Investigation strategies and methods Viral cultures May 2007

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

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

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)

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

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

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)

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

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%)

Potentially infectious specimen forms

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

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