Principles of Virology

Transcription

Welcome
to the lecture series
Principles of
Virology
Thomas Kietzmann
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Principles in Virology
Thomas Kietzmann
Viruses are everywhere
TK
• Viruses infect all living things
• We eat and breathe billions of virions regularly
• We carry viral genomes as part of our own genetic material
Thomas Kietzmann
TK
The number of viruses on Earth is staggering
More than 1030 bacteriophage particles in the world’s waters!
• A bacteriophage particle weights about a femtogram (10‐15 grams) ! 1030 X 10-15
= the biomass on the planet of BACTERIAL VIRUSES ALONE exceeds the
biomass of elephants by more than 1000-fold! !
•The length of a head to tail line of 1030 phages is 100 million light years!
http://www.phagehunter.org/2008/09/how‐far‐do‐those‐phages‐stretch.html
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There are ~1016 HIV genomes on the planet today
Thomas Kietzmann
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How infected are we?
• HSV-1, HSV-2, VZV, HCMV, EBV, HHV-6, HHV-7, HHV-8
• One infection persists the entire life
• HERVs; human endogenous retroviruses
HSV, Herpes simplexvirus; VZV, Varizella zoster virus; HCMV, Human cytomegalie virus; EBV, Ebstein-Barr virus; HHV,
Human herpes virus
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Viral infections in humans
Thomas Kietzmann
Viral reservoirs in humans
TK
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Thomas Kietzmann
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Viral entry sites in humans
Endogenous viral traces
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Thomas Kietzmann
The majority of the viruses that infect us have little or no
impact on our health
TK
Thomas Kietzmann
The “good” viruses
Virus group
Hosts
Beneficial effect
Type of mutualism
Polydnaviruses
Parasitoid
wasps
Required for survival of the wasp egg in its insect host
Symbiogenic
Retroviruses
Mammals
Involved in the evolution of the placenta
Symbiogenic
Pararetroviruses
Plants
Protect against pathogenic viruses
Symbiogenic
Herpesviruses
Humans
Suppress HIV infection
Conditional mutualism
Mice
Protect against bacterial infection
Parvoviruses
Aphids
Required for the development of wings
Phages
Bacteria
Allow the invasion of new territory by killing off competitors
Allow the invasion of mammalian hosts
Mutualism
Yeast viruses
Fungi
Allow the suppression of competitors
Fungal viruses
Fungi and
plants
Confer thermal tolerance to fungal endophytes and their plant hosts
Mutualism
Plant viruses
Plants
Confer drought and cold tolerance
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The “good” viruses
Thomas Kietzmann
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What is a virus?
An infectious, obligate intracellular parasite comprising genetic
material (DNA or RNA) surrounded by a protein coat and/or an
envelope derived from a host cell membrane
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The virion and the virus
A virus is an organism with two phases:
Virion
Virus and virus infected cell
Thomas Kietzmann
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The virion and the virus
Virion
The infectious particle that is designed for transmission of the
nucleic acid genome among hosts or host cells
Viruses
They are distinct biological entities with the following properties:
1. A virus is an infectious, obligate intracellular parasite.
2. The genetic material of a virus is either DNA or RNA.
3. The genetic material of a virus enters a host cell and directs the
production of the building blocks of new virus particles (called
virions).
4. New virions are made in the host cell by assembly of these
building blocks.
5. The new virions produced in a host cell then transport the viral
genetic material to another host cell or organism to carry out
another round of infection.
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Virions are passive molecular parasites!
They require the host as well as the virus
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Virions/Viruses are small
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Thomas Kietzmann
Virions/Viruses are small
Meter
Centimeter
Millimeter
Micrometer
Nanometer
Angstrom
100 m
10-2 m
10-3 m
10-6 m
10-9 m
10-10 m
10-12 m
1m
0.01 m
0.001 m
0.000001 m
0.000000001 m
0.0000000001 m
0.000000000001 m
1/100 m
1/1,000 m
1/1,000,000 m
1/1,000,000,000 m
1/10,000,000,000 m
1/1,000,000,000,000
m
hundreth of a
meter
thousandth of a
meter
millionth of a meter
billionth of a meter
ten billionth of a
meter
trillionth of a meter
Picometer
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How many viruses can fit on the head of a pin?
2 mm = 2000 microns
• 500 million Rhinoviruses
• 1x sneezing releases an aerosol containing enough viruses
to infect thousands of people
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How old are viruses?
