VIRUSES

Smallpox was endemic in China by 1000BC. In response, the practice of variolation was developed. Recognizing
that survivors of smallpox outbreaks were protected from subsequent infection, variolation involved inhalation of
the dried crusts from smallpox lesions like snuff, or in later modifications, inoculation of the pus from a lesion
into a scratch on the forearm of a child.

Dimitri Ivanovsky in 1890, who demonstrated that sap of the leaves infected with tobacco mosaic disease
retains its infectious property even after its filtration through a Chamberland filter. He is regarded as FATHER OF
VIROLOGY.

The third scientist who plays an important role in the development of the concept of viruses was Martinus
Beijerinck (1851-1931), he extended the study done by Dimitri Ivanofsky and showed that filterable agent
form the infectious sap could be diluted and further regains its strength after replicating in the living host; he
called it as “contagium vivum fluidum” .

Loeffler and Frosch discovered the first animal virus, the foot and mouth disease virus in 1898 and
subsequently Walter Reed and his team discovered the yellow fever virus, the first human virus from Cuba
in1901. Poliovirus was discovered by Landsteiner and Popper in 1909 and two years later Rous discovered
the solid tumor virus which he called Rous sarcoma virus.

Felix d'Herelle discovered bacteriophage and developed the assay to titrate the viruses by
plaques. Wendell Stanley (1935) first crystallized the TMV in 1939. In 1933 Shope described the first
papillomavirus in rabbits. The vaccine against yellow fever was made in 1938 by Thieler and after 45 years of
its discovery, polio virus vaccine was made by Salk in 1954.

Acellular Agents

• Viruses – protein and nucleic acid

• Viroids – only RNA. Viroids are infectious agents composed of closed, circular ssRNAs. They require host cell
DNA-dependent RNA polymerase to replicate & cause plant diseases.

• Satellites – only nucleic acids. Satellite RNAs/DNAs do NOT encode their own capsid proteins. Encode one or
more gene products. Require a helper virus for replication. Ex. human hepatitis D is satellite requires human
hepatitis B virus

• Prions – proteins only. Cause a variety of degenerative diseases in humans and animals. Ex. scrapie in sheep,
bovine spongiform encephalopathy (BSE) or mad cow disease, Creutzfeldt-Jakob disease (CJD) and variant CJD
(vCJD) in humans & kuru in humans.

Current Model of Disease Production by Prions: PrP C (prion protein) is present in “normal” form (abnormal form
of prion protein is PrP Sc). PrP Sc causes PrP C protein to change its conformation to abnormal form. Newly
produced PrP Sc molecules convert more normal molecules to the abnormal form through unknown mechanism.
Interaction of PrP Sc with PrP C may cause PrP C to crosslink and trigger apoptosis. PrP C conversion causes
neuron loss.

General Concepts: Virus diversity

1. Viruses are obligate intracellular parasites. Viruses cannot make energy or proteins independent of a
host cell. Virus genomes consist of nucleic acid, and which obligately replicate inside host cells using
host metabolic machinery and ribosomes to form a pool of components which assemble into particles
called VIRIONS. Viruses lack the enzymes necessary for protein and nucleic acid synthesis

2. Viruses are filterable Agents. Typically viruses are made up of coat (or capsid) that protects its
information molecule (RNA or DNA). Viral genome are RNA or DNA but not both.

3. Viruses are acellular organisms whose occupy the twilight zone that separates the ‘living’ from the
‘nonliving’

4. Major cause of disease. Also importance as a new source of therapy. New viruses are emerging

5. Important members of aquatic world. Move organic matter from particulate to dissolved
 6. Important in evolution. Transfer genes between bacteria, others.

7. Important model systems in molecular biology

Virology as a discipline is merely 100 years old. To group the new emerging viruses in a specific group by
specifying certain parameters was initiated in 1966 when international committee on the taxonomy of viruses
(ICTV) was formed with the aim to classify the viruses. The ICTV has adopted a norm for the description of the
viruses. Name for genera, subfamilies, families, and orders must all be a single word, ending with the
suffixes -virus , -virinae , -viridae , and -virales respectively. In written usage, the name should be
capitalized and italicized.

Viruses are obligate parasite which means their absolute dependence on living host system. Adenovirus is
an example of DNA virus that enters the host nucleus but remains separated from the host genome and at the
same time use host cell machinery for its replication. On the other hand influenza is a RNA virus that carries its
own enzyme to replicate its genome while the viral proteins are synthesized by using the host cell machinery.
Human immunodeficiency virus (HIV) is a retrovirus ; it contains RNA as a genetic material but it converts into
DNA after entering the host cell by an enzyme called reverse transcriptase . It also contains enzymes in its
virion namely, integrase and viral protease which helps HIV during maturation process inside the infected
cells. It was first erroneously thought that all virions lacked enzymes. Now accepted that a variety of virions
have enzymes. Some are associated with the envelope or capsid but most are within the capsid. Outer surface
of HIV virion contains two surface glycoproteins called as gp120 and gp41 which helps in the attachment of
virus to the cell surface.

General Concepts: Virus Shapes

Early study with tobacco mosaic virus (TMV) strongly suggested that viruses were composed of repeating
subunits of protein which was later supported by crystallization of TMV. A major advancement in determining the
morphology of virus was the development of negative stain electron microscopy . Another modification of
classical electron microscopy is cryo-electron microscopy where the virus containing samples were rapidly
frozen and examined at a very low temperature; this allows us to preserve the native structure of the viruses.

A virion is a complete virus particle that is surrounded by the capsid protein and encapsidates the viral genome
(DNA or RNA). Sometime structure without nucleic acid can be visible under the electron microscope those
structures are called as empty capsids . In some of the viruses like paramyxoviruses the nucleic acid is
surrounded by the capsid proteins and the composite structures are referred as nucleocapsid . Some of the
viruses contain the lipid envelope which surrounds the nucleocapsids. The envelopes are derived from the host
cell membrane during the budding process. As the envelopes are derived from host cell membrane they
contain many of the surface proteins present in the host cells.

There are two kinds of symmetry found among the viruses: icosahedral and helical . Icosahedral symmetry
has 12 vertices , 30 edges , and 20 faces . An icosahedron is a geometric shape with 20 sides, each
composed of an equilateral triangle, and icosahedral viruses increase the number of structural units in each face
to expand capsid size. An icosahedron has what is referred to as 2–3–5 symmetry, which is used to describe the
possible ways that an icosahedron can rotate around an axis. Two types of capsomers constitute the icosahedral
capsid 1. Petagonal capsomers at the corners (pentons) 2. Hexagonal capsomers making up the sides (hexons).

In helical symmetry
the genomic RNA forms a spiral within the core of the nucleocapsids. The viruses of this kind
look rodlike or filamentous . There are several perceived advantages to forming a helical capsid. First, only
one type of capsid protein is required. This protein subunit is repeated over and over again to form the capsid.
This structure is simple and requires less free energy to assemble than a capsid composed of multiple proteins.
In addition, having only one nucleocapsid protein means that only one gene is required instead of several,
thereby reducing the length of nucleic acid required. Because the helical structure can continue indefinitely,
there are also no constraints on how much nucleic acid can be packaged into the virion: the capsid length will
be the size of the coiled nucleic acid.

Helical viruses can be enveloped or naked. The first virus described, tobacco mosaic virus, is a naked helical
virus. In fact, most plant viruses are helical, and it is very uncommon that a helical plant virus is enveloped. In
contrast, all helical animal viruses are enveloped. These include well-known viruses such as influenza virus,
measles virus, mumps virus, rabies virus, and Ebola virus

Figure: An icosahedral virion structure & Virus structure with helical symmetry.

Virion: The complete infectious unit of virus particle.

Capsid: The protein shell, or coat, that encloses the nucleic acid genome. Functions: Protect the viral nucleic
acid, Participate in the viral infection, Antigenic and specific for each virus type & Provides structural symmetry
to the virus particle. Made of protein subunits called protomers. The protomers are composed of several
different polypeptide chains. The protomers make up capsomeres, and capsomeres form the capsid. Protomers
are the structural units that make oligomeric proteins while capsomeres are the morphological units of viral
capsids.

