virus lect

7/28/2019 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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virus lect

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