
revolutionary technical developments
