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VIRAL MULTIPLICATION
Virus particle outside host cell have no independent metabolic activity and are incapable of reproduction by the
process characteristic of other microorganisms. Multiplication takes place by replication in which the viral protein
and nucleic acid components are reproduced within susceptible host cell.
The essential features common to the replication cycle of all viruses include entry into host cell, intracellular
reproduction to produce progeny virion, escape of these into the environment and survival there. All viruses that
are obligate intracellular parasitic pass through a superficially similar cycle, but there are fundamental
differences. Viruses do not reproduce by division rather they are replicated by a process in which all molecular
components are synthesized separately. Then, these are assembled into intact virion.
For a specific virus to replicate within host cell:
1. The host cell must be permissive and the virus must be compatible with the host cell,
2. The host cell must not degrade the virus,
3. The viral genome must possess the information for modifying the normal metabolism of the host cell,
4. The virus must be able to use the metabolic capabilities of the host cell to produce new virus particles
containing replicated copies of viral genome.
The steps of viral infection and replication are: a) Adsorption, b) Penetration and uncoating, c) Component
replication and biosynthesis, d) Assembly and e) Release.
Multiplication of T-Even Phages, Animal viruses and Plant viruses:
The infection cycle of Teven bacteriophage lasts
about 20 minutes,
culminating in lysis
(bursting-open) of the
host cell, E. coli. The
whole process can be
classified into, (i)
adsorption or infection.
(ii) Penetration or
injection, (iii) the eclipse
or the latent period (iv)
maturation and (v) lysis or
release.
(i) Adsorption or
infection:
Attachment of the virus
particle onto the surface
of the host cell is
adsorption or infection.
The virus particles possess
one or more proteins on
the outside that interact
with cell surface components called receptors; the receptors are normal surface components of the host (e.g.,
proteins, carbohydrates, glycoproteins, lipids, lipoproteins, etc.). In fact, these are the receptors that determine
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which cells will be susceptible to infection. In the absence of the receptor site, the virus cannot adsorb and hence
cannot infect. If the receptor site is altered, the host may become resistant to virus infection.
The animal virus particles get absorbed to the plasma membrane of the host cells by binding to specific sites
where the receptor proteins (usually glycoproteinsl are situated. The presence of these receptor proteins is
crucial in the viral infection and it may determine host resistance or susceptibility. The receptor proteins are
usually surface proteins necessary for the host cell, as these proteins are also receptors for hormones and other
important molecules which get into the cell and are essential to the cell's function. The virus mimics these
essential molecules and manages to get into the cell by endocytosis. Many host receptor proteins are related to
immunolgobulins. For example HIV CD4 receptor, and the polio ICAM (intercellular adhesion molecule) receptor.
Envelope glycoproteins may also be involved in adsorption in enveloped viruses. The herpes simplex virus has
two glycoproteins that are involved in adsorption. Spikes of some enveloped viruses also play similar roles
(myxovirus). For example, the influenza virus has two kinds of spikes, haemagglutinin, and neutraminidase. The
haemogglutinin (H spike) attach to the host cell receptor site and recognize siatic acid (N-acetyl neuraminic acid).
The N spike (Neuraminidase) helps the virus in penetrating the nasal and respiratory tract secretions by
degrading mucosal polysaccharides. However the receptor sites vary from person to person. Plant viruses enter
their host through the cell wall.
(ii) Penetration or Injection:
After the tail fibres get adsorbed, an enzyme-system is supposed to make a pore or hole in the cell wall of the
host. It is believed that the enzyme-system consists of a phage-lysozyme, which is synthesized during the
multiplication of the parent phage inside the host cell and its molecules remain attached to the extreme tip of
the tail-fibres of the new progeny phages.
In the T-phage penetration is activated when:
i) the tail fibers of the virus attach to the cell and hold the tail firmely against cll wall.
ii) The sheath contracts driving the core into cell through the cell wall and cell membrane ,
iii) The virus injects its DNA the way a syringe injects.
This enzymes system becomes active when the released phage particles infect the new host cell.