Nucleocapsid: The capsid with the enclosed nucleic acid

Figure Schematic diagram of Coronavirus

Shape

Icosahedral: Herpesviridae Adenoviridae

Complex: Bullet shaped- Rhabdoviridae

Filamentous: TMV
Envelope: A lipid-containing membrane surrounds some viral particles. It is derived from the plasma membrane
of the host cell during there release by budding from the cell surface. Envelope Proteins, which are viral
encoded, may project from the envelope surface as spikes or peplomers. They are involved in viral attachment
to host cell. e.g., hemagglutinin of influenza virus. It can be used for identification of virus. Envelopes confer
chemical, antigenic and biological properties on viruses. It may have enzymatic or other activity. e.g.,
neuraminidase of influenza virus and may play a role in nucleic acid replication. A virus may have more than one
type of peplomer. Not all viruses have the envelope, and viruses can be divided into 2 kinds: enveloped virus
and nonenveloped (naked) virus. Enveloped viruses appear spherical in shape.
General Concepts: Virus Size

Viruses are generally much smaller than the bacteria and its average size varies from 25-300 nm in diameter.
They are visible under electron microscope and only the largest and complex viruses are seen under light
microscope with high resolution . Among all, the smallest viruses belong to the
families Circoviridae, Parvoviridae and Picornaviridae which measure about 20 - 30 nm in diameter while
the largest one belongs to Poxviridae that measures around 250-300 nm in diameter. Recently, scientists
isolated a new form of virus that infects amoeba and grouped it under a separate family Mimiviridae. The
members of the family Mimiviridae range from 400-800 nm in diameter.

On an average a bacterial cell is about 1400 nm in diameter while an average epithelial cell is about 20,000 nm.
Considering both viruses and bacteria to be nearly spherical a bacterial cell has a volume about 30,000 times
greater than a virus while an epithelial cell is about 60 million times larger.

Methods of size analysis:

1. Passing through collodion membrane filters of graded porosity (gradocol membranes)

2. Ultracentrifuge

3. Electron microscope

4. X-ray crystallography

General Concepts: Components of genomes

In general the viruses are made up of nucleic acids (genome), proteins (capsid), and lipids (envelope).

Diverse nature of genomes- Viral genomes can be either DNA or RNA. A virus may have single or double
stranded DNA or RNA. The length of the nucleic acid also varies from virus to virus. Genomes can be segmented
or circular.

When once inside a host cell, it directs synthesis of new viral proteins, and replication of new viral genomes.
Capsid is a protein covering that surrounds and protects the viral genome. It is made up of many small subunits
called as capsomeres which determine the shape of the virus. The arrangement and composition of the
capsomeres varies among the virus families. Envelopes are the lipid bilayer membranes that are derived from
the host cell membrane when virus “buds” out from the plasma membrane or passes through a
membrane-bound organelle (such as the Golgi body or endoplasmic reticulum). The envelope contains
sometimes glycoprotein (protein with carbohydrate) in the form of spikes which helps them in the attachment
during the time of infection to the host cell surface (gp120 in HIV). In non-enveloped viruses, grooves present in
the capsid and specific capsid proteins may bind to the cell surface receptor.

The most important and characteristic feature of a living organism is replication of its genetic information. The
mechanism of genome replication is done with greater economy and simplicity among different viruses. Different
families of viruses have their genome made of either double stranded (ds) DNA or single stranded (ss) DNA or
RNA. The viruses that contain RNA genome may have either positive, negative, or mixed polarity. In addition,
they either have single or multiple segments in their genome with linear or circular topology. Each of the above
parameters have their consequences for the pathways of viral genome replication, viral gene expression, and
virion assembly.

Among the families of viruses that infect animals and human, those containing RNA genome outnumber those
containing DNA genome. This disparity is even more in case of plant viruses (no double stranded DNA virus that
infect plant is known).

Isolation of Viruses

Unlike bacteria, many of which can be grown on an artificial nutrient medium, viruses require a living host cell
for replication. Infected host cells (eukaryotic or prokaryotic) can be cultured and grown, and then the growth
medium can be harvested as a source of virus. Virions in the liquid medium can be separated from the host cells
by either centrifugation or filtration. Filters can physically remove anything present in the solution that is larger
than the virions; the viruses can then be collected in the filtrate

Cultivation of Viruses

Virus lacks its independent metabolism and they can only replicates inside host cell, so viruses cannot be
cultured in non-living medium as bacteria and fungi. Virus can only be cultured in embryonated egg, cell line
culture and animal inoculation.

Techniques of virus cultivation

● Animal inoculation

● Embryonated egg culture

● Cell culture

1. Animal inoculation:

Animal inoculation is one of the primary method for isolation of certain viruses and for study of pathogenesis of
certain viral diseases. Lab mice (white mice) particularly suckling one are animal of choice for virus cultivation.
Suckling mice of age less than 48 hrs are used for culture of Toga virus and Coxsackie virus. Other animals such
as hamsters, Guinae pig, Chimpanzee etc are sometimes used as alternative for virus culture. After inoculation
of virus sample, the animals are observed for symptoms of disease till death. And finally virus is isolated from
tissue of animal.

2. Embryonated egg culture:

For virus cultivation, an egg embryo of 7-12 days is used. At first egg is kept in incubator for embryo
development up to 7-12 days and then virus sample is inoculated into the egg. Opening in egg should be shield
with paraffin and it is incubated for sufficient time. Virus can be cultured in different parts of embryonated egg,
such as choriallontoic membrane, amniotic sac, allantoic cavty or yolk sac depending upon types of virus.

3. Cell culture (tissue culture) technique:

This technique is most commonly used technique for cultivation of virus.

There are three types of cell culture technique.

i. Organ culture:Small bits of organ from human or animal is maintained in tissue culture media.This technique is
used in specific purposes only. For eg, to culture Corona virus tracheal ring culture is done.

ii. Explant culture:In this small fragment of tissue is extracted from human or animal and used for virus
culture.This technique is very rarely used.

iii. Cell line culture:This is the most commonly used technique.Cell line culture is routinely used in lab for virus
culture, isolation and identification.In cell line culture, at first growth media is prepared by maintaining balanced
salt concentration, all essential aminoacids, glucose, buffering agents, some antibiotic, serum etc.Some tissue
fragment is obtained, it is trypsionised to dissociate cells.The dissociated cell are washed and suspended in
culture media in a tube or petriplates and incubated for sufficient time.On incubation, cell divides and spread out
on the glass surface to form a confluent mono-layer of cells, which is now used for virus culture.On the basis of
origin, chromosomal characteristics and number of generation through which cell culture can be maintained, cell
line culture are of three types.

I. Primary cell line: These are normal cells, obtained from fresh organ of animals or human and cultured.Once
the cell attached to the surface of culture vessel, they divides by mitosis until confluent mono-layer of cells
covers the surface.These cells are capable of limited growth for limited generation. They cannot be maintained
in serial sub culture.These primary cell line culture is used for isolation of virus and for preparation of
vaccines.Examples: Monkey kidney cell line, Human amnion cell line, etc

II. Semi-continuous cell line (Diploid cell):These cell are fibroblastic cell.They are diploid cell containing same
number of chromosome as the parent cell.Fibroblastic cells are obtained from embryo tissue.These diploid cell
can be sub cultured for limited generation.There is a rapid cell division and after 50 serial sub culture, they
undergoes senescence.The diploid cell are susceptible for wide range of Human virus culture and also used for
vaccine production.Examples: Rhesus embryo cell, human embryonic lung strain, etc

III. Continuous cell line:These are cells of single type capable of infinite growth in vitro.These are usually cancer
cell derived from cancerous tissue. These cells grow faster and they are haploid cells.They are termed as
continuous cell line as they can be serially sub culture for infinite generation without going
senescence.Examples; HeLa cell is obtained from cervical cancer, HEP-2 (Humman Epithelioma of larynx cell
line), Vero (Vervet monkey) kidney cell lines, BHK-21 (Baby Hamster Kidney cell line).Continuous cell line is
maintained by serial sub culture or by deep freezing at -70C, so that these cell be reused when
necessary.Continuous cell line is used for virus culture but it is not used for vaccine preparation because vaccine
prepared by continuous cell culture are not considered safe to Human use.

Viruses are obligate intracellular parasites that require living cells in order to replicate. Generally cell culture,
embryonated eggs and small laboratory animals are used for the isolation of viruses. Embryonated eggs are very
useful for the isolation of influenza and paramyxoviruses. Although laboratory animals are useful in isolating
different kind of viruses, cell culture is still a preferred way for virus isolation in many of the laboratories.

For primary cell cultures, tissue fragments are first dissociated into small pieces with the help of scissors and
addition of trypsin. The cell suspension is then washed couple of times with minimal essential media and seeded
into a flat-bottomed glass or plastic container bottle after resuspending it with a suitable liquid medium and fetal
calf serum. The cells are kept in incubator at 37°C for 24 to 48hrs depending on the cell type. This allows the
cells to attach the surface of the container and its division following the normal cell cycle.

Specimens containing virus should be transported to the laboratory as soon as possible upon being taken. Oral
or cloacal swabs should be collected in vials containing virus transport medium. Body fluids and tissues should
be collected in a sterile container and sealed properly. If possible all the samples should be maintained and
transported in a cold condition for higher recovery rates.

