Virus cultivation, detection and genetics

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Virus cultivation, detection and genetics

• 9/4/03

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Laboratory animals

• Laboratory animals - animal models of human
  infection. Historically the only way to study
  viruses was from animal to animal.
• Problems - 1) inconvenient and expensive, 2) not
  a defined system - leads to generation of virus
  mutants, 3) animal welfare issues,
• Advantages - 1) some viruses can only be studied
  in this way, 2) gives unique insight into virus
  pathogenesis
• Embryonated eggs

From Principles of Virology , Flint et al ASM
press
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Cell culture I

• Currently the most common way to study viruses.
• Sterility is crucial
• Cells can be infected synchronously and viruses
  grown on a large scale
• Primary cells - these are derived directly from
  animal tissue (e.g. monkey kidney, human
  foreskin, chick embryo)
• Diploid cell lines - these maintain the diploid
  no. of chromosomes but can divide up to 100 times
  (usually from human embryos)
• Continuous cell lines - can be propagated
  indefinitely. Usually from tumor tissue or by
  treating primary of diploid cells with mutagens
  or tumor viruses. Little resemblance to original
  cell, abnormal chromosome no. (aneupolid), can be
  tumorigenic e.g. HeLa, Vero, L929, CHO.
• Diploid and continuous cells can be frozen in
  liq. N2

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Cell culture II

• Monolayer cells . These grow on a solid surface ,
  (e.g. glass, plastic). - most common
• Suspension cells. - useful for large scale
  culture (spinner culture)
• All cells need food - chemically defined medium -
  isotonic solution of salts, glucose, vitamins,
  coenzymes, amino acids, buffered to 7.3 (with
  CO2) and antibiotics. The magical ingredient is
  serum, added to provide growth factors.
• Most cell lines double every 24-48 h and must be
  passaged (divided) into new cultures every 3-4
  days. Adherent (monolayer) cells are removed from
  the vessel by treatment with proteolytic enzymes
  (trypsin) and EDTA (versene)

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Cytopathic effects (cpe)

• Some viruses kill the cells in which they
  replicate, often easily visible as cpe, other
  viruses produce little or no cpe
• Typically rounding or detaching of cells and cell
  fusion (syncytia)

From Principles of Virology , Flint et al ASM
press
From Principles of Virology , Flint et al ASM
press
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Examples of cpe
Often variable from virus to virus Can be
diagnostic, even on routine pathology
Herpesvirus cpe
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Detection and quantitation of infectious viruses

• Detection of the amount of virus that causes
  infection in the system studied
• A measure of the virus is usually referred to as
  its titer -- (note the titer can vary depending
  on the assay used)
• The virus titer is a measure of the concentration
  of the virus
• This relies on one of several assays - plaque
  assay, fluorescent focus assay, infectious center
  assay, transformation assay, endpoint dilution
  assay.
• Virus titers can be very high ( e.g 1010
  infectious units/ml), or much lower

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The plaque assay

• Monolayers of cells exposed to a defined dilution
  of virus, such that the virus is adsorbed.
• The inoculum is removed and the cells covered
  with medium that includes a gelling substance
  (agar)
• The gel prevents long range spread of virus but
  allows viruses to infect neighboring cells -
  hence a localized infection
• With time the plaque becomes visible with the
  naked eye, or can be seen after staining the cell
  (neutral red, crystal violet).
• A plaque assay will only work with viruses that
  cause cpe (cell death) - even then plaques can be
  very difficult to visualize and count
• Visualization can be markedly improved with
  immunohistochemical stains
• Measured as plaque-forming units (PFU)

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Phase contrast /herpesvirus X-gal /
herpesvirus
From Principles of Virology , Flint et al ASM
press
Influenza / crystal violet
Poliovirus/crystal violet
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Other common assays for infectivity - I

• Fluorescent focus assay - infection scored by
  addition of virus-specific antibody, and
  fluorescent secondary antibody and visualization
  under the microscope - after a single round of
  infection
• Infectivity can be scored as FFU/cell - not
  especially accurate, but can be very useful
• Infectious center assay - a modification of the
  plaque assay where infected cells are mixed with
  non-infected cells before plating - used for
  persistently infected cells
• Transformation assay - an inverse plaque assay
  that measures the production of foci of
  transformed cells (small piles) - often referred
  to as focus-forming units (FFU)

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Other common assays for infectivity - II

• Endpoint dilution - often used in animals. Virus
  is diluted into replicate animals and disease or
  death measured. The endpoint is measured as the
  dilution that causes 50 death (ID50)

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Direct measurement of virus particles