However, the tail-fibres attached on the surface of the host cell bend to bring the end-plate in contact
with the cell wall surface. Now, the protein sheath of the tail longitudinally contracts pushing the central
tubular core through the pore inside the wall of the host cell and the phage DNA molecule is released or
injected into the cytoplasm. After the DNA is released, the empty protein coat becomes of no use.
In animal viruses direct penetration or fusion with plasma membrane occurs during penetration.Some nonenveloped viruses such as the polio virus, undergo changes in capsid structure on adsorption to the plasma
membrane, and release only their nucleic acids intothe host cell. In the paramyxoviruses, and some other
enveloped viruses, the capsids fuse with host cell plasma membrane. Fusion occurs between the envelope
glycoprotins and the host plasma membrane proteins. Then the membrane lipids rearrange forming a
proteinaceous fusion pore. The nucelocapsid enters the host cell where uncoating take place.
Endocytosis: Enveloped viruses may enter the host cell in another way. The virions attach to specialized regions
on the membrane coated on the cytoplasmic side with protein clathrin. The coated regions pinch off to form
coated vesicles filled with virus particles. The vesicles fuse with lysosomes after the coating is removed. The
lysosomal enzymes help in theuncoating process.
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Uncoating: This is a process of separation of viral nucleic
acid from the protein coat. This process is not fully
understood. In some viruses the coating is done by
lysosomal enzymes of the host cell which degrade protein
coat and make the nucleic acid free in cytoplasm. In Pox
virus the viral DNA synthesizes a specific protein after
infection. Thus it varies with virus groups.
Plant viruses penetrate host cells through transient pores
(ectodesmata) which protrude through the cell wall at
intervals and communicate to the exterior to the cells.
Uncoating occurs inside the plant cells.
(iii) The Eclipse or the Latent Period/ Component
replication and biosynthesis:
When the DNA molecule is released in the host cytoplasm, it
is not degraded by the nuclease enzymes of the host cell. It
has been studied, particularly in T4, phage, that the phage
DNA contains glucosylated hydroxymethyl cytosine instead
of cytosine, which prevents the nucleases of the bacterium
from degrading the phage DNA. The phage DNA, first makes
the host cell immune against infection by genetically similar phage particles. Secondly, it immediately takes over
the charge of the cell machinery and suppresses all cellular activities such as synthesis of cellular DNA, RNA,
proteins, etc. This is the parasitism of a virus at the genetic level.
Transcription occurs in several stages leading to the formation of immediate early, delayed early and late gene
products.
i) The bacterial m-RNA and
protein stop being
synthesized within a few
minutes after the entry of
phage DNA.
ii) Bacterial DNA is quickly
degrade to small fragments
and the nucleoids
fragments of bacterium
become dispersed.
iii) Some phage m-RNA is
made immediately after
infection and the amount of
phage DNA increases after
the brief delay.
iv) Virus specific proteins appear somewhat late followed by appearance of organized capsid precursors and
resulting in the formation of mature infectious capsid.
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Immediate early phage genes are translated using the existing bacterial RNA polymerase and these genes code
for nucleases that break down host DNA and for the enzymes that alter bacterial RNA polymerases .
Delayed early genes code for phage enzymes which produce unique phage DNA constituent such as 5-hydroxy
methyl cytosine, which glycosates these nucleotides and which destroy cytosine deoxynucleotides so that no
bacterial cytosine will be incorporated into phage DNA. These alterations enable the phage to survive because
bacterial restriction enzymes are unable to degrade phage DNA modified by the substitution of 5-hydroxymethyl
cytosine.
Delayed early genes also code for a altered DNA polymerase that will transcribe the late genes.
Late genes products include the structural components of new phage particles such as heads, tail fibers and also
include lysozyme which will lyse the bacterial cell
releasing the mature virion.
The virulent animal viruses arrest all the functions
of the host cell such as DNA,RNA and protein
synthesis. The virus DNA replication usually takes
place in the host nucleus using host DNA
polymerase-II, except in the poxviruses (such as
vaccinia) whose genomes replicate in the
cytoplasm. In most viruses, early transcription
occurs using host enzymes (polymerases) except in
poxvirus where early mRNA is transcribed by a
viral polymerase.