Upon receipt, the samples should be inoculated into cell culture depending on the history and symptoms of the
disease. The infected cell culture flask should be observed every day for any presence of cytopathic effect (CPE).
Certain kind of samples, such as faeces and urine are toxic to the cell cultures and may produce a CPE-like
effect. When virus specific CPE is evident, it is advised to passage the infected culture fluid into a fresh cell
culture. For cell-associated viruses such as cytomegaloviruses, it is required to trypsinize and passage the intact
infected cells. Viruses such as adenovirus can be subcultured after couple of time freezing and thawing of the
infected cells.

Susceptible cell lines:

Influenza virus- MDCK cells, Vero cells.

Paramyxoviruses- DF-1 cells, Vero cells.

Adenoviruses- HEK cells, HuH7 cells.

Herpesviruses- LMH cells.

Respiratory syncytia virus- Hep2 cells, Vero cells.

Viruses can be grown in vivo (within a whole living organism, plant, or animal) or in vitro (outside a living
organism in cells in an artificial environment, such as a test tube, cell culture flask, or agar plate).
Bacteriophages can be grown in the presence of a dense layer of bacteria (also called a bacterial lawn) grown in
a 0.7 % soft agar in a Petri dish or flat (horizontal) flask. The agar concentration is decreased from the 1.5%
usually used in culturing bacteria. The soft 0.7% agar allows the bacteriophages to easily diffuse through the
medium. For lytic bacteriophages, lysing of the bacterial hosts can then be readily observed when a clear zone
called a plaque is detected. As the phage kills the bacteria, many plaques are observed among the cloudy
bacterial lawn.
Animal viruses require cells within a host animal or tissue-culture cells derived from an animal. Animal virus
cultivation is important for 1) identification and diagnosis of pathogenic viruses in clinical specimens, 2)
production of vaccines, and 3) basic research studies. In vivo host sources can be a developing embryo in an
embryonated bird’s egg (e.g., chicken, turkey) or a whole animal. For example, most of the influenza vaccine
manufactured for annual flu vaccination programs is cultured in hens’ eggs.

The embryo or host animal serves as an incubator for viral replication. Location within the embryo or host
animal is important. Many viruses have a tissue tropism, and must therefore be introduced into a specific site for
growth. Within an embryo, target sites include the amniotic cavity, the chorioallantoic membrane, or the yolk
sac. Viral infection may damage tissue membranes, producing lesions called pox; disrupt embryonic
development; or cause the death of the embryo.

Figure a shows a technician injecting a tray of eggs with a syringe. Figure b shows an egg with syringes in
various region such as an outer layer (the chorioallantoic membrane), an inner region called the amniotic cavity
and another inner region called the yolk sac. The embryo is connected to the yolk sac and is within the amniotic
cavity. Outside the chorioallantoic membrane is albumin and around that is the shell.

Figure a begins with induvial cells isolated from lung tissue. These few cells are put on a plate and are the
primary cell culture. These cells will grow to fill the plate and will stop when the plate is full; this is called
contact inhibition. In order to grow more cells some of these cells are transferred to a new plate; this is now
called a secondary cell culture. Figure b begins with transformed cells or individual cells isolated from a tumor
that are put on a plate. These cells form a continuous culture because they continue to grow on top of each
other even after the plate is full.
 : Cells for
culture are prepared by separating them from their tissue matrix. (a) Primary cell cultures grow attached to the
surface of the culture container. Contact inhibition slows the growth of the cells once they become too dense
and begin touching each other. At this point, growth can only be sustained by making a secondary culture. (b)
Continuous cell cultures are not affected by contact inhibition. They continue to grow regardless of cell density.
(credit “micrographs”: modification of work by Centers for Disease Control and Prevention)

An example of an immortal cell line is the HeLa cell line, which was originally cultivated from tumor cells
obtained from Henrietta Lacks, a patient who died of cervical cancer in 1951. HeLa cells were the first
continuous tissue-culture cell line and were used to establish tissue culture as an important technology for
research in cell biology, virology, and medicine. Prior to the discovery of HeLa cells, scientists were not able to
establish tissue cultures with any reliability or stability. More than six decades later, this cell line is still alive and
being used for medical research. See The Immortal Cell Line of Henrietta Lacks to read more about this
important cell line and the controversial means by which it was obtained.

Detection of a Virus

Regardless of the method of cultivation, once a virus has been introduced into a whole host organism, embryo,
or tissue-culture cell, a sample can be prepared from the infected host, embryo, or cell line for further analysis
under a brightfield, electron, or fluorescent microscope. Cytopathic effects (CPEs) are distinct observable cell
abnormalities due to viral infection. CPEs can include loss of adherence to the surface of the container, changes
in cell shape from flat to round, shrinkage of the nucleus, vacuoles in the cytoplasm, fusion of cytoplasmic
membranes and the formation of multinucleated syncytia, inclusion bodies in the nucleus or cytoplasm, and
complete cell lysis. Further pathological changes include viral disruption of the host genome and altering normal
cells into transformed cells, which are the types of cells associated with carcinomas and sarcomas. The type or
severity of the CPE depends on the type of virus involved.

This is a table of cytopathic effects of specific viruses. The first example is paramyxovirus which causes
syncytium and faint basophilic cytoplasmic inclusion bodies. Small structures are seen within a cell. Next,
Poxyvirus results in pink eosinophilic cytoplasmic inclusion bodies (seen as small structures) and cell swelling.
Next, Herpesvirus causes cytoplasmic stranding (seen as an eleongation of the cytoplasm) and nuclear inclusion
bodies (seen as structures within the nucleus). Finally, Adenovirus causes cell enlargement, rounding, and
distinctive grape-like clusters.
Hemagglutination Assay

A serological assay is used to detect the presence of certain types of viruses in patient serum. Serum is the
straw-colored liquid fraction of blood plasma from which clotting factors have been removed. Serum can be used
in a direct assay called a hemagglutination assay to detect specific types of viruses in the patient’s sample.
Hemagglutination is the agglutination (clumping) together of erythrocytes (red blood cells). Many viruses
produce surface proteins or spikes called hemagglutinins that can bind to receptors on the membranes of
erythrocytes and cause the cells to agglutinate. Hemagglutination is observable without using the microscope,
but this method does not always differentiate between infectious and noninfectious viral particles, since both can
agglutinate erythrocytes.

To identify a specific pathogenic virus using hemagglutination, we must use an indirect approach. Proteins called
antibodies, generated by the patient’s immune system to fight a specific virus, can be used to bind to
components such as hemagglutinins that are uniquely associated with specific types of viruses. The binding of
the antibodies with the hemagglutinins found on the virus subsequently prevent erythrocytes from directly
interacting with the virus. So when erythrocytes are added to the antibody-coated viruses, there is no
appearance of agglutination; agglutination has been inhibited. We call these types of indirect assays for
virus-specific antibodies hemagglutination inhibition (HAI) assays. HAI can be used to detect the presence of
antibodies specific to many types of viruses that may be causing or have caused an infection in a patient even
months or years after infection

This chart has three columns labeled components, interactions and mictotiter results. In row A the components
are the red blood cells which do not interact with anything and show no reaction in a microtiter result. The lack
of reaction is seen as a small red dot in the center of the well. In row B the components are viruses and red
blood cells. The viruses and red blood cells clump together and this is seen in a microtiter result as redness
throughout the well. This is called hemagglutination. In row C, the components are viruses, red blood cells and
antibodies. The viruses and antibodies clump together but the red blood cells do not clump with anything. This
is again seen as no reaction; this is called hemagglutination inhibition.

This chart shows the possible outcomes of a hemagglutination test. Row A: Erythrocytes do not bind together
and will sink to the bottom of the well plate; this becomes visible as a red dot in the center of the well. Row B:
Many viruses have hemagglutinins that causes agglutination of erythrocytes; the resulting hemagglutination
forms a lattice structure that results in red color throughout the well. Row C: Virus-specific antibody, the viruses,
and the erythrocytes are added to the well plate. The virus-specific antibodies inhibit agglutination, as can be
seen as a red dot in the bottom of the well. (credit: modification of work by Centers for Disease

Nucleic Acid Amplification Test

Nucleic acid amplification tests are used in molecular biology to detect unique nucleic acid sequences of viruses
in patient samples. Polymerase chain reaction (PCR) is used to detect the presence of viral DNA in a patient’s
tissue or body fluid sample. Using PCR, short nucleotide sequences called primers bind to specific sequences of
viral DNA, enabling identification of the virus. Reverse transcriptase-PCR (RT-PCR) is used to detect the presence
of RNA viruses. RT-PCR differs from PCR in that the enzyme reverse transcriptase (RT) is used to make a cDNA
from the small amount of viral RNA in the specimen. The cDNA can then be amplified by PCR. Both PCR and
RT-PCR are used to detect and confirm the presence of the viral nucleic acid in patient specimens

Enzyme Immunoassay

Enzyme immunoassays (EIAs) rely on the ability of antibodies to detect and attach to specific biomolecules
called antigens. The detecting antibody attaches to the target antigen with a high degree of specificity in what
might be a complex mixture of biomolecules. Also included in this type of assay is a colorless enzyme attached
to the detecting antibody. The enzyme acts as a tag on the detecting antibody and can interact with a colorless
substrate, leading to the production of a colored end product. EIAs often rely on layers of antibodies to capture
and react with antigens, all of which are attached to a membrane filter. EIAs for viral antigens are often used as
preliminary screening tests. If the results are positive, further confirmation will require tests with even greater
sensitivity, such as a western blot or an NAAT.

