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The Viruses Part I: Introduction & General Characteristics
Lecture #11Bio3124
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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
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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
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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
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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
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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 virusesEnveloped vs naked viruses
enveloped viruses: surrounded by membrane naked viruses: do not have envelope
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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
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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
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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
Capsid proteins
DNA or RNA coiled inside tube
Helical Capsids
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Influenza Virus – Enveloped Virus with a Helical Nucleocapsid
Helical symmetry Segmented
genome 8 RNA genome
segments
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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
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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
Vaccinia virus
200x400x250 nm, enveloped virus DNAWith double membrane envelope.
Binal symetry: head icosahedron, tail helicalTail fibers and sheath used for binding and pins for injecting genome
Phage T4
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Viral Life Cycles
All viruses must:1. Attach to host cell
2. Get viral genome into host cell
3. Replicate genome
4. Make viral proteins
5. Assemble capsids
6. Release progeny viruses from host cell
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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
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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
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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
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Lambda phage Life Cycle
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Lytic and Lysogenic life cycles
Animation: Lysis and Lysogeny
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Use cell components to synthesize capsids Assemble progeny phages Exit from cell Lysis:
Makes protein to depolymerize peptidoglycanBursts host cell to release progeny phage
Slow releaseFilamentous phages can extrude individual
progeny through cell envelope
Bacteriophage Life Cycles
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Eukaryotic Virus Life Cycles
Attachment to host cell receptor Entry into cell
Taken up via endocytosisBrought into cell in an endosome
Fuses envelope to plasma membraneReleases capsid into
cytoplasm
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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
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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
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Eukaryotic Virus Life CyclesRelease 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
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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
The Cultivation of Viruses
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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
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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
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Particle counts
direct countsdirect counts made with an
electron microscope
indirect countsindirect counts e.g., hemagglutination assay
determines highest dilution of virus that causes red blood cells to clump together
virus particles
Latex bead
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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
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Hemagglutination Titre
1:11:21:41:81:161:321:641:1281:5121:10241:20481:4096
Titre is 512 HU
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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)
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Titre of Infectious Viruses: Plaque Assay
Infecting cellular monolayers or bacterial lawn with
different viral dilutions.
Counting the number of plaques from different
dilutions
RationalRational: Each plaque is formed when a host cell
has been infected by a viral particle
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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.
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330 33 3
Dilution factor
33 PFU/0.1ml from a dilution of 10-4. Thus the titer of the original suspension is?
3.3 X 103.3 X 1066 PFU PFU//mLmL
Calculation of virus titre:
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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