The exact mechanisms for the replication of new
copies of the viral genome vary with different
types of viruses i.e; whether their nucleic acid is
DNA or RNA and their type of strands. Generally
the double stranded DNA viruses include those
capable of using host cell polymerases and RNA
viruses require viral coded polymerases.
(iv) Maturation/ Assembly: Assembly of the
various components to constitute a new phage
particle within the host cell is called maturation.
Head the tail formation start separately, the
protein components aggregate around the DNA
and form the head of the phage. End-plate is formed first followed by the formation of tubular core. Tail fibres
are formed later. Hundreds (about 200) of new phage particles are produced from each bacterium by the time of
lysis.
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The late expressing genes direct the synthesis of capsid proteins. Once enough protein and DNA are synthesized
the two will spontaneous assemble to form virus particles, as in the case plant viruses. In icosahedral virus
assembly, it appears that the
empty procapsids are first formed and then the nucleic acid is then inserted into the empty capsid in some
unknown way. The assembly of enveloped viruses follows the same pattern except in the case of pox viruses
which follow more complicated pattern and assemble in the cytoplasm rather than the nucleus.
v) Release.
After all components of virion
particles are assembled, the
bacterial cell bursts, releasing
new phages to infect other
bacteria and begin the cycle all
over again.
In
animal
viruses
,the
mechanism of virion release
differ between non-enveloped
(naked) and enveloped viruses.
The virions of naked viruses are
released by the lyse of the host
cell. In the enveloped viruses,
the formation of the envelope
and the release of the virus
particle is
a concurrent process. The viral
capsid proteins are first
attached to the plasma
membrane,
and
the
nucleocapsid is formed on the
membrane. The nucelocapsid is released by membrane budding, and the capsids carry the membrane in the
process of budding andreleased. Active filaments of the host cytoskeleton can aid in virion release.
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Lytic life cycle of T-even Bacteriophage
Picture Source: classes.midlandstech.edu
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The One-Step Growth Experiment
The development of the one-step growth experiment in 1939 by Max Delbrück and Emory Ellis marks the
beginning of modern bacteriophage research. In a one-step growth experiment, the reproduction of a large
phage population is synchronized so that the molecular events occurring during reproduction can be followed.
A culture of susceptible bacteria such as E. coli is mixed with bacteriophage particles, and the phages are allowed
a short interval to attach to their host cells. The culture is then greatly diluted so that any virus particles released
upon host cell lysis will not immediately infect new cells. This
strategy works because phages lack a means of seeking out host
cells and must contact them during random movement through the
solution. Thus
phages are less likely to contact host cells in a dilute mixture. The
number of infective phage particles released from bacteria is
subsequently determined at various intervals by a plaque count. A
plot of the bacteriophages released from host cells versus time
shows several distinct phases. During the latent period, which
immediately follows phage addition, there is no release of virions.
This is followed by the rise period or burst, when the host cells
rapidly lyse and release infective phages. Finally, a plateau is
reached and no more viruses are liberated. The total number of
phages released can be used to calculate the burst size, the
number of viruses produced per infected cell. The latent period is
the shortest time required for virus reproduction and release.
During the first part of this phase, host bacteria do not contain any complete, infective virions. This can be shown
by lysing them with chloroform. This initial segment of the latent period is called the eclipse period because the
virions were detectable before infection but are now concealed or eclipsed. The number of completed, infective
phages within the host increases after the end of the eclipse period, and the host cell is prepared for lysis. The
one-step growth experiment with E. coli and phage T2 provides a well-studied example of this process. When the
experiment is carried out with actively growing cells in rich medium at 37°C, the growth curve plateau is reached
in approximately 30 minutes. Bacteriophage reproduction is an exceptionally rapid process, much faster than
animal virus reproduction, which may take hours.
This sequence of events initiated by the infection of the phage nucleic acid and culminating in the release of
newly synthesized virion is termed viral multiplication cycle and can be used to follow the kinetics of synthesis of
viral components as well as intact virions
LYSOGENY
Lysogeny, or the lysogenic cycle, is one of two methods of viral reproduction (the lytic cycle is the other).