6.2. Purification of virus and components:

6.2.1. Ultracentrifugation:

The viruses are usually purified with the help of ultracentrifugation. The machine is capable of rotating the
samples at 20,000-100,000 rpm under the density gradient of CsCl2 or sucrose. Density at which viruses neither
sink nor float when suspended in a density gradient is called as buoyant density . The rate at which viral
particles sediment under a defined gravitational force is called as sedimentation coefficient . The basic unit is
the Svedberg (S) which is 10 -13 sec. The S value of a virus is used to estimate its molecular weight.
Types of sedimentation medium:

A. Sucrose cushions or gradient - A fixed concentration or a linear gradient of sucrose is used. Increasing
the density and viscosity of the medium decreases the rate at which virus sediments through them. In g eneral a
"cushion" of sucrose is prepared at the bottom of the centrifuge tube and the sample containing virus is overlaid
over the cushion. Since most viruses have greater densities than sucrose, separation is based on S values. This
method can be used to separate molecules with relatively close S values. Sometime glycerol is also used in place
of sucrose.

B. CsCl 2 gradient centrifugation - A linear gradient of CsCl 2 in buffer is prepared in the ultracentrifuge
tube. As the concentration of the CsCl 2 is increased the density of the medium increases in the tube so that
density is low at the top and high at the bottom. Viral particle centrifuged through this medium will form a band
at a position equal to their buoyant density. These are useful to separate viruses of different densities. Limitation
of this method is that CsCl 2 can permanently inactivate some viruses.

6.2.2. Other techniques for separation:

Viruses can also be separated by electrophoresis and column chromatography but these are not the preferred
way to separate virus while sometimes they are used to separate viral nucleic acids or proteins. Both the
methods separate the virus on the basis of charge and/or size. Virus contains a variety of charged
macromolecule on its surface which contributes to its electrophoretic mobility or ion-exchange characteristics.
Viruses are sometimes ligated with the charged group to be separated by ion exchange chromatography.
Molecular sieve chromatography can also be used to purify the viruses where large pores are formed with the
help of special agarose through which virus particles can enter.

6.3. Purity of viruses:

Many methods are used to assess the purity of virus. The ratio of UV absorption at 260 and 280 nm during a
spectrophotometric analysis (260/280) is a characteristic feature to measure the purity of a virus sample and is
dependent on the amount of nucleic acid and protein present in the virion. Serological methods such as
enzyme-linked immunosorbent assay (ELISA), radioimmuno precipitation assay (RIPA), western blot, virus
neutralization test (VNT), and complement fixation are also used to check the puirity of a virus sample. These
methods require antibodies specific to viral proteins that may be monoclonal (single type of antibody specific to
a single viral protein) or polyclonal (several different antibodies that may recognize several viral proteins or
epitopes). Plaque assay is also performed in order to isolate the single colony from a pool of quasispecies
viruses.

Figure A general approach for purifying a virus from tissue culture cells

Viral classification
The classification of viruses is in a much less satisfactory state than that of cellular microorganisms, in part due
to a lack of knowledge of their origin and evolutionary history. In 1971 the International Committee on
Taxonomy of Viruses (ICTV) developed a uniform classification system. Since then the number of viruses and
taxa has continued to expand. In its ninth report, the ICTV describes over 2,000 virus species and places them
in 6 orders, 87 families, 19 subfamilies, and 349 genera. The committee places greatest weight on these specific
properties to define families:

1. nucleic acid type,

2. presence or absence of an envelope,

3. symmetry of the capsid, and

4. dimensions of the virion and capsid.

Nomenclature

● Virus order names end in -virales;

● virus family names in -viridae;

● subfamily names in -virinae; and

● genus names in -virus.

Although the ICTV reports are the official authority on viral taxonomy, many virologists find it useful to group
viruses using a scheme devised by Nobel laureate David Baltimore. The Baltimore system complements the ICTV
system but focuses on the viral genome and the process used to synthesize viral mRNA.

All four nucleic acid types can be found in viruses: double-stranded (ds) DNA, single-stranded (ss) DNA, dsRNA,
and ssRNA. The characterization of the genome of a ssRNA virus is further complicated by the sense of the
ssRNA. Some ssRNA viruses have an RNA genome that is identical in base sequence to that of mRNA produced
by the virus. Such viruses are said to have plus-strand or positive-strand RNA. Other ssRNA viruses have
genomes that are complementary to the mRNA they produce. These viruses are said to have minus-strand or
negative-strand RNA. Baltimore's system organizes viruses into seven groups.
Virus contains its genetic material in the form of nucleic acid (DNA/ RNA) surrounded by a protein coat called as
capsid. Viruses are the obligatory intracellular parasites of cells. This means that the viruses can only replicate
within a living host cell. The virus does this by subverting the biosynthetic pathways and protein synthesizing
capacity of the cell. This helps the virus to replicate its viral nucleic acid, make viral proteins, and facilitate its
escape from the parasitized cell.

In order to know the outcome of virus infection on the animal cells two factors play an important role
-- virulence of the virus and the susceptibility of the host.

Virulence – It may be defined as the ability of the virus to cause disease or in other words it gives the relative
degree of pathogenicity of the infecting virus. Viral virulence differs greatly among the strains depending on the
pathogenic nature of the virus. Virus may be categorized as pathogenic or non- pathogenic. The pathogenicity
of the virus range from mild to severe depending on the virulence of the viral strains. The term virulence is used
as a quantitative measure of its pathogenicity. The degree of virulence is usually related with the ability of the
pathogen to multiply within the host and depends on other factors such as host environment and its immune
status.

Terms describing infections of an organism

Lytic infection - When virus enters the cell and hijacks its cellular machinery to rapidly multiply and in the
process kills the cell is termed as lytic infection (many influenza viruses).
Lysogenic infection - It is the process characterized by the incorporation of viral DNA to the cellular DNA.
Once incorporated, the viral DNA replicates along with the host DNA. The incorporated viral DNA permits the
host cell to undergo normal cell cycle.

Acute infection - It is a rapid onset of disease symptoms resulting in severe illness or death of the infected
animal (influenza, viral hemorrhagic fever).

Chronic Infection - It is a prolonged infection in which the organism is not immediately killed and may carry
the virus for long period of time (hepatitis, HIV).

A cell is said to be permissive when it supports the virus multiplication. Viruses infecting the permissive cells
are usually cytocidal (kill the host cell) while infection to non- permissive cells do not produce any effect upon
infection hence called abortive. When the virus replication gets completed, no more viral mRNA or protein are
produced in the infected cells and is referred as restricted . In some cases viral DNA or RNA may sequester
indefinitely inside a host cell and this condition is called as persistent infection .

9.1 Cytolytic infections

Cytolytic infections can be clearly visualized under a light microscope. The characteristic of CPE effect is an
important parameter for a virologist to identify the virus species. In some viral infections inclusion bodies which
are formed upon viral infection are identified after specific staining methods and are used as a tool for
identifying the virus. Seller's stain is used to visualize the Negri bodies in the cells infected with Rabies virus.
Inclusion bodies are the remnants of viral structural and non-structural proteins. Alternatively, inclusion bodies
may be formed by a host cell macromolecule upon virus infection. For example, Cytomegalovirus infection to a
cell changes the cytoskeleton of infected cell which are then visible as inclusion bodies. Viral infection to a
permissive cell is often associated with changes in cellular biosynthetic pathways, its morphology, and cell
physiology.

Table Viral inclusion bodies in some human diseases

9.1.1 Effects on biosynthetic pathways

Virus infection to a host cell inhibits its DNA and/or RNA, and its protein synthesis. Sometimes it also causes
breakage and fragmentation of host chromosome. Moreover it also changes the growth characteristics, shape,
and surface protein expression of the infected host cell. Viruses often subvert the host biosynthetic pathway for
their own benefits at the cost of cellular macromolecules.

