Download - Virus Lect
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General Virology
BCH-411
Dr.Kalsoom sughra
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OVER VIEW OF COURSE CONTENTS
Introduction, living or non living History, evolutional history, discovery
Isolation, purification- safety labs
Structure of viruses- schematic illustration of complete virus, capsid,
Capsomere , defective virus, envelop, nucleocapsid, structural unit, virions
Replication
Classification
Bacteriophages
Picornavirus
Reoviruses
Retroviruses- AIDS
Adenoviruses
Cancer or tumor viruses
Vaccines
Antiviral drugs
Major viral diseases
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WHAT IS VIRUS Viruses are simple, acellular obligate parasite.
They possess only one type of nucleic acid, either DNA or RNA, and only
reproduce within living cells.
Viruses [Latin word virus means poison or venom].Until nineteenth
century, harmful agents were often grouped together as viruses.
Study of all the characteristics of viruses, diseases caused by them along
with mechanism of infection is called virology.
Viruses can infect all forms of life (bacteria, plants, protozoa, fungi, insects,
fish, reptiles, birds, and mammals)
Common diseases
HIV AIDS, polio, small pox, mumpscommon cold, Chickenpox, rabies,
hepatitis
Tobacco mosaic, tomato wilt
Yellow dwarf of wheat, Leaf curl, Stenosis in cotton
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Discovery of virus
In 1982 Ivanowski experiments on tobacco mosaic diseaseshowed that leaf extracts from infected plants would inducedisease even after filtration to remove bacteria. The filtrate iscalled magic filtrate.
He attributed this to the presence of a toxin and proposed thatthe disease was caused by an entity different from bacteria, afilterable particle later called virus.
He observed that the virus would multiply only in living plantcells, but could survive for long periods in a dried state.
German scientists in 1899 Friedrich Loeffler and Paul Froschdiscovered Foot and mouth disease virus of farm and otheranimals.
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1901Yellow fever virusWalter Reed (the first human
virus) 1903Rabies virus (Remlinger, Riffat-Bay)
1906Variola virus (Negri)
1908 - Poliovirus (Karl Landsteiner and E. Popper); chicken
leukemia virus (Ellerman, Bang) 1911Rous sarcoma virus (Peyton Rous)
1915Bacteriophages -Frederik Twort, Felix DHerelle
1931Swine influenza virus (Shope)
1933Human influenza virus (Smith)
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Evolution of viruses
The exact origin of virus is difficult to speculate. The main problem is lack of fossil viruses
There are three main hypotheses regarding the originsof viruses:
The progressive hypothesis, or escape, hypothesisstates that viruses arose from genetic elements that
gained the ability to move between cells.
The regressive hypothesis, or reduction, hypothesis
asserts that viruses are remnants of cellular organisms. The virus-first hypothesis states that viruses coevolved
with their current cellular hosts.
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living or non living
Biological status of virus is an enigma as they have no cellar structure and
do not respond to the external stimuli. However they replicate and produce
progeny maintaining their genetic continuity.
Viruses are living because
(1) The presence of genetic material either DNA or RNA,(2) Have protein coat and necessary enzymes to reproduce.
(3) Their ability to reproduce in host cells
Viruses differ from living cells in at least three ways:
(1) Their simple, acellular organization
(2) The presence of either DNA or RNA, but not both, in almost all virions
(3) Their inability to reproduce independent of cells and carry out cell
division as prokaryotes and eukaryotes do.
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Characteristics
A complete virus particle or virion consists of one or more molecules of
DNA or RNA enclosed in a coat of protein, and sometimes also in other
layers.
These additional layers may be very complex and contain carbohydrates,
lipids, and additional proteins.
Viruses can exist in two phases:
Extracellular, possess few if any enzymes and cannot reproduce
independent of living cells
Intracellular, exist primarily as replicating nucleic acids that induce host
metabolism to synthesize virion components; eventually complete virusparticles or virions are released.
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Structure of virus
Viruses are extremely small, approximately 15 - 25 nanometers indiameter.
A virion consist of
nucleic acid core (DNA or RNA)
protein coat or capsid
Some viruses have envelop
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Isolation and Purification
Isolation and purification of viruses is important to and study theirstructure, reproduction, and other aspects of their biology. Thesemethods are so important that the growth of virology as a moderndiscipline depended on their development.
Virions are very large relative to proteins, more stable than normal
cell components, have surface Many techniques useful for the isolation of proteins and
organelles can be employed in virus isolation.