Lysogeny is characterized by integration of the bacteriophage nucleic acid into the host bacterium's genome. The
newly integrated genetic material, called a prophage can be transmitted to daughter cells at each subsequent cell
division, and a later event (such as UV radiation) can release it, causing proliferation of new phages via the lytic
cycle. The distinction between lysogenic and lytic cycles is that the spread of the viral DNA occurs through the
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usual prokaryotic reproduction, while the lytic phage is spread through the production of thousands of individual
phages capable of surviving and infecting other bacterium.
The process of lysogeny:
i) After penetration the viral chromosome directs the production of repressor protein that specifically binds to
the viral chromosome and turn off replication of viral DNA.
ii) The repressed viral DNA then integrates into and becomes the physical part of the host chromosome. The
integrated virus DNA is now called a prophage.
iii) The virus DNA replicates whenever the bacterial chromosomes doubles so all the progeny cells inherit one
copy of the prophage in the chromosome and this carry the potential for producing the temperate phage.
iv) An induction occurs occasionally when prophage detaches from bacterial DNA, produces progeny viruses
eventually lysing the host cells.
v) Induction occurs spontaneously but its frequency in increased by irradiation with UV light.
Certain types of viruses replicate by the lysogenic cycle, but also partly by the lytic cycle (mixed cycles). Some
DNA phages, called temperate phages, only lyse a small fraction of bacterial cells; in the remaining majority of
the bacteria, the phage DNA becomes integrated into the bacterial chromosome and replicates along with it. In
this lysogenic state, the information contained in the viral nucleic acid is not expressed. The model organism for
studying lysogeny is the lambda phage. Roughly 50-60 nucleotides are taken out of the lysogenic pathway and
used.
Advantages of Lysogeny to the Bacterial Cells:
i) The cells get protection from the lytic infection by the viruses of the same type as prophage.
ii) Bacteriophages may bring in new genes either by transduction or by lysogenic conversion .These new genes
may increase the bacterial ability to survive .E.g. Resistance to antibiotics.
Medical Use of Virulent Phage:
The primary uses of bacteriophages are: i) the identification of bacterial strains, ii) used as
genetic model in molecular biology.
The lytic phages have been used in the detection and identification of pathogenic bacteria.
Strains of bacteria may be characterized by their resistance and susceptibility to lysis by
specific virulent phage. The resulting pattern of lysis from visible plaques on a lawn of
bacterial growth by different phages gives an indication to the identification of strain.
Picture source: classes.midlandstech.edu
VIRUS CLASSIFICATION
In all viral taxonomic individual viruses are grouped by:
i) The nucleic acid they contain ( DNA or RNA )
ii) Single or double strandedness of nucleic acid
iii) Capsid morphology
iv) Presence or absence of envelope.
v)Host range.
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In some single stranded RNA viruses, the chromosome also serve as viral m-RNA termed as plus strand and while
in others transcribe RNA , complementary to virus NA is termed as minus strand RNA.
Animal, plant and bacterial viruses are usually classified separately. Animal viruses are often grouped into
families, genera and species which are given Latin names but English names are also used. A taxonomy
employing Latin family names have also been proposed for bacterial viruses but this system is rarely use. Plant
viruses are grouped on the basis of the structure of virion, whether they contain DNA or RNA and its mode of
transmission. In most cases the groups of plant viruses are named after a prominent representative. E.g. A group
of plant virus closely related to Tobacco mosaic is termed the Tobacco group.
Viruses have been traditionally named by adding the word 'virus' after the disease caused in the major host, e.g.
poliovirus, the causative agent of poliomyelitis.
Classification of Viruses on the basis of differentiations in their transcription process
Group
Description
I
II
III
IV
V
VI
VII
Double stranded DNA genome
Genome replication:dsDNA→ dsDNA
mRNA synthesis: dsDNA→mRNA
Single stranded DNA genome
Genome replication:ssDNA→ dsDNA→ ssDNA
mRNA synthesis: ssDNA→ dsDNA→ mRNA
Double stranded RNA genome
Replication: dsRNA→ssRNA→RNA
mRNA synthesis: dsRNA→mRNA
Plus stranded RNA genome
Replication:+RNA→-RNA→+RNA
mRNA synthesis::+RNA→=mRNA
Negative stranded RNA genome
Replication:-RNA→+RNA→-RNA
mRNA synthesis::-RNA→=mRNA
Single stranded RNA genome
Replication:ssRNA→dsDNA→ssRNA
mRNA synthesis::ssRNA→dsDNA→mRNA
Double stranded gaped DNA genome
Replication: gapped dsDNA → dsDNA→+RNA→-DNA → gapped dsDNA
mRNA synthesis: gapped dsDNA →dsDNA →mRNA
Bacteriophages were named after laboratory code symbols e,g. φX174, P22, T7 etc.
