Medical Virology

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Medical Virology

• Spring 2005

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Reference

• Medical Microbiology Murray?Rosenthal fourth
  edition
• Principles of VirologyMolecular Biology,
  Pathogenesis, and Control 2000 Flint
• Field's Fundamental Virology 4/e 2001 Knipe

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Topics

• Definition
• Structure
• Classification
• Replication
• Methods of study
• Sterilization and disinfection

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DEFINITION OF A VIRUS

• Viruses are organized associations of
  macromolecules-nucleic acid (which carries the
  blueprint for the replication of progeny virions)
  contained within a protective shell of protein
  units.
• On its own, a virus may be considered as an inert
  biochemical complex since it cannot replicate
  outside of a living cell. Once it has invaded a
  cell it is able to direct the host cell machinery
  to synthesize new intact infectious virus
  particles (virions). Because viruses are
  non-motile, they are entirely dependent on
  external physical factors for chance movement and
  spread to infect other susceptible cells.

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Structure 1components

• Viruses are very small in size (20 - 300
  nanometers) and contain either DNA or RNA (not
  both as in higher forms of life)
• The genome ( DNA or RNA) codes for the few
  proteins necessary for replication.
• Some proteins are non-structural, eg. nucleic
  acid polymerases
• Some are structural, ie. they become incorporated
  and form part of the virion.

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Structure 2 building block

• Protein building blocks are assembled according
  to general Principles of virus architecture to
  form a tight "shell" (capsid) inside which the
  nucleic acid genome lodges for protection. This
  shell may take the form of a polyhedron (usually
  icosahedral) or it may be spiral (helical
  symmetry), or it may be more complex.

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Structure 3

• Some viruses acquire an outer lipoprotein coat by
  "budding" through the host cell membranes
  (nuclear membrane or cytoplasmic membrane) and
  are thus called enveloped viruses.
• All the viral proteins have reactive epitopes
  which are important for interaction with cellular
  components during the process of infection and
  replication. The host's defence mechanisms
  (cellular and humoral mediated responses) are
  directed against the viral antigenic epitopes.

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Structure

• Identical subunits
• Cubic symmetry
• Icosahedral symmetry

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• An ICOSAHEDRON
• is composed of 20 facets, each an equilateral
  triangle, and 12 vertices, and because of the
  axes of rotational symmetry is said to have532
  symmetry
• There are, in fact, six 5-fold axes of symmetry
  passing through the vertices, ten 3-fold axes
  extending through each face and fifteen 2-fold
  axes passing through the edges of an icosahedron.

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DNA viruses
adenovirus
Papillomavirus
Parvo virus
Hepatitis B virus
Molluscum contagiosum
(Pox virus)
Herpesvirus
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RNA viruses
Rift valley fever
Enterovirus
Rotavirus
Influenza viruses
Paramyxovirus
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CLASSIFICATION

• Viruses are broadly classified primarily upon the
  type of genomic nucleic acid, eg. DNA or RNA
• The number of strands of nucleic acid (eg.
  double-stranded DNA, double-stranded RNA or
  single-stranded RNA, with a positive or negative
  "sense" of that single strand)
• Retroviruses are a special category of RNA
  viruses that require reverse transcription of
  their RNA to DNA and then integration of that DNA
  into the host cell genome before replication can
  take place. They carry a reverse transcriptase
  enzyme as part of the virion.

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RNA-dependent RNA polymerases Protein assembly
required to carry out RNA synthesis, not just
the polypeptide that just catalyzes chain
elongation Synthesis of viral RNA in the
presence of actinomycin D (a drug inhibits
cellular DNA-dependent RNA polymerase) Animal
virus RNA-dependent nucleic acid polymerases that
are purified and characterized polioviral
3Dpol, vesicular
stomatitis virus L protein,
and retroviral reverse transcriptase

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Replication 1

• 1. Adsorption
• Viruses have reactive sites on their surface
  which interact with specific receptors on
  suitable host cells.
• This is usually a passive reaction (not
  requiring energy) and the specificity of the
  reaction between viral protein and host receptor
  defines and limits the host species as well as
  the type of cell that is infected (although
  transfected nucleic acid can by-pass this
  limitation and extend the host range).
• Damage to these binding sites (eg. by
  disinfectants or heat), or blocking by specific
  antibodies (neutralizing antibodies) can render
  virions non-infectious.

