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Baedah Madjid

DEPART. OF MICROBIOLOGY, MEDICAL


FACULTY, HASANUDIN UNIVERSITY
2007
1. Identification of the virus in the cell culture
2. Direct microscopic identification
3. Antibodies detection in the serum
(serologic procedures)
4. Viral antigens detection in blood or other
body fluids
5. Viral nucleic acid detection in blood or the
patients cell.
Virus only replicate only in living cell
grow only in cell culture
Most virus inactivated at room temperature
specimen must be inuculated as soon
as possible
Brief transport & storage at 4oC I
acceptable.
Presumptive
identification
Cytopathic Effect (CPE):a change in the
appearance of the virus-infected cells.
The changes: in size, shape, fusion of
cells to form multinucleated giant cells
(syncytia).
CPE: usually manifestation of virus-
infected cells that are dying or dead.
If the virus do not produce CPE, its present can be
detected by several other technique:
1. Hemadsorption: attachment of erythrocytes to the
surface of virus-infected cells. This technique limited to
the viruses with a hemaggultinin protein on their
envelope such as mumps, parainfluenza, & influenza
viruses.

2. Interference with the formation of CPE by a second


virus. Foe examp. rubella virus, which does not cause a
CPE, can be detected by interference with the formation
of CPE by certain enteroviruses such as echovirus.
3. A decrease acid production by infected, dying
cells. Can be detected visually by a color change of a pH
indicator in the culture medium. Yhe indicator remain red
(alkaline) in the presence of virus-infected cells but turn to
yellow in the presence of normal cell due to the acid
production.
Definitive Identification
A. Complement Fixation
B. Hemagglutination Inhibition
C. Neutralization
D. Fluorescent-Antibody Assay
E. Radioimmunoassay
F. Enzyme-Linked Immunosorbent Assay
G. Immunoelectron Microscopy
Definitive Identification
A. Complement Fixation
Ag of unknown virus in culture medium

+
homologous
Known antibody

Complement will be fixed


to Ag-Ab complex

The indicator system not lyse


(sensitized erythrocytes)
Definitive Identification
B. Hemagglutination Inhibition
Virus + antibody (known) homologous

The virus can not attach to the erythrocytes

No heagglutination
Definitive Identification
C. Neutralization
Virus + antibody (known) homologous

Ab will bound to the surface of the virus

The virus can not entry in to the cell

This neutrizes viral infectivity


Definitive Identification

D. Fluorescent-Antibody Assay

Virus-infected cells + Fluorescein-tagged Ab


homologous
Ultraviolet microscope

Typical apple-green color of fluorescein


Definitive Identification
E. Radioimmunoassay

Virus + antibody (known) homologous

Less Ab left to bind to known radio-labeled virus


Definitive Identification
F. Enzyme-Linked Immunosorbent Assay
(ELISA)
Known Ab + Culture
If virus is presence
Virus will bind to the Ab

Enzyme linked to Ab to Immnuoglobulin


Substrate

Color change measured


Definitive Identification
G. Immunoelectron Microscopy

Virus + antibody (known) homologous

Aggregates of the virus-antibody complexes

Can be seen in the electron microscope


Viruses can be detected & identified by
direct microscopic examination of specimen
such as biopsy material or skin lesion.
Procedure can be used:
1. Light Microscopy: characteristic inclusion
bodies or multinucleated giant cells.
2. UV Microscopy: used for fluorescent-Ab
staining of the virus-infected cells. (a rapid,
specific diagnosis).
3. Electron Microscopy: to detect virus
particles, which can be identified by their size
and morphology. (Not used for clinical diagnosis)
Light Microscopy
Inclusion bodies
- form by aggregates of many virus particles
- can be seen in either the nucleus of cytoplasm of
infected cells.
- are not specific
- cytoplasmic inclision formed by rabies virus
(Negri bodies)
Multinucleated Giant cells
formed by several viruses: certain herpes viruses,
measles virus, and respiratory syncytial virus.
The rise in the titer of Ab to the virus can be
used to diagnose current infection.
A serum sample is obtain as soon as a viral
etiology is suspected (acute-phase).
A second sample is obtained 10-14 days later
(convalescent-phase).
If the Ab titer of the convalescent-phase serum
sample is at least 4-fold higher than the titer of
the acute-phase serum sample infected.
Ab titer on a single sample does not distinguish
between a previous and a current infection.
The Ab titer can be determine by many of the
immunologic test mention above
These serologic diagnoses are usually made
retrospectively: the disease has frequently run its
course by the time the result are obtain.
The presence of IgM; used for current infection.
The present of IgG cannot be used to diagnose
current infection (Ab may be due to an infection in
the past. an acute and convalescent serum
sample should be analized.
Specimens:
Blood or body fluids.

Methods
Various test but mostly by ELISA

Examples:
Test for the p24 antigen of HIV
Test for the surface antigen of hepatitis B
Viral nucleic acid, either the viral genome or
viral mRNA can be detected in the blood or
tissues with complementary DNA or RNA (cDNA
or cRNA) as a probe.
The presence of viral DNA or RNA is
increasingly becoming the gold standard in
viral diagnoses.
Small amount of viral nucleic acid amplified
(PCR).