Transcript The Viruses Part I - Université d`Ottawa

The Viruses
Part I: Introduction & General Characteristics
Lecture #11
Bio3124
Viruses are ancient
 many epidemics of viral diseases occurred before anyone understood
the nature of their causative agents.
 measles and smallpox viruses were among the causes for the decline
of the Roman Empire
Paralytic infection by Poliovirus
Discovery of Viruses
 Charles Chamberland (1884)
 developed porcelain bacterial filters, viruses can pass
through
 Dimitri Ivanowski (1892)
 demonstrated that causative agent of tobacco mosaic
disease passed through bacterial filters
 thought agent was a toxin
 Martinus Beijerinck (1898-1900)
 showed that causative agent of tobacco mosaic disease was
still infectious after filtration
 referred to as filterable agent
 Loeffler and Frosch (1898-1900)
 showed that foot-and-mouth disease in cattle was caused by
filterable virus
Discovery of Viruses…
 Walter Reed (1900)
 yellow fever caused by filterable virus transmitted by
mosquitoes
 Ellerman and Bang (1908)
 leukemia in chickens was caused by a virus
 Peyton Rous (1911)
 muscle tumors in chickens were caused by a virus
 Frederick Twort (1915)
 first to isolate viruses that infect bacteria
(bacteriophages or phages)
 Felix d’Herelle (1917)
 firmly established the existence of bacteriophages
 devised plaque assay
 bacteriophages only reproduce in live bacteria
What is a Virus?
 Not living
 Are intracellular parasites
 Depends on host metabolism
 Energy, materials, enzymes
Virion: a complete virus particle
 has a genome
 DNA or RNA, single- or double-stranded
 has a protein coat
 “Capsid”
 Protects genome
 Mediates host attachment
The Structure of Viruses
 ~10-400 nm in diameter ; too small to be seen with the light
microscope
 Contain a nucleocapsid which is composed of nucleic acid
(DNA or RNA) and a protein coat (capsid)
 some viruses consist only of a nucleocapsid, others
have additional components
 Enveloped vs naked viruses
 enveloped viruses: surrounded by membrane
 naked viruses: do not have envelope
Viral Envelopes and Enzymes
 Envelope: outer, flexible, membranous layer
 spikes or peplomers virally encoded proteins, may
project from the envelope
 Neuraminidase
releases mature virions
from cells
 Hemagglutinin binds
cellular receptor
 RNA dependent RNA pol
Replicates – sense genome
Influenza virus
Capsids
 large macromolecular structures which serve as
protein coat of virus
 protect viral genetic material and aids in its transfer
between host cells
 made of protein subunits called protomers
 Protmers form capsomers that arrange
symmetrically to form the coat
 Symmetry in capsid
 Helical
 Icosahedral
 complex
Helical Capsids
 Filamentous capsids
 Long tube of protein, with genome inside
 Tube made up of hundreds of identical protein
subunits
 Tube length reflects size of viral genome
DNA or RNA coiled
inside tube
Capsid proteins
Influenza Virus – Enveloped Virus with a Helical Nucleocapsid
 Helical
symmetry
 Segmented
genome
 8 RNA genome
segments
Icosahedral Capsids
 Icosahedral capsids




20 triangular sides
Each triangle made up of at least 3 identical capsid proteins
Arranged in 2,3 and 5 fold symmetry
Many animal viruses
Viruses with Capsids of Complex Symmetry
 some viruses do not fit into helical or icosahedral capsids
symmetry groups
 examples are the poxviruses and large bacteriophages
Phage T4
Vaccinia virus
200x400x250 nm, enveloped virus DNA
With double membrane envelope.
Binal symetry: head icosahedron, tail helical
Tail fibers and sheath used for binding and
pins for injecting genome
Viral Life Cycles
 All viruses must:
1.
2.
3.
4.
5.
6.
Attach to host cell
Get viral genome into host cell
Replicate genome
Make viral proteins
Assemble capsids
Release progeny viruses from host cell
Bacteriophage Life Cycles
 Attach to host cell receptor proteins
 Inject genome through cell wall to cytoplasm
 Replicate genome
 Lytic vs. lysogenic cycle
 Synthesize capsid proteins
 Assemble progeny phage
 Lyse cell wall to release progeny phage
 “Blows apart” host cell
 Some phages use slow, non-lytic release
Bacteriophage Life Cycles
 Attachment to host cell
proteins
 receptors normally used
for bacterial purposes
 Examples: sugar
uptake, iron uptake,
conjugation
 Virus takes advantage of
host proteins
 Injects genome through cell
wall to cytoplasm
Bacteriophage Life Cycles
 Lytic cycle
 Phage quickly replicates, kills host cell
 Generally lytic when host cell conditions are good
– Bacteria divide quickly, but phage replicates
even faster
 Or conditions are very bad (e.g., cell damaged)
 Lysogenic cycle
 Phage is quiescent
 May integrate into host cell genome
 Replicates only when host genome divides
 Generally lysogenic in moderate cell conditions
 Phage can reactivate to become lytic, kill host
Lambda phage Life Cycle
Lytic and Lysogenic life cycles
Animation: Lysis and Lysogeny
Bacteriophage Life Cycles
Use cell components to synthesize capsids
 Assemble progeny phages
 Exit from cell
 Lysis:

