E P I D E M I C A L E R T A N D R E S P O N S E Laboratory Training for Field Epidemiologists

Viral cultures

Investigation strategies and methods

May 2007

E P I D E M I C A L E R T A N D R E S P O N S E Laboratory Training for Field Epidemiologists

Learning objectivesLearning objectives

At the end of the presentation, participants should:

• Understand the principle of cultivating viruses

• Understand the methods and problems with cultivating viruses

E P I D E M I C A L E R T A N D R E S P O N S E Laboratory Training for Field Epidemiologists

Techniques to identify virusesTechniques 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

E P I D E M I C A L E R T A N D R E S P O N S E Laboratory Training for Field Epidemiologists

Virus cultureVirus cultureIs 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)

E P I D E M I C A L E R T A N D R E S P O N S E Laboratory Training for Field Epidemiologists

Virus cultureVirus culture

Long process

• Not always possible for front-line diagnosis

Primary objective for the diagnosis of an unknown disease

No generic protocol

E P I D E M I C A L E R T A N D R E S P O N S E Laboratory Training for Field Epidemiologists

How to go about virus culture?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

E P I D E M I C A L E R T A N D R E S P O N S E Laboratory Training for Field Epidemiologists

Culture procedureCulture procedureUse 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)

E P I D E M I C A L E R T A N D R E S P O N S E Laboratory Training for Field Epidemiologists

Limitations of cultures to identify Limitations of cultures to identify virusesviruses

Absence of detection system for the agent

Inappropriate culture systems

Viruses that cannot be cultured

A negative viral culture results does not mean that theagent is absent

• Need of other tests

• PCR can detect the viral genome in absence of the complete virus

E P I D E M I C A L E R T A N D R E S P O N S E Laboratory Training for Field Epidemiologists

Specimens used to culture virusesSpecimens 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%)

E P I D E M I C A L E R T A N D R E S P O N S E Laboratory Training for Field Epidemiologists

Potentially infectious specimen formsPotentially infectious specimen forms

E P I D E M I C A L E R T A N D R E S P O N S E Laboratory Training for Field Epidemiologists

SequencingSequencing

Analysis of sequence of nucleic acid fragment afterPCR amplification

Comparison of the alignment of nucleotides with other sequences present in different data bases for theidentification of an agent

Confirmatory analysis

• Final DNA fingerprint is molecular signature of the micro-organism

E P I D E M I C A L E R T A N D R E S P O N S E Laboratory Training for Field Epidemiologists

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

Investigation strategies and methods