Holmes (1948) followed the Linnaeus system of binomial nomenclature. Viruses were grouped under the order
Virales, which was divided into three suborders:
Phaginae - infecting bacteria.
Phytophaginae - infecting plants.
Zoophaginae - infecting animals.
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Classification by the Provisional committee on nomenclature of Viruses ( Linnaeus classification scheme with
nomenclature).
Phylum: Vira
Subphylum: Deoxy vira ( DNA virus)
Class: Deoxyhelica (Helical Symmetry)
Order: Chitoviraes ( In Greek: Chitn – tunica or envelope)
Family: Poxviridae.
Phylum:Vira
Subphylum: Deoxy vira ( DNA virus)
Class: Deoxcubica (Cubical Symmetry)
Order: Haplovirales
Family: Microviridae-12 capsomeres.
Phylum: Vira
Sub phyum:Ribovira
Class: Ribo helica (Helical Symmetry)
Order:Rhabdovirales
Suborder:Rigidovirales
Family:Protovidae.
Suborder: Flexiviridales- Plant viruses
Family: Leptoviridae 10-11 nm
Phylum: Vira
Sub phyum:Ribovira
Class: Ribo cubica (cubical Symmetry)
Order:Gymnovirales
Family:Napoviridae
Properties that determine genera within family include i)capsomere: structure, antigenic properties, molecular
weight, ii)Capsid: number of capsomeres, antigenic properties, reaction to heat, pH, other physical and chemical
agents.
Additional features for generic and species differentiation include data on envelope or mantle enzymes, mode of
development, sensitivity to interferon, specificity for host virulence and clinical effect.
Family names end in –viridae, subfamily-virinae, genera like specis-virus.
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Typical Classification of Animal and Plant Viruses on the basis of their inherent Properties
Nucleic
Capsid
Acid Symmetry
RNA
RNA
RNA
RNA
DNA
DNA
DNA
Icasohedral
Icasohedral
Helical
Helical
Icasohedral
Icasohedral
Complex
Presence
of
Envelope
+
+
+
Coat
Virion
size in
nm
28
35-80
175-300
80-120
50
180-200
230-300
Family or Group
Typical Member
Poty virus
Toga viridae
Tobamo virus
Orthomyxoviridae
Cauliovirus
Herpes viridae
Poxviridae
Turnip yellow mosaic virus
Napha virus
Tobacco mosaic virus
Influenza virus
Cauliflower mosaic virus
Herpes virus
Pox virus
CULTIVATION OF VIRUSES
viruses cannot be cultured in the same way as bacteria and eukaryotic microorganisms. For many years
researchers have cultivated animal viruses by inoculating suitable host animals or embryonated eggs—fertilized
chicken eggs incubated about 6 to 8 days after laying .To prepare the egg for virus cultivation, the shell surface is
first disinfected with iodine and penetrated with a small sterile drill. After inoculation, the drill hole is sealed with
gelatin and the egg incubated. Viruses may be able to reproduce only in certain parts of the embryo;
consequently they must be injected into the proper region. For example, the myxoma virus grows well on the
chorioallantoic membrane, whereas the mumps virus prefers the allantoic cavity. The infection may produce a
local tissue lesion known as a pock, whose appearance often is characteristic of the virus.
More recently animal viruses have been grown in tissue (cell)
culture on monolayers of animal cells. This technique is made
possible by the development of growth media for animal cells
and by the advent of antibiotics that can prevent bacterial and
fungal contamination. A layer of
animal cells in a specially
prepared petri dish is covered
with a virus inoculum, and the
viruses are allowed time to
settle and attach to the cells.
