Introduction to Virology

1
Introduction to Virology

• Learning Objectives
• Understand what a virus is.
• Summarize the history of virology.
• Describe techniques used to study viruses.

2
Virus Diversity

• There is much biological diversity between
  viruses.
• Viruses are successful parasites.
• Understanding diversity is the key to
  understanding viruses.
• At a molecular level
• protein-protein
• protein-nucleic acid
• protein-lipid interactions

3
Viruses are distinct from living organisms

• Viruses are submicroscopic, obligate
  intracellular parasites.
• Rickettsiae and Chlamydiae are obligate
  intracellular parasitic bacteria.
• Therefore it is necessary to add further define
  what constitutes a virus.

4
Virus Definition

• Virus particles are produced from the assembly of
  pre-formed components, other agents grow and
  reproduce by division.
• Virus particles (virions) do not grow or undergo
  division
• Viruses lack the genetic information necessary
  for the generation of energy or for protein
  synthesis (ribosomes)

5
Are viruses are alive?

• One view is that inside the host cell, viruses
  are alive, but outside it they are complex
  assemblages of inert chemicals.
• Chemical changes occur in extracellular virus
  particles, but these are not the 'growth' of a
  living organism.

6
The History of Virology

• First record of virus infection poliovirus in
  ancient Egypt (3700 BC).
• Pharoh Ramses V died from smallpox in 1196 BC.
• Smallpox was endemic in China by 1000 BC.
• The Chinese invented variolation early
  vaccination.
• breathed in dry infectious material or pus on a
  cut, prevented from getting sick
• Edward Jenner Smallpox vaccination.

7
The History of Virology

• Antony van Leeuwenhoek (1632-1723) constructed
  the first microscopes and saw bacteria.
• Robert Koch and Louis Pasteur (1880s) jointly
  proposed the 'germ theory' of disease.

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Kochs Postulates

• The agent must be present in every case of the
  disease.
• The agent must be isolated from the host and
  grown in vitro.
• The disease must be reproduced when a pure
  culture of the agent is inoculated into a healthy
  susceptible host.
• The same agent must be recovered once again from
  the experimentally infected host.

9
Discovery of bacteriophages

• Frederick Twort (1915) and Felix d'Herelle (1917)
  were the first to recognize bacteriophages
  ("eaters of bacteria").
• In the 1930s and subsequent decades, Salvador
  Luria, Max Delbruck and many others used these
  viruses as models.
• Important to understanding all types of virus.
• The history of virology is the development of
  experimental tools.

10
Living Host Systems

• 1881 Louis Pasteur, attenuation of rabies.
• 1885, inoculation with the first artificially
  produced virus vaccine.
• Whole plants have been used to study plant
  viruses ever since Tobacco mosaic virus was
  discovered by Iwanowski.

11
Living Host Systems

• 1900 Walter Reed demonstrated that yellow fever
  was caused by a virus and spread by mosquitoes.
• 1937 Max Theiler produced an yellow fever
  attenuated vaccine.
• 1930s-1950s animal systems to identify and
  propagate pathogenic viruses.

12
Tissue Culture

• Eukaryotic cells grown in vitro ("tissue
  culture").
• Embryonated hens eggs Influenza virus,
  Vaccinia virus.
• Counting the 'pocks' on the chorioallantoic
  membrane of eggs was the first quantitative assay
  for a virus.

13
Animal hosts still have their uses in virology

• For viruses which cannot be propagated in vitro
• Pathogenesis experiments
• Vaccine safety
• Nevertheless, they are increasingly being
  discarded
• Expensive
• Complex and difficult to interpret
• Host variation
• Ethical?
• Overtaken by cell culture and molecular biology

14
Cell Culture Methods

• Began early in the twentieth century with
  whole-organ cultures, then progressed to methods
  involving individual cells, either
• Primary cell cultures
• or
• Immortalized cell lines

15
Cell Culture Methods

• 1949 John Enders and colleagues propagate
  Poliovirus in primary human cell cultures.
• 1950s and 1960s many viruses.
• 1952 Renato Dulbecco - plaque assay.

16
Plaque Assays
17
Serological/Immunological Methods

• 1941 George Hirst - haemagglutination of red
  blood cells by influenza virus.
• An important tool for influenza and other
  viruses, for example, Rubella virus.
• Can measuring the titre (i.e. amount) of virus
  and determine the antigenic type.

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Serological/Immunological Methods

• Complement fixation tests
• Radioimmunoassays
• Immunofluorescence (direct detection of virus
  antigens in infected cells or tissue)
• Enzyme-linked immunosorbent assays (ELISAs)
• Radioimmune precipitation
• Western blot assays

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Serological Methods
20
MonoclonalAntibodies
21
Ultrastructural Studies

• Physical methods
• Chemical methods
• Electron microscopy
• 1930s Physical measurements of virus particles.
• 1960s Sedimentation properties of viruses in
  ultracentrifuges.

22
Differential Centrifugation
23
Physical Methods

• Spectroscopy
• Electrophoresis
• X-ray diffraction by crystalline forms of
  purified virus

24
Physical Methods

• Complete structures of many viruses at a
  resolution of a few angstroms (Å).
• Not all viruses!
• Nuclear magnetic resonance (NMR).
• Only relatively small molecules can be analysed
  with NMR technology.

25
Chemical Methods

• Stepwise disruption of particles.
• Electrostatic interactions
• Non-ionic, hydrophobic interactions.
• Proteins which interact with lipids.
• Surface labelling.
• Cross-linking reagents.

26
Denaturation of TMV
27
Electron Microscopy

• Electron microscopes.
• The first electron micrograph of a virus (TMV)
  was published in 1939.
• Direct examination of viruses at magnifications
  of over 100,000 times.
• Two types of electron microscope, the
  transmission electron microscope (TEM) and the
  scanning electron microscope (SEM).

28
ElectronMicroscopy
29
Molecular Biology

• The terms 'molecular biology, 'genetic
  engineering' and 'genetic manipulation' have
  taken on the meaning of manipulating nucleic
  acids in vitro.
• Virus infection has long been used to probe the
  working of 'normal' (uninfected) cells.
• This new technology shifted the emphasis from
  proteins to nucleic acids.

30
Hybridization Techniques

• Nucleic acid-centred technology offers
  significant advances in detection of viruses and
  virus infection.
• A labelled hybridization probe is allowed to
  react with a crude mixture of nucleic acids.
• The specific interaction of the probe with
  complementary virus-encoded sequences reveals the
  presence of virus genetic material.

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HybridizationTechniques
32
The PolymeraseChain Reaction(PCR)
33
Bioinformatics

• Computers are the ideal means of storing and
  processing nucleotide sequences.
• 'Bioinformatics' is a broad term used to describe
  any application of computers to biology.
• Specifically, the term applies to computer
  manipulation of biological sequence data.
• Bioinformatics permits the inference of function
  from a linear sequence.

34
Bioinformatics
35
Bioinformatics

• Computers are used to make predictions based on
  nucleotide sequences, including
• open reading frames
• amino acid sequences of proteins
• control regions of genes
• secondary structure of proteins and nucleic acids
• molecular modelling
• Vast databases of nucleotide and protein sequence
  information.

36
Three-dimensional structure of the DNA binding
domain of SV40 T-antigen
37
Summary

• Investigations of viruses - from particles via
  genomes back to proteins again - have come full
  circle.
• The present pace of research in virology shows
  that there is still far more we need to know.