• Estimates of molecular evolution place
some viruses among the dinosaurs
• Likely originated billions of years ago
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How old are viruses?
Ramses V, ~1196BC with smallpox
Ancient Egypt ~3700BC, the temple priest
Ruma shows typical clinical signs of
paralytic poliomyelitis.
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Thomas Kietzmann
Virus discovery
• Prevention of virus infections in practice
since the 11th century without
knowledge of agent
• Survivors of smallpox protected against
disease
• Variolation ‐ inoculation of healthy
individuals with material from a
smallpox pustule (Lady Montagu)
• 1790s --‐ experiments by Edward Jenner
in England establish vaccination
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Thomas Kietzmann
The 'germ theory' of disease
Robert Koch (1843-1910)
Louis Pasteur (1822-1895)
• The agent must be present in every case of the disease.
• The agent must be isolated from the host & 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.
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Thomas Kietzmann
Virus discovery
Martinus Beijerinick
(1851-1931)
Dmitri Iwanowski
(1864-1920)
• 1892 Ivanovsky
• 1898 Beijerinck: contagium vivum fluidum
• Virus: (latin: slimy liquid, poison)
Friedrich Loeffler (1852-1915)
• 1898 Loeffler & Frosch: agent of foot &
mouth disease is filterable
• Key concept: agents not only small, but
replicate only in the host, not in media
TK
Thomas Kietzmann
Virus discovery
Karl Landsteiner (1868-1943)
Erwin Popper (1879-1955)
Walter Reed (1851-1902)
1901 first human virus
• yellow fever virus
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•
•
•
•
•
In 1908 poliomyelitis was found to be
caused by a 'filterable agent'
1903 rabies virus
1906 variola virus
1908 chicken leukemia virus, poliovirus
1911 Rous sarcoma virus
1915 bacteriophages
1933 influenza virus ….and many more
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Virus classification
• Nature and sequence of nucleic acid in virion
• Symmetry of protein shell (capsid)
• Presence or absence of lipid membrane
(envelope)
• Dimensions of virion & capsid
Classical hierarchical system:
• Kingdom
• Phylum
• Class
• Order (--‐virales)
• Family (--‐viridae) Filoviridae (filovirus
family)
• Genus (--‐virus) Ebolavirus
• Species: Zaire ebolavirus
International Committee on Taxonomy
of Viruses (ICTV); Viruses in 7 orders, 96
families, 420 genera, 2618 species
BUT‐1030 virus particles in the oceans!
Thomas Kietzmann
TK
Two simple facts
• All viral genomes are obligate molecular parasites that
can only function after they replicate in a cell
• All viruses must make mRNA that can be translated by
host ribosomes: they are all parasites of the host protein
synthesis machinery
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The infectious cycle
Thomas Kietzmann
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The lytic viral life cycle
Phase
1. Adsorption/attachement
2. Penetration
3. Uncoating
Event
Specific binding between viral capsid proteins or envelope
proteins and specific receptors at the host cellular surface.
This specificity determines the host range of a virus.
Virions enter the host cell through receptor mediated
endocytosis or membrane fusion.
A process in which the viral capsid is removed and the viral
genomic nucleic acid is released
4. Component synthesis
Expression of the viral genome
Replication of the viral genome
5. Morphogenesis
Self assembly of the nucleocapsid from nucleic acids and
capsid and nucleo proteins
Budding: virus acquires its envelope, which is a modified piece
of the host's plasma or other, internal membrane
or
Lysis: a process that kills the cell by bursting its membrane
releasing virions without envelope
6. Release
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The infectious cycle
• A susceptible cell has a functional receptor for a given virus - the cell
may or may not be able to support viral replication
• A resistant cell has no receptor -it may or may not be competent to
support viral replication
• A permissive cell has the capacity to replicate virus - it may or may not
be susceptible
• A susceptible AND permissive cell is the only cell that can take up a
virus particle and replicate it
Thomas Kietzmann
TK
Propagation of viruses in the lab
• At first animal viruses could not be routinely propagated in cultured
cells
• Most viruses were grown in laboratory animals
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TK
Thomas Kietzmann
Studying the infectious cycle in cells
• Not possible before 1949 (animal viruses)
• Enders, Weller, Robbins propagate poliovirus in primary
cultures of human embryonic tissues
• Nobel prize, 1954
John F. Enders,
Thomas H. Weller,
Frederick C. Robbins
Thomas Kietzmann
TK
Virus-dependent cellular changes
1. Morphological changes (Cytopathic effect: CPE)
1.1
Altered shape
1.2
Appearence of inclusion bodies
1.3
Formation of syncytia
1.4
Nuclear shrinking
1.5
Rounding and vacuoles
1.6
Detachement of cells
1.7
Inclusion bodies
1.8
Clumps of ribosomes or chromatin
Formation of syncytia
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Cytopathic effect: CPE
Rounding and vacuoles, detachement of cells, dead cells
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Detection and quantification methods
How many viruses are in a specific sample?