 Makes
protein to depolymerize peptidoglycan
 Bursts host cell to release progeny phage

Slow release
 Filamentous
phages can extrude individual
progeny through cell envelope
Eukaryotic Virus Life Cycles
 Attachment to host cell receptor
 Entry into cell
 Taken up via endocytosis
 Brought into cell in an endosome
 Fuses envelope to plasma membrane
 Releases capsid into
cytoplasm
Eukaryotic Virus Life Cycles
 Genome replication
 DNA viruses must go to cell nucleus to use host
polymerase
 Or replicate in cytoplasm with viral polymerase
 RNA viruses must encode a viral polymerase
 Host cells cannot read RNA to make more RNA
 dsRNA and (+)ssRNA genome can be translated
 (-)ssRNA and retrovirus genomes must be
replicated to be translated
–Only (+)ssRNA can be used as mRNA
Eukaryotic Virus Life Cycles
 All viruses make proteins with host ribosomes
 Translation occurs in cytoplasm
 Assembly of new viruses
 Capsid and genome
 Assembly may occur in cytoplasm
 Or in nucleus
 Capsid proteins must move into nucleus
 Envelope proteins are inserted in host
membrane
 Plasma membrane or organelle membrane
Eukaryotic Virus Life Cycles
Release of progeny viruses from host cell
 Lysis of cell, similar to bacteria
 Budding
 Virus passes through membrane
 Membrane lipids surround capsid to form
envelope
 All enveloped viruses bud
from a membrane
 Plasma membrane
or organelle membrane
The Cultivation of Viruses
 Infection of a living host (animal or plant)
 embryonated eggs
 tissue (cell) cultures
 monolayers of animal cells
 plaques
 localized area of cellular destruction and lysis
 cytopathic effects
 microscopic or macroscopic degenerative changes
or abnormalities in host cells and tissues
Hosts for Bacterial and Archael Viruses
 usually cultivated in broth or agar cultures
actively growing bacteria
 broth cultures lose turbidity as viruses
reproduce
 plaques observed on agar cultures
Virus Assays
 used to determine quantity of viruses in a sample
 two types of approaches
 direct
 count particles
 indirect
 measurement of an observable effect of the
virus
Particle counts
 direct counts
virus particles
 made with an
electron
microscope
 indirect counts
Latex bead
 e.g., hemagglutination assay
 determines highest dilution of virus
that causes red blood cells to
clump together
Indirect Counts: Hemagglutination Test
 Measures minimal viral quantity needed for agglutination of RBC
 Relative Concentration.
 Good for viruses that express hemagglutinin on the envelope; e.g.
Influenza virus, paramyxoviruses, adenovirus.
 Doesn’t distinguish between infectious and non-infectious particles.
 Simple and Fast.
 Dilution series of virus is prepared and mixed with chicken RBC in a
microtitre plate
 Hemagglutination is detected by RBC/virus lattice formation that does
not sink to the bottom of the wells
Hemagglutination Titre
1:4096 1:2048 1:1024 1:512 1:128
1:64 1:32
1:16
Titre is 512 HU
1:8
1:4
1:2
1:1
Measuring concentration of infectious units
 plaque assays
 dilutions of virus preparation made and plated
on lawn of host cells
 number of plaques counted
 results expressed as plaque-forming units (PFU)
Titre of Infectious Viruses: Plaque Assay
 Infecting cellular monolayers or bacterial lawn with
different viral dilutions.
 Counting the number of plaques from different
dilutions
 Rational: Each plaque is formed when a host cell
has been infected by a viral particle
Plaques assay: virus titre
 Localized cytopathic effect.
 Results in death or cell lysis
 Virions released from the infected cell
infect the nearby cells and infection
spreads radially
 Cleared areas (plaques) become visible
within uninfected monolyer or bacterial
lawn
 Each plaque represents a focus of
infection.
 Each focus of infection is initiated by an
infected cell.
Calculation of virus titre:
3.3 X
Dilution factor
330
6
10
PFU/mL
33
 33 PFU/0.1ml from a dilution of 10-4.
 Thus the titer of the original suspension is?
3
Culturing Viruses
 Viruses grown with host cells as food

Viruses bound to host
 Free
virus
concentration drops

Eclipse period
 Viruses
making
proteins, genomes,
assembling

Rapid rise period
Burst of bacteriophage = bacterial lysis
 Rapid release of eukaryotic viruses
