Download Viral Diseases - OUR SITE

Viral Diseases
HOW TO DIAGNOSE
By: Dr. Amr
Special characters of viruses
- Viruses are prokaryotes; Not cellular, No
ribosomes, No membrane-bound organells.
- Can reproduce ONLY inside host cells
“Obligate intracellular”.
- Reproduction is the only characteristic of life.
- Very small size: 20-300 nm ‘diameter’.
- Only 1 kind of nucleic acids (DNA or RNA)
Laboratory
diagnosis
1- Direct
detection
AParticles
BInclusion
bodies
CAntigens
DNucleic
acid
Virus
1- Direct
detection
A- Viral
particles
By Electron
Microscope
1- Direct
detection
By Inverted
Microscope
B- Inclusion
bodies
=
Site of viral
replication
In the”
- nucleus or
- cytoplasm of
infected cells
1- Direct a- ELISA
detection
C- Viral
antigens
ELISA plate
Color change is detected by Spectrophotometer
1- Direct
detection
b- RIA ‘Radio Immune Assay
C- Viral
antigens
Gamma
counter
1- Direct
detection
c- IF ‘Immunofluorescence’
C- Viral
antigens
Solid phase
Seen with Fluorescent microscope ‘UV’
1- Direct
detection
D- Viral
nucleic acid
PCR & RT-PCR, probes
2- Virus
isolation
A- Tissue
culture
Viruses are ‘Obligate intra-cellular’
Cell culture:
1- Primary cell line:
- Somatic cells from animal ‘monkey kidney cells’
or human.
- Maintained for short period in culture.
2- Semi-continuous cell line:
- Human embryo lung ‘fibroblasts’
- Limited passage number (30)
- Susceptible to many viruses
3- Continuous cell line:
- Tumor cells ‘HELA’ ( human cervical cancer cells)
- Indefinite passage number (300)
- but susceptible to few viruses
Culture media
 FCS ‘foetal calf serum’  Growth factors
 Amino acids
 Vitamins
 Antibiotics & Antifungals
Passage: dilution of cells to keep them growing
(e.g: twice / week).
Detection:
CPE ‘Cytopathic effect’
(Cell changes that can be seen by the microscope)
- Cell death & detachment from surface (polio v.)
- Rounding & grape-like cluster formation (adeno v.)
- Syncytium ‘giant cell formation’ (measles, mumps)
If the virus doesn’t produce a CPE, its presence
can be detected by:
1- Haemadsorption: attachment of erythocytes to
the surface of virus infected cells e.g. in mumps,
parainfluenza and influenza viruses
2-Haemagglutination: The HA test can be used to
detect and quantitate the virus in vitro. Using sheep
RBCs.
3- Hemagglutination inhibition: It is a test used for
detection of specific antibodies that could prevent
haemagglutination by the virus.
4- Detection of the virus antigens or its genome in
infected cells.
5- Inclusion bodies in some infected cells.
2- Virus
isolation
BEmbryonated
egg
• Some viruses will replicate in the
living tissues and membranes of
developing embryonated hen’s eggs,
such as influenza virus.
• Egg-adapted strains of influenza
virus replicate well in eggs and very
high virus titers can be obtained.
3- Indirect
detection
Serologic
detection of
‘antiviral
antibodies’
- Detection of IgM / or at least (4fold increase) of IgG.
- By IF, ELISA, RIA
IF
patient
serum ?
= ‘Host
response’
Solid phase
4- Animal
- One of the earliest ways of detecting
pathogenicity
a virus.
- Animals with actively replicating
cells give more observed response as
suckling mice.
- limited by virus ‘species specificity’,
human viruses may need primates
for replication.
5- Viral quantitation
1- Physical ‘EM’
- Does not
differentiate bet.
‘infective, non-inf.’
3- Biological
“plaque assay”
‘infectious only’
2- Biochemical
- Enzymes (RT)
- Ag (p24 of
retrovirus)
4- Molecular
“Q. PCR”
5- Viral
quantitation
“Plaque
assay”
• Dilutions of the virus are used
to infect a cultured cell
monolayer,
which
is
then
covered with soft agar to restrict
diffusion of the virus, resulting in
localized cell killing and the
appearance of plaques after the
monolayer is stained.
• Counting the number of
plaques directly determines the
number of infectious virus
particles applied to the plate.
*Number of plaques =
number of infectious v. particles
5- Viral
quantitation
“Q PCR”