62325 Microbiology 1 M Kotiw Lecture 2

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62325 Microbiology 1M Kotiw Lecture 2

• Brief review of Virology
• Virus structure
• Replication
• Classification

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Brief overview of virology

• Lecture aims
• To become aware of methods used to detection
  viral presence
• To review virus structure
• To review the basic steps in virus replication
  virus
• To review how viruses are classified with
  particular emphasis on the Baltimore System
• Reference Sherris p69-104

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http//www.tulane.edu/dmsander/WWW/109/genomes.ht
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Demonstration of viruses

• Viruses can be demonstrated by
• Centrifugation and EM
• Plaque assays
• Eg bacteriophages on bacterial lawns
• Viral lesions (cytopathology)
• eg HSV and RSV forming syncytia in cellular
  components and HSV forming inclusion bodies
• Cytopathogenic effect (CPE) in permisive cell
  lines
• eg RSV in Hep2 cells HSV in BHK2 cells
• Some difficult to demonstrate Immunostains can
  be useful eg IFT for CMV

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• Isolation in some cases may be very difficult
• eg animal isolation may be only method, some
  arboviruses (insect borne) are grown in newborn
  mice
• Alternatives to isolation
• Immunology
• generally provides historical evidence
  IgG and IgM for EBV
• although virus proteins can be used to
  demonstrate HIV (WB) HBV sAg
• PCR (amplification) and/or Probes (hybridisation)

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http//www.tulane.edu/dmsander/WWW/109/genomes.ht
ml
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Virus structure

• Basic components
• Genome A single molecule of DNA or RNA but never
  both
• (can be fragmented as found in rotaviruses RNA)
• NA may be ss or ds or partially
• eg HBV partially ss and mainly ds DNA (refer
  Baltimore system)
• Coat protein (capsid) made up of discrete and
  standard number of capsomer units specific for a
  virus
• eg 162 for HSV
• Lipid envelope /-
• eg HSV (can be demonstrated by ether
  susceptibility)
• Spikes /- present on the outside termed
  peplomers
• often important in attachment to specific host
  tissues eg HIV

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Structure contin...

• Viruses generally fall into 2 morphological
  groups
• Helical ie rod shaped
• eg TMV
• rod shaped viruses tend to be described in three
  dimensions of the protein coat and number of
  capsomere/turn

http//www.tulane.edu/dmsander/WWW/109/structure.
html
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Structure contin...

• Isometric ie largely spherical
• eg HSV
• spherical viruses -generally icosahedral (body
  with 20 equal triangles with 12 vertices or
  points) Often characterized by number of
  capsomeres in each triangle

http//www.tulane.edu/dmsander/WWW/109/structure.
html
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Virus replication

• Many varieties of replicative modes
• Often best understood in terms of how the viral
  genome gets to generate mRNA
• Basis of Baltimore system of classification
• RNA viruses may be /- sense
• is infectious

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Replication contin...

• 5 recognized steps
• Adsorption
• Penetration
• Replication
• Maturation
• Release

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Adsorption

• Tends to be a very host specific event
• May be mediated by attachment sites (host and
  viral receptors)
• In some cases only single glycoproteins involved
  eg Influenza
• Lipid coats if present may facilitate
  attachment/diffusion through plasma membrane

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Penetration

• Non enveloped viruses may enter cell by a process
  similar to phagocytosis (vacuole)
• Enveloped viruses may diffuse through lipid based
  plasma membrane
• Some host cell proteases disrupt the viral
  lipid/glycoprotein coat inducing fusion with the
  lipid plasma membrane
• Viral coat is generally degraded by host based
  proteases freeing nucleic acid either into
  cytoplasm or into nucleus (as in HSV)

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Replication

• Can occur immediately or be delayed
• host cell genesis is inhibited by viral dependent
  proteins
• mechanism of transcription is dependent on the
  specific virus mRNA mode
• Viral genome usually codes for both structural eg
  coat and NA acid and enzymatic proteins eg virus
  DNA dependent RNA polymerase

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Replication contin...

