MICROSCOPIC

IDENTIFICATION
• Viruses can be detected & identified by direct microscopic examination of clinical
specimens. Three different procedures can be used.

1. Light microscopy can reveal characteristic inclusion bodies or multinucleated giant

cells.

2. fluorescent microscopy is used for detected of the virus fluorescent antibody

Assays.

3. Electron microscopy detects virus particles, which can be characterized by their

size and morphology.
 Light Microscopy

• The size of most viruses is beyond the resolution limits of light

microscopes.

• However, light microscopy is useful for detecting of virus-infected cells

• For example by observing cytopathic effects or by detecting a fluorescent

dye linked to antibody molecules that have bound to a virus antigen
(fluorescence microscopy).
 HISTOLOGY/CYTOLOGY
• Direct microscopic examination of stained histology or cytology specimens can
on occasion provide the first indication that a virus may be responsible for a
pathological process, for example the intranuclear (early) or basophilic (late)
inclusions seen in interstitial nephritis in renal transplant biopsies due to BK
virus, changes in cervical cytology seen in association with human papilloma
virus (HPV) and the nuclear inclusions seen in erythroid precursor cells in
Parvovirus B19 infection.
Characteristic changes in the appearance of the
cells resulting from virus-induced damage. A
change of this type is known as a cytopathic
effect (CPE); examples of CPEs induced by
poliovirus and herpes simplex virus can be
seen in Fig. The poliovirus-infected cells have
shrunk and become rounded, while a
multinucleated giant cell known as a syncytium
(plural syncytia) has been formed by the fusion
of membranes of HSV-infected cells.
Fig. 48.1a Uninfected Graham 293 cells.
Fig. 48.1b Graham 293 cells showing adenovirus cytopathic effect.
 FLUORESCENT Microscopy

Fluorescent Antibody Assay

Immunofluorescence tests (IF or IFT)

FLUORESCENCE MICROSCOPY
 Fluorescent Antibody Assay
Immunofluorescence tests (IF or IFT)

• IFT can detect either viral antibody or antigen in the patient

specimen.

• Fluorescein-labelled antibody is used which fluoresce apple-green

under a Fluorescent microscope.
• To look for viral antigen, cells from the patient’s secretions (e.g.
nasopharyngeal aspirate) are fixed on a glass slide

• fluorescein-labelled monoclonal antibody against the virus (RSV,

influenza A etc.) is added.

• A mixture of these monoclonal antibodies can be added at the same

time to detect a panel of viruses (e.g. respiratory viruses, all at one
go).
 Direct Immunofluorescence Assay
• Viruses growing in cell culture can be identified by direct or indirect IFT by

virus-specific antibodies.

• In direct IFT, specific antibodies labelled with fluorescein isothiocyanate

(FITC) dye are reacted with virus-infected cells fixed on microscope slides.

• Specific antigen-antibody interactions are detected using fluorescence

microscope.
 Indirect Immunofluorescence Assay
• In indirect immunofluorescence, the specific antibody is
unlabeled.

• Binding of specific antibody to antigen is detected after a

second incubation with a secondary antibody labeled with FITC.
 Virus-infected cells detected using a virus-
specific antibody labelled with a fluorescein dye
• IFT is rapid serological test, but labor intensive to carry out

and requires subjective interpretation dependening upon

operator expertise.
electron Microscopy
• Viruses are below the resolution of light microscopy and therefore require an electron
microscope for visualization.

• A limiting factor of electron microscopy (EM) is that viruses belonging to the same family
can not be distinguished from each other as they will have the same morphology (size,
shape and surface characteristics).

• Therefore EM can not be used to make the differential diagnosis of a herpes simplex or
chickenpox lesion, as both will contain a ‘herpes’ virus with exactly the same morphology
(Fig. 48.2).
• On the other hand EM is a useful tool in making a differential
diagnosis of viral gastroenteritis, as rotavirus, norovirus and enteric
adenoviruses all belong to a different family and can be distinguished
from each other on the basis of their morphology.
• It is a ‘catch all’ technique and many viruses (rotavirus, norovirus)
were discovered by EM.
 ELECTRON MICROSCOPY
• Electron microscopy is the only technique available for direct
visualization of viruses, and therefore has many applications
beyond the diagnostic range.

• With the advent of alternative diagnostic methods, EM retains a

limited role in for the diagnosis of viral gastroenteritis and
examination of skin lesions for herpes and pox viruses.
• Preparation of specimens for EM and the technique of negative staining
are straight forward and quick, and the method is a ‘catch-all’ approach
to detecting viruses.

• However, it has a limit of sensitivity of approximately 106 viral particles

per millilitre of fluid, making negative results unreliable.
• Vast numbers of virions are present during acute skin and gastrointestinal
disease and a diagnosis is easily made, but later in the course of infection
viral shedding is reduced below the level of detection.

• Although sensitivity can be enhanced by antibody-induced clumping of

virus (immune EM) or ultracentrifugation, it is unrealistic to undertake
these methods routinely. The advantages and disadvantages of EM are
summarized in Table 1.1.
• The survival of EM within the routine clinical virology laboratory hinges on the
emergence of alternative, more sensitive methods of diagnosis.

• Many centres now use latex agglutination for rotavirus diagnosis, and PCR is
more sensitive than EM for detection of herpesviruses in vesicular fluid
(Beards et al., 1998) and for the detection of noroviruses (previously called
Norwalk-like viruses ) (O’Neill et al., 2001).

• Thus, the future of EM in clinical virology is in some doubt. However, one of

the first indications for EM was for the rapid diagnosis of smallpox and, in the
era of bioterrorism, EM may continue to play a role in specialist centres in the
event of a bioterrorist attack.
 Immunoelectron Microscopy

• If the antibody is homologous to the virus,

• aggregates of virus– antibody complexes are seen in the electron

microscope.