1. Investigation strategies and methods
Viral cultures
May 2007

2. Learning objectives
At the end of the presentation, participants should:
Understand the principle of cultivating viruses
Understand the methods and problems with cultivating viruses

3. Techniques to identify viruses
It can take a few hours to weeks to identify a virus
Techniques include:
PCR (single round) or nested/semi-nested PCR
Real-time PCR
Direct electronic microscopy
Antigen capture
Isolation
Long process
Gold standard for viruses that can be cultured

4. Virus culture
Is based upon amplification of potentially infectious pathogens
Implies intracellular replication of viruses in the cytoplasm or
in the nucleus
Is controlled by regulations (i.e. bio-safety level 2, 3 or 4)
Allows for:
Identification
Further studies (e.g., Pathogenicity, antiviral sensitivity, research)

5. Virus culture Long process
Not always possible for front-line diagnosis
Primary objective for the diagnosis of an unknown disease
No generic protocol

6. How to go about virus culture?
Obtain suitable specimens
Identified specimens with suitable information
Evaluate of chances of success of the process before start
Make sure transportation used cold chain
4°C
-20°C
Dry ice (-79°C)
Use suitable culture protocol
In vitro/in vivo cell cultures

7. Culture procedure Use of a variety of cell sources and techniques
Treatment of the specimen prior to inoculation
Follow-up
Viral detection:
Non specific
Cytopathogenic effect (microscope)
Electronic microscopy identification (morphology)
Specific
Immunological detection: antigen detection, PCR, IFA…
Viral load estimation (titration, plaque assay)

8. Limitations of cultures to identify viruses
Absence of detection system for the agent
Inappropriate culture systems
Viruses that cannot be cultured
A negative viral culture results does not mean that the
agent is absent
Need of other tests
PCR can detect the viral genome in absence of the complete virus

9. Specimens used to culture viruses
Blood specimens
EDTA
Heparin
Serum
Stool
Throat swabs
Naso-paryngeal aspirates
Stools, rectal swabs
Urine
Saliva
Cerebro-spinal fluid
Biopsy
Skin (filoviridae)
Organs (fixation with formaldehyde 10%)

10. Potentially infectious specimen forms

11. Sequencing Analysis of sequence of nucleic acid fragment after
PCR amplification
Comparison of the alignment of nucleotides with other
sequences present in different data bases for the
identification of an agent
Confirmatory analysis
Final DNA fingerprint is molecular signature of the micro-organism

12. Investigation strategies and methods
Developed by the Department of Epidemic and Pandemic Alert and Response of the World Health Organization with assistance from: European Program for Intervention Epidemiology Training Canadian Field Epidemiology Program Thailand Ministry of Health Institut Pasteur