Four of the most widely used approaches are
(1) differential and density gradient centrifugation, (2) precipitation of viruses,
(3) denaturation of contaminants,
(4) enzymatic digestion of cell constituents.
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Differential centrifugation
Viruses can be isolated by differential centrifugation. Sepratedon the basis of size and density
Infected tissue or cells are first disrupted in a buffer to producean aqueous suspension or homogenate consisting of cellcomponents and viruses.
The homogenate is first centrifuged at high speed to sedimentviruses and other large cellular particles, and the supernatant,which contains the homogenates soluble molecules, isdiscarded.
The pellet is next resuspended and centrifuged at a low speedto remove substances heavier than viruses. Higher speedcentrifugation then sediments the viruses. This process may berepeated to purify the virus particles further.
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Differential centrifugation
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Gradient centrifugation
Viruses also can be purified based on their size and density by use ofgradient centrifugation.
A sucrose solution is poured into a centrifuge tube so that itsconcentration smoothly and linearly increases between the top andthe bottom of the tube making a gradient.
The virus preparation, often after purification by differentialcentrifugation, is layered on top of the gradient and centrifuged.
The particles settle under centrifugal force until they come to rest atthe level where the gradients density equals theirs (isopycnicgradient centrifugation).
Viruses can be separated from other particles only slightly differentin density. Gradients also can separate viruses based on differencesin their sedimentation rate (rate zonal gradient centrifugation).
Usually the largest virus will move most rapidly down the gradient
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Gradient centrifugation
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Precipitation with salts:
Viruses can be purified through precipitation with concentrated ammonium
sulphate. Initially, sufficient ammonium sulphate is added to raise its concentration to
a level just below that which will precipitate the virus.
precipitated contaminants are removed, more ammonium sulphate is added
and the precipitated viruses are collected by centrifugation.
Viruses sensitive to ammonium sulphate often are purified by precipitationwith polyethylene glycol.
Removal of cellular proteins by enzymes:
Viruses usually are more resistant to attack by nucleases and proteases than
are free nucleic acids and proteins.
Cellular proteins and nucleic acids can be removed from many virus
preparations through enzymatic degradation.
For example, ribonuclease and trypsin often degrade cellular ribonucleic
acids and proteins while leaving virions unaltered.
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Removal of cellular proteins by organic solvents
Viruses frequently are less easily denatured than manynormal cell constituents.
Some viruses also tolerate treatment with organic solventslike butanol and chloroform, solvent treatment can be usedto both denature protein contaminants and extract any
lipids in the preparation. The solvent is thoroughly mixed with the virus preparation,
then allowed to stand and separate into organic andaqueous layers.
The unaltered virus remains suspended in the aqueousphase while lipids dissolve in the organic phase.
Substances denatured by organic solvents collect at theinterface between the aqueous and organic phases
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Quantification of viruses The quantity of viruses in a sample can be determined by two
methods1. counting particle numbers
2. measurement of the infectious unit concentration.
Electron microscopy:
Virus particles can be counted directly with the electron microscope.
The virus sample is mixed with a known concentration of small latexbeads and sprayed on a coated specimen grid.
The beads and virions are counted
Virus concentration is calculated from these counts and from the bead
concentration .
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Advantage: This technique often works well withconcentrated preparations of viruses of known morphology.
Viruses can be concentrated by centrifugation beforecounting if the preparation is too dilute.
Disadvantage:
If the beads and viruses are not evenly distributed (assometimes happens), the final count will be inaccurate.
Although most normal virions are probably potentiallyinfective, many will not infect host cells because they donot contact the proper surface site.
Thus the total particle count may be from 2 to 1 milliontimes the infectious unit number depending on the nature ofthe virion and the experimental conditions
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Hemagglutination assay The most popular indirect method of counting virus particles.
Many viruses can bind to the surface of red blood cells. If theratio of viruses to cells is large enough, virus particles will join
the red blood cells together, forming a network that settles out of
suspension or agglutinates.
Red blood cells are mixed with a series of virus dilutions and
each mixture is examined.
The hemagglutination titer :
highest dilution of virus that still
causes hemagglutination.
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Advantage:
Accurate, rapid method for determining the relative
quantity of viruses such as the influenza virus. Can be used for quantitative measurements, if the
actual number of viruses needed to causehemagglutination is determined by any othertechniques.
