Practical Applications in Virology

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Practical Applications in Virology

• Thomas Klimkait
• Institut für medizinische Mikrobiologie
• Jan. 22, 2004

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Viruses

• are harmless Bystanders
• cause economical Damage
• cause Disease
• have utility in Research

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Where do we find them?

• ... everywhere!
• As phages in bacteria
• (T-series, Lambda, ?X,..)
• As pests or ornamenting tools in plants
• (various mosaic viruses)
• In vertebrates
• (multiple forms of DNA- und RNA-viruses)

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attractive Simplicity!

• simple nucleid Acid
• - DNA or RNA, ss or ds

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Variation is broad (everything is possible)
Classification is tricky(no natural systematic)
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what is practical?

• genetic material can be exchanged
• Genes/proteins can be highly amplified
• (information can become inheritable)

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Viruses as vectors

• for laboratory cloning, expression
• for therapy
• delivery system for antigens (vaccine)
• delivery system for lost genetic information
  (gene therapy)
• delivery system for new genes/active intervention

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Conzepts of vectors
retrovirus
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Gene Therapy

• Adenoviruses
• Adeno-associated Virus
• Retroviruses

• Caveats
• immunogenicity
• prior immunity
• lacking integration into host genome
• chromosomal integration

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properties of persisting Viruses
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• Diagnositics employing Nucleic Acids
• qualitative determination
• after signal amplification by
• PCR/LCR/b-DNA
• gt is an RNA/DNA detectable? pos/neg
• quantitative determination
• competitive PCR/real-time PCR
• gt how much RNA/DNA is present in sample

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DNA-detection via Agarose Gel
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real-time PCR
Q
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applications for virology

• diagnostic need of real time PCR for
  quantification
• need for new tools for higher sensitivity
• test systems for novel pathogens
• systems for new properties (therapy escape)

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break
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US - AIDS Tote seit 1980
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properties of retroviruses

• envelope (gp120, gp41 trimer)
• retrotranscription in the cell
• genome integration (gt Provirus)
• gene activation
• separate expression for Gag and glycoproteins
• RNA and Env transport to viral buds
• viral protease mediates maturation
• intracellular recombination!
• high mutability, high error rate of RT

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HIV-life cycle
host genome
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Primoinfection with HIV
read- out
CD4-cells
Virus
time
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A patient under HAART-therapy
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drug-dosing
dosing suboptimal
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• per cycle

? 1 Virus gt gt103 particle/cell

• sloppy copying

? 1 mutation / 1 genome
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HAART (Highly Active Anti-Retroviral Therapy)
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Escape-Mutations - HIV Protease
saquinavir
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1. Problem assignment of complex mutations
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Genotyping
mutations need to be translated into Resistances!
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2. Problem ambiguities in the interpretation
different algorithms use different rules
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3. Problem mixed virus populations, viral
minorities (lt10)

• single sequences will not exhaustively evaluate
  complex virus populations.
• mixtures can be analyzed in parallel by the
  employment of culture systems.

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from patient
rev
5LTR
gag
vpu
tat
nef
pol
env
3LTR
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Phenotyping
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PhenoTect
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4. Problem HIV subtypes
HIV is regionally quite different - different
sequence different response to therapy! -
resistances and metabolic parameters have to
be clarified regionally!