The cells are then covered with
a thin layer of agar to limit
virion spread so that only
adjacent cells are infected by
newly produced virions. As a
result localized areas of cellular destruction and lysis called
plaques often are formed and may be detected if stained with dyes, such as neutral red or trypan blue, that can
distinguish living fromdead cells.
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Viral growth does not always result in the lysis of cells to form a plaque. Animal viruses, in particular, can cause
microscopic or macroscopic degenerative changes or abnormalities in host cells and in tissues called cytopathic
effects Cytopathic effects may be lethal, but plaque formation from cell lysis does not always occur.
Bacterial viruses or bacteriophages (phages for short) are cultivated in either broth or agar cultures of young,
actively growing bacterial cells. So many host cells are destroyed that turbid bacterial cultures may clear rapidly
because of cell lysis. Agar cultures are prepared by mixing the bacteriophage sample with cool, liquid agar and a
suitable bacterial culture. The mixture is quickly poured into a petri dish containing a bottom layer of sterile agar.
After hardening, bacteria in the layer of top agar grow and reproduce, forming a continuous, opaque layer or
“lawn.” Wherever a virion comes to rest in the top agar, the virus infects an adjacent cell and reproduces.
Eventually, bacterial lysis generates a plaque or clearing in the lawn, plaque appearance often is characteristic of
the phage being cultivated.
Plant viruses are cultivated in a variety of ways. Plant tissue cultures, cultures of separated cells, or cultures of
protoplasts may be used. Viruses also can be grown in whole plants. Leaves are mechanically inoculated when
rubbed with a mixture of viruses and an abrasive such as carborundum. When the cell walls are broken by the
abrasive, the viruses directly contact the plasma membrane and infect the exposed host cells. (The role of the
abrasive is frequently filled by insects that suck or crush plant leaves and thus transmit viruses.) A localized
necrotic lesion often develops due to the rapid death of cells in the infected area . Even when lesions do not
occur, the infected plant may show symptoms such as changes in pigmentation or leaf shape. Some plant viruses
can be transmitted only if a diseased part is grafted onto a healthy plant.
VIRUS (ENUMERATION OF VIRUSES) ASSAYS:
The quantity of viruses in a sample can be determined either by counting particle numbers or by measurement of
the infectious unit concentration. Although most normal virions are probably potentially infective, many will not
infect host cells because they do not contact the proper surface site. Thus the total particle count may be from 2
to 1 million times the infectious unit number depending on the nature of the virion and the experimental
conditions. Despite this, both approaches are of value. Virus particles can be counted directly with the electron
microscope. In one procedure the virus sample is mixed with a known concentration of small latex beads and
sprayed on a coated specimen grid. The beads and virions are counted; the virus concentration is calculated from
these counts and from the bead concentration. This technique often works well with concentrated preparations
of viruses of known morphology. Viruses can be concentrated by centrifugation before counting if the
preparation is too dilute. However, if the beads and viruses are not evenly distributed (as sometimes happens),
the final count will be inaccurate. the hemagglutination assay. Many viruses can bind to the surface of red blood
cells . If the ratio 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. In practice, red blood cells are mixed with a series of virus
preparation dilutions and each mixture is examined. The hemagglutination titer is the highest dilution of virus (or
the reciprocal of the dilution) that still causes hemagglutination. This assay is an accurate, rapid method for
determining the relative quantity of viruses such as the influenza virus. If the actual number of viruses needed to
cause hemagglutination is determined by another technique, the assay can be used to ascertain the number
of virus particles present in a sample. A variety of assays analyze virus numbers in terms of infectivity, and many
of these are based on the same techniques used for virus cultivation. For example, in the plaque assay several
dilutions of bacterial or animal viruses are plated out with appropriate host cells. When the number of viruses
plated out are much fewer than the number of host cells available for infection and when the viruses are
distributed evenly, each plaque in a layer of bacterial or animal cells is assumed to have arisen from the
reproduction of a single virus particle. Therefore a count of the plaques produced at a particular dilution will give
the number of infectious virions or plaque-forming units (PFU), and the concentration of infectious units in the
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original sample can be easily calculated. Suppose that 0.10 ml of a 10–6 dilution of the virus preparation yields 75
plaques. The original concentration of plaque-forming units is PFU/ml _ (75 PFU/0.10 ml)(106) _ 7.5 _ 108.