Renato Dulbecco in 1952 was the first to accurately quantify
animal viruses using a plaque assay – Nobel prize 1975
• Dilutions of the virus are used to infect a cultured cell monolayer
• The cells are then covered with soft agar to restrict diffusion of the virus
• This results in localized cell killing and appearance of plaques after the
cell monolayer is stained
• Counting the number of plaques directly determines the number of
infectious virus particles applied to the plate.
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How many viruses are needed to form a plaque?
Thomas Kietzmann
TK
How many viruses are needed to form a plaque?
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Particle-to-PFU ratio
•
•
•
•
Number of virus particles in sample/number of infectious particles
~1 for many bacteriophages
High for many animal viruses
Complicates study of animal viruses
Thomas Kietzmann
TK
Particle-to-PFU ratio
A single particle can initiate infection (how do we know this?)
High particle-to-pfu ratio: not all viruses are successful. Why
not? -Damaged particles, Mutations
Complexity of infectious cycle: failure at any step prevents
completion
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Multiplicity of infection (MOI)
Number of infectious particles ADDED per cell
Not the number of infectious particles each cell receives
Add 107 virions to 106 cells = MOI of 10 but each cell does NOT
receive 10 virions
Infection depends on the random collision of virions and cells
When susceptible cells are mixed with virus, some cells are
uninfected, some receive one, two, three or more particles
The distribution of virus particles per cell is best described by the
Poisson distribution
Thomas Kietzmann
TK
P(k) = e‐mmk/k!
P(k): fraction of cells infected by k virus particles
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•
•
•
m: multiplicity of infection (moi)
uninfected cells: P(0) = e‐m
cells receiving 1 particle: P(1) = me‐m
cells multiply infected: P(>1) = 1-e-‐m(m+1)
[obtained by subtracting from 1 {the sum of all probabilities for
any value of k} the probabilities P(0) and P(1)]
Examples:
If 106 cells are infected at moi of 10: 45 cells are uninfected 450 cells receive 1
particle the rest receive >1 particle
If 106 cells are infected at moi of 1: 37% of the cells are uninfected 37% of the
cells receive 1 particle 26% receive >1 particle
If 106 cells are infected at moi of .001: 99.9% of the cells are uninfected
00.099% of the cells receive 1 particle (990) 00.0001% receive >1 particle
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Detection of viruses
• Hemagglutination
• Electron microscopy
• Viral enzymes
• Serology (RIA, ELISA…etc)
• Nucleic acids
• Crystallography
Thomas Kietzmann
TK
Hemagglutination
Influenza virus can bind to erythrocytes (red blood cells), causing the formation
of a lattice- this process is called hemagglutination
The red blood cells that are not bound by influenza virus sink to the bottom and
form a button.
The red blood cells attached to virus form a lattice that coats the well
Rapid quantitative assay, often first choice
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Immunostaining and immuno (Western) blotting
Thomas Kietzmann
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Enzyme linked immunosorbent assay (ELISA)
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PCR
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Deep, high throughput sequencing
• Metagenomics
• Identification of new viruses in environmental samples
• Identification of new pathogens
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Thomas Kietzmann
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Key terms and questions
What is a virion and what is a virus?
Given that viruses are part of the biosphere in which other
organisms exist, what/how may viruses exert pressure on
evolution?
How can viruses be detected and quantified?
Why is size not a good feature to classify a virus; what might
be a better feature?
What kind of molecular process is necessary and common in
every virus?
Viruses are called obligate intracellular parasite“; For which
step of gene expression do all viruses completely depend on
the host cell?
What kind of cellular changes are caused by viral infection?
What are the steps of a lytic viral infection cycle?
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Principles of Virology

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