Unit 5 bacteria and viruses

Investigation strategies and methods Viral cultures May 2007 ie Xà World Health ` Nh;Z # Organization... Learning objectives At the end of the presentation, participants should: ¢

Investigation strategies and methods

Viral cultures

May 2007

ie Xà World Health

` Nh;Z # Organization

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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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

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Virus culture

ls 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:

¢ Further studies

(e.g., Pathogenicity, antiviral sensitivity, research)

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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

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How to go about virus culture?

Obtain suitable specimens

Evaluate of chances of success of the process before start

Make sure transportation used cold chain

© 42°C

© -20°C

¢ Dry ice (-79°C)

Use suitable culture protocol

¢ In vitro/in vivo cell cultures

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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

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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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Specimens used to culture viruses

°® Serum

Cerebro-spinal fluid

formaldehyde 10%)

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Potentially infectious specimen forms

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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

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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

6 = Thailand Ministry of Health

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