Virus infection to a cell forms many early proteins that mediate the changes in cellular biochemical pathways.
Viral nucleic acid contains specific signal sequences that help in migration of nucleic acids to different cellular
locations. In addition, it also contains some motifs that bind to regulators of cellular transcriptional machinery.
Therefore many viral early proteins contain binding sites for a wide range of cellular transcriptional factor. These
interactions and bindings are very important for the activation of virus protein synthesis and production of
progeny viruses. The biochemical events sometimes include glycosylation and phosphorylation of viral proteins.
These modifications are often associated with the increase or decrease of the pathogenicity and virulence of the
viruses. Generally virus alters the cascades that are involved in the synthesis of protein kinases and secondary
messengers (cyclic AMP,cyclic GMP, etc). Occasionally virus triggers the cells to overproduce regulatory proteins
that changes the cellular biochemical pathways. These regulatory proteins may be transforming growth factors,
interleukins, cytokines, NF-kβ or TNFα and TNFβ (HIVand Herpesviruses). In some viral infections cellular mRNA
get degraded (Influenza virus). Alternatively herpesviruses and reoviruses inhibit the cellular DNA synthesis.
Interestingly Pox virus degrades the host DNA with the help of virus associated DNase.

9.1.2 Effects on cell morphology

Changes evident in a cell following the virus infection are called cytopathic effects (CPE). There are various kinds
of CPE depending on type of infection. For example, detachment of cells from monolayer, rounding of cells,
formation of syncytia (multinucleated cells formed after fusion of nuclei) and nuclear or cytoplasmic inclusion
bodies formation.

Figure Cytopathic effects in the cells infected with viruses

9.1.3 Effects on cell Physiology

Virus infection to a cell changes many of the physiological events, including changes in cellular metabolism,
alteration in the ATP synthetic pathways, and deviation in the ion channel system. Physiological condition of a
viable cell has a great effect on the outcome of a virus infection because the host cell provides the cellular
machinery, regulatory proteins, and source for the viral nucleic acid, and protein synthesis. Attachment of virion
with the receptors present on cell membrane leads to a series of events that are associated with the changes in
morphological, physiological and biochemical characteristics of the cell. The receptor present on the cell surface
determines the host range as well as tissue tropism of a viral species. Influenza virus infects the cell after
binding to the sialic acid receptor present on the cell membrane. Similarly HIV infects the T-cells upon binding to
the chemokine receptors of the cell. Usually virus infection alters the intracellular ion concentration that affects
the cell membrane permeability (For example picornaviruses).

9.1.4 Effect on host chromosome

Virus infection to a cell directly or indirectly leads to the damage of the host cell chromosome that may be lethal
to the cell. If the cell does not die, viral genome may persist within the cell causing instability of cellular genome
and alteration in the expression of proteins.

10.1 Persistent infection

 In persistent infection virus is not eliminated from the cell. Persistent infection may be chronic, latent, and
transforming. In chronic infection the spread of virus is checked by host immune system while in latent infection
only few cells express the viral protein and virus replication is largely restricted. In transforming persistent
infection cell undergoes genetic changes that results in malignancy.

Persistent infection may sometime cause autoimmune disease condition in the host cell. Newly viruses bud out
from the cell membrane following the virus infection, this leads to change in the antigenicity of the host cell.
Immune system recognizes it as a nonself and produces an immune response which eventually causes death of
the cell. The immune response also causes formation of viral antigen-antibody (Ag-Ab) complexes which may
get deposited into vital organs like brain and kidney. Deposition of Ag-Ab complex elicits inflammatory condition
in those organs (nephritis and encephalitis)

10.2 Transforming infection

TTransformation refers to the ability of cells to multiply indefinitely that leads to cancerous condition. Mostly
DNA viruses like Epstein Barr virus and polyoma virus can cause transformation in permissive cells.
Transformation is essentially orchestrated by the viral proteins that may inactivate tumor suppressor proteins
(Retinoblastoma proteins and p53) of the host cell.

Cellular transformation usually involves two stages namely

1. 1. Immortalization and

2. Tumor production

Figure List of changes associated with transformed cells

Influenza virus – orthomyxovirus – negative strand RNA virus

Minus-Strand RNA Viruses – Influenza virus

https://youtu.be/tB5FQZi4HKY
https://youtu.be/oXzwtGFyBik

Negative-strand viruses are found in many families, including Rhabdoviridae (e.g., rabies virus), Filoviridae
(e.g., Marburg and Ebola viruses), Paramyxoviridae (e.g., measles and mumps viruses), Bunyaviridae (e.g.,
hantaviruses), and Orthomyxoviridae (e.g., influenza viruses).

Influenza viruses belong to the Orthomyxoviridae family and are classified as either type A, B, C, or the
recently identified type D. Influenza A viruses and type B viruses contain 8, negative-sense,
single-stranded viral RNA gene segments, which encode transcripts for 10 essential viral proteins, as well
as several strain-dependent accessory proteins. In comparison, influenza type C and D viruses only possess
seven vRNA gene segments. Influenza viruses are acquired by inhalation or ingestion of virus-infected
respiratory secretions. The virion adheres to the epithelium of the respiratory system with the aid of two
viral envelope proteins: neuraminidase and hemagglutinin.
 1. Neuraminidase (NA) is thought to hydrolyze the mucus that covers the epithelium. This allows
hemagglutinin (HA) to interact with receptors on the surface of epithelial cells, and the virion
enters by receptor-mediated endocytosis. This encloses the virus in an endosome. The
hemagglutinin molecule in the virion envelope undergoes a dramatic conformational change when
the endosomal pH decreases. The hydrophobic ends of the hemagglutinin spring outward and
extend toward the endosomal membrane. After they contact the membrane, fusion occurs and the
nucleocapsids are released into the cytoplasm.
2. Each genome segment is associated with nucleocapsid proteins NP and PB1 to form the
nucleocapsid. The nucleocapsids enter the nucleus, where synthesis of viral mRNA and genomes
occurs.
3. The endonuclease activity of the PB1 protein cleaves the cap and about 10 nucleotides from the
5' end of host mRNA (cap snatching). The fragment is used to prime viral mRNA synthesis by the
RNA-dependent RNA polymerase activity of the PB1 protein.
4. Viral mRNA is translated. Early products include more NP and PB1 proteins.
5. RNA polymerase activity of the PB1protein synthesizes +ssRNA from genomic -ssRNA molecules.
Some of new genome segments serve as templates for the synthesis of more viral mRNA. Later in
the infection, they will become progeny genomes.
6. Viral mRNA molecules transcribed from other genome segments encode structural proteins such as
hemagglutinin (HA) and neuraminidase (NA). These messages are translated by ER-associated
ribosomes and delivered to the cell membrane.
7. Viral genome segments are packaged as progeny virions bud from the host cell and thus acquire
their envelope. NA facilitates release of the progeny virions by cleaving the sialic acid groups from
glycoproteins on the host cell surface.

Virion properties

Orthomyxovirus virions are pleomorphic in shape and around 80–120 nm in diameter. The nucleocapsids are
helical in symmetry and contains eight (influenza virus A, B, and isavirus), seven (influenza virus C), or six
(Thogotovirus) RNA segments. Genome is single stranded negative sense RNA of 10 – 14Kb in size. The viruses
also contain surface glycoproteins over the lipid envelope. The two envelope glycoproteins are hemagglutinin
protein (H) and neuraminidase protein (N). There are 15 different subtypes of “H” and 9 different subtypes of
“N”. This provides a total of 135 (15 x 9) possible combinations. Hemagglutinin is the most important protein of
the virus and determinant of its virulence while neuraminidase helps in the budding and release of the progeny
virions. Both hemagglutinin and neuraminidase frequently undergo genetic modifications decreasing the
effectiveness of the host immune response. Influenza virus “C” lacks the “N” protein. Envelope is lined by Matrix
protein “M1” and an ion channel matrix protein “M2”. Three proteins namely PB1, PB2 and PA form the viral RNA
polymerase complex which is associated with genomic RNA and nucleoprotein.
Figure Schematic representation of an influenza virus:

Virus Replication

Influenza virus enters the cell after binding to sialic acid receptor present on the cell membrane. Different cells
of the body contain different sialic acid receptor types which largely determine the host range of these viruses.
The respiratory tract epithelium of humans contains α 2-6 linkage sialic acid receptor while birds contain α 2-3
sialic acid receptor. The types of sialic acid receptor in the epithelium determines the tropism of human as well
as avian influenza viruses. A single amino acid change in the hemagglutinin protein [E (Glutamic acid) 190D
(Aspartic acid)] changed the binding efficiency of influenza virus from α2,3 to α2,6 which caused the outbreak of
1918 influenza virus (Spanish flu). Virus enters the cell by receptor mediated endocytosis and uncoats under the
low pH condition of endosome. RNA synthesis of influenza virus takes place in the nucleus of the cell.
Nucleoprotein of influenza virus contains nuclear localization signals (NLS) that help in transportation of
ribonucleoprotein complex into the nucleus. Negative sense RNA genome of influenza viruses serve as a
template for the synthesis of positive sense RNA. Positive sense replicative intermediate RNA acts as a template
for progeny RNA genome. Interestingly, viral endonuclease activity of PB2 protein cleaves the 5' cap and 10-13
nucleotides from a cellular mRNA in order to transcribe the viral RNA. The phenomenon is called as cap
snatching . All orthomyxoviruses undergo splicing phenomena to produce two proteins from one gene such as
influenza virus A uses gene segment 7 to produce M1 and M2 protein. Similarly 8 th segment of influenza virus
produces NS1 and NS2 protein after undergoing splicing. In certain influenza viruses, frame shift mutation leads
to formation of PB1-F2 protein. Viral protein synthesis occurs in the cytoplasm and its maturation takes place in
endoplasmic reticulum and Golgi apparatus. Viral nucleoproteins are required for replication of genomic RNA
which then enters the nucleus along with polymerase protein for transcription. Progeny virus is released by
budding through the plasma membrane.
Figure Schematic representation of an influenza virus replication cycle:

Influenza viruses have caused major pandemics. This is related to the nature of their RNA-dependent RNA
polymerases and the fact that they have segmented genomes. RNA-dependent RNA polymerases lack
proofreading activity and are unable to correct errors made during replication of the viral genome. Thus
mutations arise fairly frequently, periodically producing new influenza virus strains for which humans have
no immunity. A more dramatic method for creating new influenza virus strains occurs when two different
strains infect the same host cell. As new virions are assembled, the RNA segments of the two strains can be
mixed together in a virion, generating a recombinant virus.
Hepatitis B virus replication
https://youtu.be/VaLDGWumrVw

Member of the Hepadnavirus family. The virus particle, called Dane particle (virion), consists of an
outer lipid envelope and an icosahedral nucleocapsid core composed of protein. The genome of HBV is
made of circular DNA, but it is unusual because the DNA is not fully double-stranded and has a DNA
polymerase that has reverse transcriptase activity similar to retroviruses. One end of the full length
strand is linked to the viral DNA polymerase. The outer envelope contains embedded proteins which are
involved in viral binding of, and entry into, susceptible cells. The virus is one of the smallest enveloped
animal viruses with a virion diameter of 42 nm, but pleomorphic forms exist, including filamentous and
spherical bodies lacking a core. These particles are not infectious and are composed of the lipid and protein
that forms part of the surface of the virion, which is called the surface antigen (HBsAg), and is produced in
excess during the life cycle of the virus.
Hepatitis B virus replication
The life cycle of Hepatitis B virus is complex. Hepatitis B is one of a few known non-retroviral viruses which
use reverse transcription as a part of its replication process.
Attachment
Although hepatocytes are known to be the most effective cell type for replicating HBV, other types of cells
in the human body have be found to be able to support replication to a lesser degree. The virus gains entry
into the cell by binding to receptors on the surface of the cell and entering it by endocytosis mediated by
either clathrin or caveolin-1. There is reversible and non-cell-type specific attachment to cell-associated
heparan sulfate proteoglycans, followed by specific and probably irreversible binding to an unknown
hepatocyte-specific preS1-receptor. This step presumably requires activation of the virus resulting in
exposure of the N-terminus of the L-protein which then binds tightly to the cell surface receptor sodium
taurocolate cotransporting polypeptide (NTCP) mostly found in the sinusoidal membrane of liver cells.
Cyclosporine A inhibits NTCP.

Penetration
Two different entry pathways have been proposed: (A) endocytosis followed by release of nucleocapsids
from endocytic vesicles; (B) fusion of the viral envelope at the plasma membrane. It leads to
cytoplasmic release of the viral nucleocapsid containing the relaxed circular partially double stranded DNA
(rcDNA) with its covalently linked polymerase.
Uncoating
It is thought the capsid is transported on the microtubules to the nuclear pore. Accumulation of the capsids
at the nuclear envelope facilitates interactions with adaptor proteins of the nuclear pore complex. There is
possible trapping of the nucleocapsid in the nuclear basket and release of rcDNA into the nucleoplasm.
Partially double stranded viral DNA is then made fully double stranded (by host DNA polymerases) and
transformed into covalently closed circular DNA (cccDNA) that serves as a template for transcription of four
viral mRNAs.

Replication
Replication of the hepadnaviral genome can broadly be divided into four phases:
1. Formation of ccc DNA- Upon infection, the partially double-stranded, circular but not covalently
closed relaxed circular, or RC-DNA is converted, inside the host cell nucleus, into a plasmid-like
covalently closed circular DNA (cccDNA). This “Repair” of the incoming rcDNA is achieved by:
a) Completion of the plus strand of the rcDNA by the viral polymerase.
b) Removal of the polymerase from the 5′-end of the minus strand DNA by cellular enzymes.
c) Removal of a short RNA-primer used for the DNA-plus strand synthesis by cellular enzymes
d) cccDNA formation by covalent ligation of both DNA strands. The cccDNA molecule is organized
as a chromatin-like structure (minichromosome).
 2. Transcription- The cccDNA utilizes the cellular transcriptional machinery to produce all viral RNAs
necessary for protein production and viral replication. The (-) strand of ccc DNA serves as template
for transcription by cellular RNA polymerase II of a longer-than-genome-length RNA called the
pregenome and shorter, subgenomic transcripts, all of which serve as mRNAs (viral mRNAs-
(pregenomic, precore, HBx, PreS1, and PreS2/S RNA. The HBV mRNAs are translated into the large
(L), middle (M), and small (S) surface, precore, core, polymerase, and HBx proteins.
Several non-coding RNA elements have been identified in the HBV genome. These
include: PREalpha, PREbeta and signal epsilon.

3. Translation of the pregenomic RNA (pgRNA) to the core protein and the viral polymerase. The
regulatory X-protein and the three envelope proteins are translated from the subgenomic RNAs. The
shorter viral mRNAs are translated by ribosomes attached to the cell's endoplasmic reticulum and
the proteins that are destined to become HBV surface antigens in the viral envelope are assembled.
The pregenome RNA is translated to produce a polymerase protein, P, which then binds to a specific
site at the 3' end of its own transcript, where viral DNA synthesis eventually occurs.
4. Reverse transcription of the pgRNA followed by plus-strand DNA-synthesis within the
nucleocapsid. Occuring at the same time as capsid formation, the RNA-P protein complex is
packaged and reverse transcription begins to form new RC-DNA genomes. Maturation of the
RNA-containing nucleocapsids to DNA-containing nucleocapsids occurs within the cytoplasm.
DNA-containing nucleocapsids can be either re-imported into the nucleus to form additional cccDNA
molecules, at early times after the infection, where the process is repeated or can be enveloped for
secretion. This results in the accumulation of 10 to 30 molecules of CCC DNA.

Assembly
These four viral transcripts undergo additional processing and go on to form progeny virions. The envelope
proteins are co-translationally inserted into the ER membrane, where they bud into the ER lumen. At some
step after preS-mediated nucleocapsid envelopment translocation across the membrane occurs.

Release
They and are secreted by the cell, either as 22 nm subviral envelope particles (SVPs) or as 42 nm infectious
virions (Dane particles) if they have enveloped the DNA-containing nucleocapsids before budding.
Therapeutic agents interfering with HBV life-cycle: Nucleos(t)ide analogues (Lamivudine, Adefovir,
Entecavir, Telbivudine, Tenofovir) and interferon (IFN) α / PEG-IFN α are the only currently approved
therapeutic treatments.

The HBsAg gene is one long open reading frame but contains three in frame "start" codons that divide the
gene into three sections, pre-S1, pre-S2, and S. Because of the multiple start codons, polypeptides of three
different sizes called large, middle, and small (pre-S1 + pre-S2 + S, or S) are produced.
T4 bacteriophage

Bacteriophages are viruses that infect and replicate within bacteria. The term is derived from two words
'bacteria' and ‘phage’ meaning ‘to eat’. Phages are widely distributed wherever bacterial hosts are found
such as soil or the intestines of animals. Sea water contains up to 9×108 virions/ milliliter and up to 70% of
marine bacteria may be infected by phages. Phages have been used for over 90 years as an alternative to
antibiotics in Europe and called Phage therapy. Phage infection is highly specific and there are about 10
phages for every type of bacteria.