• Synthesis of components is highly ordered and
  staggered as required
• Enzyme synthesis repressed until required
• Small viruses (limited coding capacity) may
  utilize host derived polymerases to commence
  replication
• Most RNA viruses use a virus RNA dependent RNA
  polymerase
• eg HIV have an RNA dependent DNA polymerase
  (called reverse transcriptase) which makes a
  DNA copy of the RNA virus

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Maturation

• Initial replicative processes are early protein
  synthesis necessary for turning the host cell
  off, replicating viral genome and making
  structural proteins
• late enzyme synthesis is related to viral
  packaging and release mechanisms (if required)

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Maturation

• Evidence that late packaging occurred was
  observed in HSV DNA by restriction ez analysis
  of single clones

IR
IR
IR
IR
IR
IR
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Release

• This may occur by simple burst due to large
  number of viral numbers
• budding of mature entities from nuclear or plasma
  membrane eg HSV HIV

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Virus classification

• When ever a group is defined and given a name,
  then this always seems to be a system for
  detecting objects which dont quite fit in so we
  define another group

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Classification in brief

• Generally accepted Linnaeus model of
• Family-gtgenera -gt species
• Family names end in viridae
• sub families in virinae
• genera and species both as virus
• Bacterial virologists call their viruses
  bacteriophages
• Plant virologists dont readily follow this
  system at all
• Animal virologist tend to be systematic (ICTV)
• main characteristics used NA type, lipid
  envelope presence, character of coat proteins

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Baltimore Classification
ssDNA () dsDNA intermediate
dsDNA (/-)
dsRNA (/-)
Transcription
Transcription
Transcription
Transcription
dsDNA (/-) RNA intermediate
mRNA
direct
ssRNA ()
Transcription
Transcription
ssRNA () DNA intermediate
ssRNA (-)
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Baltimore contin...

• 1. Double-stranded DNA eg Herpesviruses
  Poxviruses
• May replicate in the nucleus using cellular
  proteins. Poxviruses replicate in the cytoplasm
  and make their own enzymes for nucleic acid
  replication

-
Translation
Protein
mRNA
Old New
Ds DNA
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Baltimore contin...

• 2. Single-stranded () sense DNA eg Parvoviruses
• Replication in the nucleus forms a (-) sense
  strand, which serves as a template for ()strand
  RNA and DNA synthesis.

-
mRNA

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Baltimore contin

• 3. Double-stranded RNA eg Reoviruses
• Have segmented genomes. Each genome segment is
  transcribed separately to produce monocistronic
  mRNAs.

-
mRNA (alternative route)
mRNA
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Baltimore contin...

• 4. Single-stranded ()sense RNA eg
  Picornaviruses, Togaviruses
• Two strategies
• I. Polycistronic mRNA eg. Hepatitis A.
• Genome RNA mRNA naked RNA is infectious
• II. Complex Transcription e.g. Togaviruses.
• Two or more rounds of translation are necessary
  to produce the genomic RNA

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Baltimore contin...

• 5. Single-stranded (-)sense RNA eg
  Orthomyxoviruses, Rhabdoviruses
• Have a virion RNA directed RNA polymerase
• Two strategies
• I. Segmented e.g. Orthomyxoviruses.
• First step in replication is transcription of the
  (-)sense RNA genome by the virus RNA-dependent
  RNA polymerase to produce monocistronic mRNAs
• The () sense RNA becomes the template for new
  (-)sense genomes replication
• II. Non-segmented e.g. Rhabdoviruses.
• Replication occurs as above and polycistronic
  mRNAs are produced

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Baltimore contin...

• 6. Single-stranded ()sense RNA with DNA
  intermediate in life-cycle (Retroviruses)
• Genome is ()sense and is diploid
• does not serve as mRNA, but as a template for
  reverse transcription
• Has ds DNA intermediate

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Baltimore contin...

• 7. Double-stranded DNA with RNA intermediate the
  Hepadnaviruses
• Also relies on reverse transcription
• Unlike Retroviruses, this occurs inside the virus
  particle on maturation