Disadvantage:
Less sensitivity
Poor reproducibility
Influenza hemagglutination inhibition assay:
To check the antibody produced in blood serum
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Plaque assay:
Analyze virus numbers in terms of infectivity,
Several dilutions of bacterial or animal viruses are
plated out with appropriate host cells.
When the number of viruses plated out are muchfewer than the number of host cells available forinfection and when the viruses are distributed evenly,
each plaque in a layer of bacterial or animal cells isassumed to have arisen from the reproduction of asingle virus particle.
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Count of the plaques produced at a particular dilutionwill give the number of infectious virions or plaque
forming units (PFU). Suppose that 0.10 ml of a 106 dilution of the virus
preparation yields 75 plaques. The originalconcentration of plaque-forming units is
PFU/ml = (75 PFU/0.10 ml)(106) = 7.5 x 108. Viruses producing different plaque morphology can be
counted separately.
Although the number of PFU does not equal the
number of virus particles, their ratios are proportional. A preparation with twice as many viruses will have
twice the plaque forming units.
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The plaque assay in embryos and plants.
Chicken embryos can be inoculated
with a diluted preparation of virus.
The number of pocks on
embryonic membranes or necrotic
lesions on leaves is multiplied
by the dilution factor and
divided by the inoculums
volume to obtain the
concentration of infectious units.
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Advantage: This method is used to find LD50 andID50
The lethal dose (LD50) is the dilution that contains adose large enough to destroy 50% of the host cells ororganisms.
the infectious dose (ID50) is the dose which, whengiven to a number of test systems or hosts, causes an
infection of 50% of the systems or hosts under theconditions employed.
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Structure of virus
Viruses are extremely small, approximately 15 - 25 nanometers indiameter.
A virion consist of
nucleic acid core (DNA or RNA)
protein coat or capsid
Some viruses have envelop
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Techniques to study virus structure: electron microscopy, X-ray
diffraction, biochemical analysis and immunology
There are four general morphological types of capsids and virionstructure.
Icosahedral ; An icosahedron is a regular polyhedron with 20
equilateral triangular faces and 12 vertices
Helical; shaped like hollow protein cylinders, which may be either
rigid or flexible
Envelope an outer membranous layer surrounding the nucleo-
capsid. Enveloped viruses have a roughly spherical but somewhat
variable shape even though their nucleo-capsid can be either
icosahedral or helical Complex viruses have capsid symmetry that is neither purely
icosahedral nor helical. They may possess tails and other structures
(e.g., many bacteriophages) or have complex, multilayered walls
surrounding the nucleic acid (e.g., poxviruses such as vaccinia).
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Structure of virus
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Helical viruses
Helical capsids are shaped much like hollow tubes with protein
walls. A single type of protomer associates together in a helical or
spiral arrangement
The RNA genetic material is wound in a spiral and positioned
toward the inside of the capsid where it lies within a grooveformed by the protein subunits.
TMV virus helix is rigid tube,
15 -18 nm in diameter and
300 nm long.
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The size of a helical capsid is influenced by both its
protomers and the nucleic acid enclosed within the
capsid.
The diameter of the capsid is a function of the size,
shape, and interactions of the protomers
The nucleic acid determines helical capsid lengthbecause the capsid does not seem to extend much
beyond the end of the DNA or RNA.
Not all helical capsids are as rigid as the TMV capsid.
Influenza virus RNAs are enclosed in thin, flexiblehelical capsids folded within an envelope.
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The icosahedron is one of natures favourite shapes
most efficient way to enclose a space
Few genes code for proteins that selfassemble to form the
capsid.
Small number of linear genes can specify a large three-
dimensional structure
Icosahedral viruses
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The capsids are constructed from ring or knobshaped
units called capsomers.
Each usually made of five or six protomers. Pentamers or pentons have 5 subunits;
hexamers or hexons have 6
Pentamers are at the vertices
of the icosahedron
hexamers form its edges and
triangular faces
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In many plant and bacterial RNA viruses, both the
pen-tamers and hexamers of a capsid are constructed
with only one type of subunit, whereas adenoviruspentamers are composed of different proteins than are
adenovirus hexamers
Protomers join to form capsomers through
noncovalent bonding.
The bonds between proteins within pentamers and
hexamers are stronger than those between separate
capsomers. Empty capsids can even dissociate into separate
capsomers.
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Most icosahedral capsids contain both pentamers and
hexamers except SV-40 that contains only pentamers.
The virus is constructed of 72 cylindrical pentamerswith hollow centers.
Five flexible arms extend from the edge of each
pentamer
h i h d i i i h
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12 pentamers at the icosahedrons vertices - associate with 5neighbors.
