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Investigation strategies and methods
Viral cultures
May 2007
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Laboratory Training for FieldEEpidemiologists
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Learning objectives
At the end of the presentation, participants should:
• Understand the principle of cultivating viruses
• Understand the methods and problems with cultivating viruses
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Laboratory Training for FieldEEpidemiologists
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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
P I D E M I C A L E R T
Laboratory Training for FieldEEpidemiologists
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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)
P I D E M I C A L E R T
Laboratory Training for FieldEEpidemiologists
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Virus culture
Long process
• Not always possible for front-line diagnosis
Primary objective for the diagnosis of an unknown disease
No generic protocol
P I D E M I C A L E R T
Laboratory Training for FieldEEpidemiologists
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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
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Laboratory Training for FieldEEpidemiologists
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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)
P I D E M I C A L E R T
Laboratory Training for FieldEEpidemiologists
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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
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Laboratory Training for FieldEEpidemiologists
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Specimens used to culture viruses
Blood specimens
Stools, rectal swabs
• EDTA
Urine
• Heparin
Saliva
• Serum
Stool
Cerebro-spinal fluid
Biopsy
Throat swabs
• Skin (filoviridae)
Naso-paryngeal aspirates
• Organs (fixation with
formaldehyde 10%)
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Laboratory Training for FieldEEpidemiologists
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Potentially infectious specimen forms
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Laboratory Training for FieldEEpidemiologists
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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
P I D E M I C A L E R T
Laboratory Training for FieldEEpidemiologists
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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
P I D E M I C A L E R T
Laboratory Training for FieldEEpidemiologists
A N D
R E S P O N S E