Bacteriophages follow two types of life cycles, lytic and lysogenic. Most of the phages follow lytic cycle
and follow four phases:

⮚ adsorption of the phage to the host and penetration of virus genetic material
⮚ synthesis of virus nucleic acid and capsid proteins
⮚ assembly of complete virions
⮚ release of phage particles from the host

Bacteriophages that follow either lytic or lysogenic cycle are called temperate phages. In the lysogenic
cycle the viral genome is integrated into the host genetic material as a prophage during lysogeny. The
temperate virus genetic material remains in the host cell and reproduces in synchrony with the host for long
periods in a relationship known as lysogeny. The lysogenic state is very stable, certain physiologic
conditions (DNA damage induced by UV light) can destabilized the prophage like UV light or chemical
mutagens, resulting in its excision from the host-cell genome and its entry into the lytic pathway.
2 types of bacterial viruses are widely used in biochemical and genetic research (both infect E.coli):
1) DNA phages of the T series- the T phages of E.coli are large lytic phages. T1, T2, T3, T4, T5,
T6, T7. These viruses are called T phages because of the presence of t gene in their genome. The t gene
product is an enzyme which causes lysis of the host cell.
2) Temperate phages- Typical of this class of phages is E.coli bacteriophage , which has one of the
most studied genomes. On entering an E.coli cell, the double–stranded  DNA can enter either the
lytic cycle (T phages) or the lysogenic cycle. In the later case, the viral DNA forms a circle and
approaches the circular host DNA at a specific site.
Enzymes break both circular molecules of DNA and then rejoin the broken ends, so that the viral DNA
becomes inserted into the host DNA. Decision of λ phages between Lytic or Lysogenic life cycle is decided
by two repressors, cI or cro. These 2 proteins compete for the same binding sites (operators) on phage
DNA

● If cI binds it represses synthesis of all genes and phage DNA enters into Lysogenic state.
● If cro binds it represses synthesis of cI and Lytic cycle favours.

T phages are divided into two groups

a) T-even bacteriophages (T2, T4, T6)

b) T-odd bacteriophages (T1, T3, T5 and T7)
Double-stranded DNA phages: There is a large variety of dsDNA phages, of which the T phages and λ
have been particularly well characterized.
T2, T4 and T6 belong to the family Myoviridae (from the Greek mys, myos, ‘muscle’, referring to phages
with contractile tails),
T1 and T5 together with λ are members of the Siphoviridae (from the Greek siphon, ‘tube’, referring to
phages with long, flexible, non-contractile tails)
T3 and T7 are in the Podoviridae (from the Greek pous, podos, ‘foot’, referring to phages with short,
non-contractile tails).

Diagrammatic representation of T4 phage

T4 is a relatively large phage and divided into head, neck, tail and tail fibers. It is approximately 90 nm
wide and 200 nm long.
The capsid comprises a multiple-protein, icosahedral head separated from the tail by the neck. The tail
consists of two helical arrangements of protein: one forming the sheath, the other the tube, with a collar and
a baseplate with pins and tail fibres. The pins at the baseplate vertices anchor the phage to the host cell.
Proteins are labelled with their corresponding gene name or number. Virus assembly requires a protein
scaffold. The dsDNA is densely packed as concentric layers in the head. Head- The T4 head is made of 24
proteins of which 16 are involved in prohead formation and maturation, 5 are responsible for DNA
packaging and 3 proteins complete and stabilize the head. Hoc (highly antigenic outer capsid protein) and
Soc (small outer capsid protein) are two nonessential proteins found in the head.
The neck is composed of gene products 13, 14, wac. The tail is made of a baseplate and two slender
cocylinders. The inner cylinder consists of 144 subunits of gp19 arranged in 24 stacked hexameric rings.
The outer cylinder consists of 144 subunits of gp18 arranged in the same manner. The baseplate is composed
of six gene products (5, 27, 29, 26, 28 and 51). The gp5-gp27 complex attached at the tip of the tube and has
lysozyme like activity. The short tail fiber is a trimer of gp12 and the long tail fiber is made of gp9.

Genome of T4
The T4 genome is linear dsDNA of about 169 kbp. The genome is AT-rich and contains modified bases in
the form of 5-hydroxy-methyl-cytosine, rather than cytosine, which protect the phage DNA from many
host restriction systems and from phage-encoded nucleases that degrade cytosine-containing host DNA
during phage infection. The majority of the hydroxy-methyl groups are glucosylated after DNA synthesis
to counteract the host Mcr (modified cytosine restriction) systems, to which the hydroxy-methylcytosine
residues confer susceptibility.
The genome is circularly permuted and terminally redundant (a sequence of about 1.6 kbp at one end is
directly repeated at the other) due to the mode of replication and packaging by the head-full mechanism.
The genome comprises about 300 probable genes assigned by the timing of their expression, with three
different types of promoter:

● early (Pe),
● middle (Pm) and
● late (Pl).

The early and middle genes encode functions for DNA replication and for regulating expression of the late
genes, which encode head and tail components for the phage particles, and functions for cell lysis. Early
genes rely on the transcription apparatus of the host, being transcribed from normal σ70 promoters. Early
gene products alter the host RNA polymerase in two ways for expression of middle genes. The enzyme is
thereby able to read through a transcription terminator by an anti-termination mechanism to express genes
downstream of early genes and is modified to recognize middle promoters. These promoters differ from
σ70 promoters in the −35 sequence, and a transcriptional activator, encoded by the T4 motA early gene,
allows the host RNA polymerase to transcribe from such promoters. Late mRNA directs the synthesis of
three kinds of proteins: Phage structural proteins, Proteins that help in phage assembly and Proteins that
help in cell lysis and release of new phage particles.
Mechanism of T4 infection

1) Adsorption and penetration

The T4 of E. coli uses cell-wall proteins and polysaccharides as receptors.

• First the tail fibers contact the appropriate receptor site.

• The baseplate next settles down on the surface.
 • Binding involves electrostatic interaction followed by ligand-receptor interaction. pH and presence
of L-tryptophan and ions like Mg2+ influence the same.
• Conformational changes occur in the base-plate and the sheath. The sheath becomes shorter and
wider.
• The central tube or core is pushed through the bacterial wall.
• The baseplate contains the protein gp5 that has lysozyme activity and facilitates digestion of the
peptidoglycan layer and aid in the penetration of the tube through the peptidoglycan layer.
• The tube possibly interacts with the plasma membrane to form a pore.
• The DNA is finally extruded from the head, through the tail tube, into the host cell.
• The trans-membrane electrochemical potential is required for transfer of the T4 DNA to the
cytoplasm.
• The empty capsid remains extracellular.

The timing of the life cycle of T4

T=0 Absorption of Phage to the bacterial cell

T=1 Synthesis of host DNA, RNA, protein turned off

T=2 Synthesis of first mRNA

T=3 Degradation of bacterial DNA

T=5 Initiation of phage DNA synthesis

T=9 Synthesis of late mRNA

T=12 Appearance of complete head, tail

T=15 Appearance of first complete phage particle
T=22 lysis of bacteria, release of progeny phages (Burst size~300)
 2) Synthesis of early proteins
39 early promoters are present in T4 genome. T4 genes are transcribed by the regular host RNAP (RNA
polymerase) and the sigma factor. gp Alt enters the host with the infecting DNA and ADP-ribosylates the α
subunit of RNAP. This inhibits the transcription of the host genes and promotes virus early gene
expression. After a short interval, 2 viral enzymes ModA and ModB are expressed which catalyses the
ADP-ribosylation of both the subunits of RNAP. This turns off some of the early T4 genes.

Two viral endonucleases gp denA and denB degrade the viral genome into short dsDNA molecules which
are furthur degraded into small pieces by viral exonucleases. The organization of the T4 genome is also
suited for efficient control of its life cycle. The early and the late genes are clustered separately on the
genome; they are transcribed in separate directions (early – counterclockwise and late – clockwise). The
early and the late genes are therefore located on different DNA strands. 30 Pm are present in the T4
genome. Two early proteins AsiA and MotA, modifies RNAP and stimulates transcription of early late or
middle genes. The expression of T4 genes is tightly regulated. The HMC present in the T4 DNA is
synthesized by two virus-coded genes before DNA replication begins.

T4 hydroxymethylase

dCMP → dHMP
HM Kinase

dHMP → dHDP

Inhibition of entry of cytosine into T4 genome

Any base should be present in its triphosphate state before entering into the growing DNA molecule. So the
base cytosine must be present as dCTP and entry of this base will make the T4 DNA susceptible to its own
endonuclease which is otherwise used to cleave the host DNA. Hence to prevent the entry of cytosine, T4
carries an enzyme called dCTPase which cleaves dCTP as soon as it is produced. T4 also carries an
enzyme, dCMP deaminase, which converts dCMP to dUMP.

dCTPase dCMP deaminase dCTP/dCDP

→ dCMP → dUMP

Following T4 DNA synthesis HMC is glucosylated by 2 enzymes

α-glycosyltransferase and β-glucosyltransferase. Glucosylated HMC protects the T4 DNA from attack by
E. coli restriction endonuclease.

Synthesis of late proteins

50 late promoters have been found in T4 genome. Transcription of late genes requires two new
phage-encoded factors, transcription activator gp33 and σ factor gp55, which replaces the host σ70. Late
transcription is responsible for the synthesis of T4 head, tail, fibre proteins, Virion assembly factors,
Recombination proteins etc. Other T4 gene products, including those of genes 44, 45 and 62, which are
involved in replication, are additionally required for late gene transcription. RNA polymerase complexed
with gp55 seems to need an actively replicating T4 DNA for effective transcription of the late genes. Such
coupling between replication and transcription ensures that phage DNA becomes available for packaging
as the capsid proteins are synthesized from the late genes.