• Electron microscopy The virus is dried onto an EM
  grid and stained. The inclusion of a known
  dilution of latex beads allows quantitation of
  the virus
• Hemagglutination (HA assay) Many viruses bind red
  blood cells and link multiple cells together.
  Presence of the virus causes agglutination and
  the formation of a lattice, absence of a virus
  leads to the presence of sharp dot or button

15 latex beads alongside 14 poxviruses (brick
shaped, and slightly smaller)
Influenza virus
From Fields Virology 4th Ed Lippincott Williaams
and Wilkins
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Other ways to measure viruses

• Enzyme activity - e.g reverse transcriptase (RT)
  assay for retroviruses
• Serological assays -can detect either virus
  antigen or virus antibody e.g enzyme-linked
  immunosorbent assay (ELISA), or Western blot
• A) Virus neutralization - can distinguish between
  different serotypes e.g with plaque assay
• B) Hemagglutination inhibition (HAI)
• C) Complement fixation Combinations of virus
  antigen and antibody can cause complement
  fixation which leads to red cell lysis

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Diagnostic virology

• This can be challenging
• Diagnosis can be the crucial factor in the
  development/usefulness of antiviral drugs
• Most common assay is ELISA-based
• Immunofluorescence is better, but expensive and
  slow
• Electron microscopy is still essential in some
  cases

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Direct and indirect immunofluorescence

• Labeling of virus antigen in infected tissue
• Needs specialized equipment
• Requires pre-existing diagnostic antibody

Herpesvirus/ICP0
From Principles of Virology , Flint et al ASM
press
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Particles vs. infectious particles

• Not all virus particles are infectious. In many
  cases the vast majority of particles are not
  infectious -
• The ratio of particles infectious particles is
  termed e.g. the particle to PFU ratio

From Principles of Virology , Flint et al ASM
press
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Multiplicity of infection (MOI)

• Infection depends on the random collision of
  cells and virus particles
• In any particular experiment some cells get no
  viruses, some get 1 virus, some 2 , 3, 4 etc
• A Poisson distribution
• See box 2.2 in Flint for the math
• Bottom line is that to get every cell infected
  (99) it is necessary to infect the cells at an
  MOI of 4.6

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The one-step growth curve

• First determined by Ellis and Delbruck in 1939
• A multiplicity of infection (moi) of 5-10 pfu/ml
  ensures that almost all cells become infected
• Virus is added to cells to allow adsorption
  (typically 1h), in a small volume to promote
  adhesion
• The inoculum is removed, cells are washed and the
  medium replaced
• At different times, samples are collected and
  titered
• The one-step growth curve is a fundamental
  feature of a virus - and distinguishes it from
  e.g. a bacterium

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Note - times can be rapid or slow Titers can
vary Intra- vs. extra-cellular periods can
vary Burst size ( yield per cell) can vary All
viruses have eclipse and latent periods
From Principles of Virology , Flint et al ASM
press
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Genetic Analysis of Viruses

• An analysis of virus mutants has told us much
  about virus life-cycles
• Spontaneous mutations - RNA viruses naturally
  contain a high proportion of mutants (1
  misincorporation per 104-105 nucleotides).,
• For DNA viruses (1 misincorporation per 108-1011
  nucleotides) exposure to a mutagen is necessary
  (e.g. hydroxylamine).
• Mutants are selected and isolated by e.g high or
  low temperature, or resistance to a
  drug/antibody, or by changes in plaque size or
  host range.
• Mutants are mapped by recombination (for
  unimolecular genomes) or reassortment (for
  segmented genomes). Cells are co-infected with
  two mutants. The recombination frequency is
  determined by the physical distance between the
  two lesions.
• Complementation. Co-infection with two viruses
  that contain different mutations results in
  growth, but no growth with the same mutation ---gt
  complementation groups

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Infectious DNA clones

• Recently is has become possible to reconstruct
  the genome of a virus into plasmid DNA - this
  allows introduction of a mutation anywhere in
  the virus genome
• Mutations can be deletions of a complete gene or
  part of a gene, insertion mutations (e.g from
  related viruses), point mutation to change
  individual amino acids

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Virus vectors - I

• Our ability to manipulate the virus genome at
  will has important implications
• 1) Gene therapy - the introduction of genes into
  terminally differentiated cells (DNA viruses,
  esp. adenovirus)
• Problems include targeting, toxicity and the
  immune response
• 2) Vaccines - the production of non-infectious
  viruses that retain the ability to induce immune
  responses. Foreign antigens can be expressed
  (e.g. malaria antigen produced from recombinant
  influenza viruses)
• Problems include ability to do booster injections

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Virus vectors - II
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Virus vectors - III

• Retrovirus vectors

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Reading material

• Flint Chapter 2
• Fields Chapter 2
• For next Tuesday Chapter 3 of Flint