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Introduction Practical 

virology 
‫إعداد‬
‫ مرتضى عبد المھدي المظفر‬.‫د‬
Molecular virology
E‐ mail : 
murtadha.ama@uokufa.edu.iq

١ Murtadha AL‐Muthafer  3/10/2016
• Viruses, are not cells , no functional
organelles and completely dependent on
their host for energy production and synthesis
of macromolecules.

• They contain only one type of functional 
nucleic acid ,either DNA or RNA  never both. 
Laboratory Diagnosis of Viral 
Infection

Detection – Isolation & identification  ‐
Serology

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Sample Type

• Blood, Urine, Stool, nasal washing, nasal


swab , throat swab, saliva , sputum, rectal
swab, vesicle fluid( scraping or swab),
tissue ,brain biopsy, cerebrospinal fluid,
et al.
It depends on three main principles:
detection Isolation & Serology using
Identification

Virus particles Tissue culture IF & IHC

Viral Chick embryo HI & HA


nucleic acid

Cytopathology Laboratory ELISA


animals

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I. Direct detection

• Virus particles
Microscopy Identification
Electron microscopy

• Viral nucleic acid

Molecular Technique 
• Cell death – Cytopathic effect

This is the end result of many lytic virus infections in


which the cell is killed following virus infection ,
Called CPE found in cell culture systems infected
with lytic viruses.
The form of virus‐induced CPE can take many forms
ranging from the lysis of the cell to a fusion event
with the formation of syncytia.
Qrt‐pcr Gradient PCR 

Fluorescent Microscope  TEM & SEM 
II. Isolation and identification
Isolation and identification of the virus from 
clinical specimens:
three main systems are used for viral isolation:
1‐ Tissue culture. 

2‐ Chick embryo.

3‐ Laboratory animals 

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Tissue culture preparation: 
From the desired tissue the following steps are followed: 

 Tissue source 1mm fragments. 

 Incubate with proteolytic enzyme (trypsin) to disperse the cells. 

 Add growth media to make a cell suspension. 

 Incubate in  flasks , microtiter plate wells  or tubes, cells settle on 


the dependent surface and grow into confluent monolayer.

 Re‐disperse monolayer cells and increase number of cultures for 
cell culture passage. 

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Isolation in embryonated 
hen’s eggs

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Laboratory  Animals 
IN‐VIVO / IN‐VITRO?
1. Biological System
a) Natural host
b) Experimental animals          In ‐ vivo
c) Transgenic animals
2. Embryonated Eggs –
In ‐ vivo & In ‐ vitro
3. Tissue Culture System – In ‐ Vitro
ANIMAL INOCULATION‐ DISADVANTAGES

Cost •
• Maintenance
• Interference of immune system
• Individual variations
• Difficulty in choosing of animals for 
particular virus
EMBRYONATED EGGS

ADVANTAGES
• Isolation and cultivation of many avian and 
few mammalian viruses
• Sterile & wide range of tissues and fluids 
•Cost‐ much less
•Maintenance‐easier
•Less labor
• Readily available
• Free from bacteria and many latent
viruses.

• Sensitive to viruses which do not 
produce infection in adult birds.
METHODS OF CULTIVATION
Various routes of inoculation 
a)Yolk sac
b) Allantoic sac
c) Chorioallantoic membrane
d) Amniotic cavity
e) Intravenous
٢١ Murtadha AL‐Muthafer  3/10/2016