Each of the 60 nonvertex pentamers associates with its 6adjacent neighbors .
An arm extends toward the adjacent vertex pentamer(pentamer 1) and twists around one of its arms. Three morearms interact in the same way with arms of other nonvertex
pentamers (pentamers 3 to 5).
The fifth arm binds directly to an adjacent nonvertex
pentamer (pentamer 6) but does not attach to one of its arms.
An arm does hold pentamer 2 in place.
Thus an icosahedral capsid is assembled
without hexamers by using flexible arms
as ropes to tie the pentamers together.
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Bounded by an outer membranous layer called an envelope .
Animal virus envelopes usually arise from host cell nuclear or
plasma membranes
Lipids and carbohydrates are normal host constituents.
Envelope proteins are coded by virus genes and may even
project from the envelope surface as spikes or peplomers.
These spikes may be involved in virus attachment to the host
cell surface.
Spikes differ among viruses, they also can be used to identify
some viruses.
Enveloped viruses
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Because the envelope is a flexible, membranousstructure, enveloped viruses frequently have a
somewhat variable shape and are calledpleomorphic.
Bullet-shaped rabies virus are firmly attached tothe underlying nucleocapsid and endow the virion
with a constant, characteristic shape . ether sensitive viruses.
RHABDOVIRUS HIV HERPES
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Influenza virus is a well studied example of an enveloped
virus.
Spikes project about 10 nm from the surface at 7 - 8 nm
intervals.
Hemagglutinin spikes
Neuraminidase spikes
Glycoproteins
A non-glycosylated protein,
the M or matrix protein.
Ribonucleoprotein
RNA
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Enzyme neuraminidase, help in penetratingmucous layers of the respiratory epithelium to
reach host cells. Hemagglutinin spikes have hemagglutinin
proteins responsible for binding to RBCmembranes and cause hemagglutination
Hemagglutinins participate in virion attachmentto host cells.
Glycoprotein -the proteins have carbohydrate
attached to them are on the outer envelop surface. M or matrix protein, is found on the inner surface
of the envelope and helps stabilize it.
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The influenza virus uses RNA as its genetic material
and carries RNA-dependent RNA polymerase that acts both as a
replicase and as an RNA transcriptase that synthesizes
mRNA under the direction of its RNA genome.
The polymerase is associated with ribonucleoprotein.
Although viruses lack true metabolism and cannot
reproduce independently of living cells, they may
carry one or more enzymes essential to thecompletion of their life cycles.
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Complex Viruses
Combination of icosahedral and helical shape and may have a
complex outer wall or head-tail morphology. The poxviruses andlarge bacteriophages
The poxviruses are the largest of the animal viruses (400 x 240 x 200nm)
visible in phase-contrast microscope or in stained preparations.
Complex internal structure with an ovoid to brick shaped exterior. dsDNA associated with proteins in the nucleoid,
A biconcave disk and surrounded
by a membrane.
Two elliptical or lateral bodiesb/w nucleoid and its outer envelope,
a membrane and a thick layer covered
by an array of tubules or fibers.
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Large bacteriophages like T2, T4, and T6 contains headresembles an icosahedron elongated by one or two rows of
hexamers in the middle and contains the DNA genome.
The tail is composed of
a collar joining it to the head,
a central hollow tube,
a sheath surrounding the tube,
a complex baseplate.
Tail fibers
Binal symmetry;
a combination of
icosahedral (the head) and
helical (the tail) symmetry.
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The sheath is made of 144 copies of the gp18 protein arranged
in 24 rings, each containing 6 copies.
In T-even phages, the baseplate is hexagonal and has a pin anda jointed tail fiber at each corner.
The tail fibers are responsible for virus attachment to the
proper site
Coliphages have true icosahedral heads. T1, T5, and lambda phages have sheathless tails that lack a
base plate and terminate in rudimentary tail fibers.
Coliphages T3 and T7 have short, noncontractile tails without
tail fibers. Viruses can complete their reproductive cycles using a variety
of tail structures.
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Nucleic acid
They employ all four possible nucleic acid types: single-stranded DNA, parvoviruses
double-stranded DNA, X174 and M13 bacteriophages
single-stranded RNA,
positive strand RNA viruses, Polio, tobacco mosaic, bromemosaic, and Rous
Negative strand RNA viruses; mumps, measles, and influenza
viruses
double-stranded RNA, reoviruses All four types are found in animal viruses.
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