Viruses producing different plaque morphology types on the same plate may 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. The same approach employed in the plaque assay
may be used with embryos and plants. Chicken embryos can be inoculated with a diluted preparation or plant
leaves rubbed with a mixture of diluted virus and abrasive. The number of pocks on embryonic membranes or
necrotic lesions on leaves is multiplied by the dilution factor and divided by the inoculum volume to obtain
the concentration of infectious units. When biological effects are not readily quantified in these
ways, the amount of virus required to cause disease or death can be determined by the endpoint method.
Organisms or cell cultures are inoculated with serial dilutions of a virus suspension. The results are used to find
the endpoint dilution at which 50% of the host cells or organisms are destroyed . The lethal
dose (LD50) is the dilution that contains a dose large enough to destroy 50% of the host cells or organisms. In a
similar sense, the infectious dose (ID50) is the dose which, when given to a number of test systems or hosts,
causes an infection of 50% of the systems or hosts under the conditions employed.
VIRUS PURIFICATION
Purification makes use of several virus properties. Virions are very large relative to proteins, are often more
stable than normal cell components, and have surface proteins. Because of these characteristics, 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, and (4) enzymatic digestion of cell constituents.
1. Host cells in later stages of infection that contain mature virions are used as the source of material. Infected
cells are first disrupted in a buffer to produce an aqueous suspension or homogenate consisting of cell
components and viruses. Viruses can then be isolated by differential centrifugation, the centrifugation of a
suspension at various speeds to separate particles of different sizes . Usually the homogenate is first centrifuged
at high speed to sediment viruses and other large cellular particles, and the supernatant, which contains the
homogenate’s soluble molecules, is discarded. The pellet is next resuspended and centrifuged at a low speed to
remove substances heavier than viruses. Higher speed
centrifugation then sediments the viruses. This process may be
repeated to purify the virus particles further. Viruses also can
be purified based on their size and density by use of gradient
centrifugation . A sucrose solution is poured into a centrifuge
tube so that its concentration smoothly and linearly increases
between the top and the bottom of the tube. The virus
preparation, often after purification by differential
centrifugation, is layered on top of the gradient and
centrifuged. The particles settle under centrifugal force until
they come to rest at the level where the gradient’s density
equals theirs (isopycnic gradient centrifugation). Viruses can be
separated from other particles only slightly different in density.
Gradients also can separate viruses based on differences in
their sedimentation rate (rate zonal gradient centrifugation.
When this is done, particles are separated on the basis of both
size and density; usually the largest virus will move most
rapidly down the gradient. It that viruses differ from one
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another and cell components with respect to either density (grams per milliliter) or sedimentation coefficient(s).
Thus these two types of gradient centrifugation are very effective in virus purification.
2. Viruses, like many proteins, can be purified through precipitation with concentrated ammonium sulfate.
Initially, sufficient ammonium sulfate is added to raise its concentration to a level just below that which will
precipitate the virus. After any precipitated contaminants are removed, more ammonium sulfate is added and
the precipitated viruses are collected by centrifugation. Viruses sensitive to ammonium sulfate often are purified
by precipitation with polyethylene glycol.
3. Viruses frequently are less easily denatured than many normal cell constituents. Contaminants may be
denatured and precipitated with heat or a change in pH to purify viruses.
Because some viruses also tolerate treatment with organic solvents like butanol and chloroform, solvent
treatment can be used to 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 and
aqueous layers. The unaltered virus remains suspended in the aqueous phase while lipids dissolve in the organic
phase. Substances denatured by organic solvents collect at the interface between the aqueous and organic
phases.
4. Cellular proteins and nucleic acids can be removed from many virus preparations through enzymatic
degradation because viruses usually are more resistant to attack by nucleases and proteases than are free nucleic
acids and proteins. For example, ribonuclease and trypsin often degrade cellular ribonucleic acids and proteins
while leaving virions unaltered.
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Pass word: virus