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Replication 2

• 2. Uptake
• After adsorption, the coat of enveloped
  viruses may fuse with the host cell membrane and
  release the virus nucleocapsid into the host
  cytoplasm. Other viruses may enter the cell by a
  process of "endocytosis" which involves
  invagination of the cell membrane to form
  vesicles in the cell cytoplasm.

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Replication 3

• 3. Uncoating
• The release of the viral genome from its
  protective capsid to enable the nucleic acid to
  be transported within the cell and transcribed to
  form new progeny virions.

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Replication 4

• 4. Genomic activation
• Messenger RNA (m-RNA) is transcribed from
  viral DNA (or formed directly from some RNA
  viruses) and codes for viral proteins that are
  translated by the host cell.
• " Early" proteins are usually non-structural
  (eg. DNA or RNA polymerases) and later proteins
  are structural, eg. capsid proteins, ie.
  building blocks of the virion. Nucleic acid
  replication produces new viral genomes for
  incorporation into progeny virions.
• In general, DNA viruses replicate mainly in
  the nucleus and RNA viruses mainly in the
  cytoplasm, but there are exceptions, eg. Pox
  viruses contain DNA but replicate in the
  cytoplasm of the host cell.

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Replication 5

• 5. Assembly
• Assembly of viral nucleocapsids may take place
  in the nucleus (eg. herpes virus, adenovirus) in
  the cytoplasm (eg. polio virus) or at the cell
  surface, eg. "budding" viruses such as influenza.
• Accumulation of virions at sites of assembly may
  form "inclusions" that are visible in stained
  cells with the light microscope.

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Release 6

• 6. Release
• Release of new infectious virions is the
  final stage of replication.This may occur by by
  budding from the cell surface, as occurs with
  many enveloped viruses. In this case capsid
  proteins and nucleic acid condense directly
  adjacent to the cell membrane and viral-coded
  envelope proteins, introduced into the cell
  membrane, concentrate in the vicinity of capsid
  aggregates. The membrane surrounding the
  nucleocapsid then bulges out and becomes "nipped
  off" to form the new enveloped virion.
• Some viruses utilize the cellular secretory
  pathway to exit the cell. Virus particles
  enclosed within Golgi-derived vesicles are
  released to the outside of the cell when the
  transport vesicle fuses with the cell membrane.
• Disintegration or lysis of the infected cell
  can also result in the release of intact
  infectious virions.

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A general scheme of viral replication
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A single-cycle growth curve of a virus
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METHODS OF STUDY OF VIRUSES

• Diagnosis of viral infections
• Viruses can be studied in a number of direct
  and indirect ways and all these methods can be
  applied in a diagnostic situation, ie. is this
  patient infected with a particular virus?
• There are two approaches
• 1. detection and demonstration of the virus
  itself
• 2. the study of the host's response to that
  virus

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Methods

• "in vitro" cell cultures
• "non-cultivable" viruses have been extensively
  studied by molecular techniques ("genetic
  engineering")
• Electron microscopy
• Negative staining, using transmission electron
  microscopy
• only electrons which pass through the
  specimen are involved in the formation of the
  final image.
• Serological techniques
• precipitation, agglutination,
  immunofluorescence, ELISA, complement fixation
  and radio immuno assays
• haemagglutination (causing red blood cells
  to stick together ) which can be used to detect
  and quantitate the virus (by haemagglutination)
  or specific antibodies to that virus
  (haemagglutination inhibition)
• neutralization
• Molecular techniques

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Molecular Techniques

• Polyacrylamide gel electrophoresis (PAGE) of
  protein
• Western blotting identification of specific
  proteins with labelled probes
• Polymerase chain reaction (PCR), to amplify
  specific segments of viral nucleic acid
• Southern blotting, and DNA hybridization with
  labelled probes
• Sequencing of portions of the viral genome
• Restriction fragment length polymorphisms of
  viral nucleic acid