Replication of T4
T4 DNA replication requires several viral-coded proteins (T4 DNA polymerase, primase, helicase, a
recombination exonuclease, SS-binding protein, etc). T4 DNA exhibits terminal redundancy – a base
sequence is repeated at both ends of the molecule. T4 DNA replicates in two phases: first phase -
Primary Origin Initiation (replication begins at multiple origin of replication and proceeds
bidirectionally); second phase - Secondary Initiation by Join-Copy Recombination. In the first phase
replication begins at multiple origin of replication and proceeds bidirectionally. Origin initiation requires
transcription from an early promoter by host RNA polymerase to provide a primer or for transcriptional
activation of the origin. RNA polymerase can synthesize a primer for leading-strand synthesis since the
leading strand is initiated normally if both the T4 and E. coli primases are inactivated by mutations

A new DNA strand (red) is always synthesized in the 5’3’ direction. The template is read in the opposite
direction, 3’5’. The leading strand is continuously synthesized in the direction taken by the replication
fork. The other strand, the lagging strand is synthesized discontinuously in short pieces (Okazaki
fragments) in a direction opposite to that in which the replication fork moves. The Okazaki fragments are
spliced together by DNA ligase.
Two different strategies for overcoming this problem:

⮚ bacteriophages T4, T7 forms concatamers,

⮚ bacteriophage lambda circularises its chromosome.

T-even bacteriophages possess linear, double stranded DNA molecule that is terminally redundant:
5’-abcdef…..wxyzabc-3’
When a terminally redundant phage DNA molecule replicates in the infected cell, genetic recombination
can proceed within the region of terminal redundancy of two daughter molecules, giving rise to molecular
concatemers.

abcdef…..wxyzabc X abcdef…..wxyzabc

abcdef…..wxyzabcdef…..wxyzabc

These concatemers are cut into phage-genome-sized pieces before incorporation into the heads of progeny
virion particles. Constant lengths are cut off the concatemer, irrespective of sequence generating permuted
molecules with repetitious ends. The cutting process start taking its measure of phage-genome length at any
randomly chosen genetic site giving rise to a collection of phage genomes whose terminal redundancy is
circularly permuted.

Second phase of recombination

Giesela Mosig first proposed this model and thus called Mosig Model for recombination dependent
replication. General recombination is essential for growth of phage T4, because origin initiation of DNA
replication is inactivated during development, and recombination dependent initiation is necessary for
continuing DNA replication. T4 maintains multiple recombination mechanisms. The 3’-OH terminal region
of the template remains uncopied because of the difficultyin synthesizing an Okazaki fragment at the
extreme terminal segment of a strand. When more than one T4 chromosome is present in the cell, the
3’-terminus invades a homologous sequence in the interior of another chromosome and provides a (DNA)
priming terminus for continuing replication. Repetition of this process is one way by which long
concatemeric DNA molecules are produced during replication in T4. One reason for the phage to switch to
replication from secondary origins is that initiation at primary origins in the infected cell is dependent upon
RNA primers to be synthesized from host RNA polymerase which by the action of the phage-coded gp55 is
diverted to transcription from late promoters.

The assembly of phage particles

The DNA is packaged into the pre-made heads. Tail and baseplate assemble separately. The base-plate is
constructed of 15 gene products. Tail fibres are next attached to the base plate. The tail tube is built on the
base-plate. The sheath is assembled around the tail tube. The phage pro-head or pro-capsid is constructed
separately of more than 10 proteins and then spontaneously combined with the tail assembly. The procapsid
is assembled with the aid of scaffolding proteins that are removed after construction is completed. A special
portal protein is present at the base of the pro-capsid where it connects to the tail. The portal proteins are a
part of the DNA translocating vertex that initiates head assembly and DNA movement into and out of the
head. DNA is drawn into the completed shell (500um long DNA in head with 0.1 um diameter). Probably a
DNA concatemer enters the head in an ATP-dependent process. Packaging is size specific, depending on the
size of the head, and is accompanied by a structural change in the procapsid. After a full T4 genome (~2%
more DNA) has entered the head the concatemer is cut and the assembly process is completed. At 370C, T4
particles appear in about 15 minutes, post-infection, from the host E.coli cells.

Release of phage particles

The lysis of host E.coli cells takes place after about 22 minutes at 370C and approximately 300 T4 particles
are released. Several T4 genes are involved in the lysis process. T4 and many other phages lyse their host
by damaging the cell membrane and cell-wall.

⮚ Holin – enzyme which destabilizes the host cell membrane (pokes holes)
⮚ Lysin – phage enzyme which breaks host cell wall (lyses host bacteria)

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. The experiment involves mixing a culture of susceptible
bacteria such as E. coli with bacteriophage particles, and the phages are allowed a short interval to attach
to their host cells. Next, the culture is greatly diluted so that the newly released virions do not immediately
infect new cells. The phages need to come into physical contact with the bacterial cells to attach
themselves, and that is prevented by the high-degree of dilution i.e. the 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 assay. 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 (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.
Morphology of Phage Lambda:
The head has 20 faces, an icosahedron. The head is made of protein of several types and contains a 46,500
bp long genomic (g) DNA. The phage λ contains double stranded circular DNA. The head is 55 nm in
diameter consisting of 300-600 capsomers.
The capsomers are arranged in clusters of 5 and 6 subunits i.e. pentamers and hexamers. The head is joined
to a non-contractile 180 µm long tail by a connector. The tail consists of 35 stacked discs. It ends in a fiber.
Unlike T-even phage, it is a simple structure devoid of the tail sheath.
Bacteriophage lambda falls under the family Siphoviridae of the Group I (dsDNA viruses). Phage lambda is a
virus of E. coli K12 which after entering inside host cell normally does not kill it in-spite of being capable of
destroying the host.
Therefore, it leads its life cycle in two different ways, one as virulent virus and the second as non-virulent.
The virulent phase is called lytic cycle and the non- virulent as temperate or lysogenic one, and the
respective viruses as virulent phage and temperate phage, respectively. The other temperate phages are
21, Ø80, Ø81, 424, 434, etc.

DNA and Gene Organization of Phage Lambda:

Lambda DNA is a linear and double stranded duplex of about 17 µm in length. It consists of 48, 514 base
pairs of known sequence. Both the ends of 5′ terminus consists of 12 bases which extend beyond the 3′
terminus nucleotide. This results in single stranded complementary region commonly called cohesive ends.
The cohesive ends form base-pairs and can easily circularize. Consequently a circular DNA with two single
strand breaks are formed. The double stranded region formed after base pairing of complementary
nucleotides is designated as COS.
The events of circularization occurs after injection of phage DNA into E.coli cell where the bacterial enzyme,
i.e., E.coli DNA ligase, converts the molecule to a covalently sealed circle.
Life Cycles of Phage Lambda:

Lambda phage J protein interaction with the

LamB porin [maltose outer membrane porin product of lamB gene]
Upon adsorption on the lamb receptor of the host cell, lambda gDNA is injected through the tail which
forms a hollow tube through which the DNA passes to the cell. The phage λ leads two life cycles, the lytic
cycle and the lysogenic cycle after injecting its DNA into E.coli cell.
In the lytic cycle, phage genes are expressed and DNA is replicated resulting in production of several phage
particles. The lytic cycle ends with lysis of E.coli cells and liberation of phage particles.

In addition, the lysogenic cycle results in integration of phage DNA with bacterial chromosome and
becomes a part of host DNA. It replicates along with bacterial chromosome and is inherited into progenies.
The phage DNA integrated with bacterial chromosome is called prophage.
The prophage is non-virulent and termed as temperate phage. The bacteria containing prophage are called
lysogenic bacteria, and the prophage stage of viruses as lysogenic viruses. After treatment of lysogenic
bacteria with UV light. X-rays or mitomycin, the prophage can be separated from bacterial chromosomes
and enter the lytic cycle. This process is known as induction.
Genetic Map of Phage Lambda:
The remarkable characteristics of the map is the clustering of genes according to their functions. For
example the head and tail synthesis, replication and recombination genes are arranged in four distinct
clusters. These genes can also be grouped into three major operons viz. right operon (head synthesis, tail
synthesis and DNA replication leading lytic cycle), left operon (integration and recombination events of
lysognic cycle) and immunity operon (interact with DNA and decide whether the phage will initiate lytic
cycle or lysogenic cycle).
Choice between Lytic and Lysogenic Cycles:
Soon after circularization of genome and start of transcription, the gpcll and gpcIII accumulate. The gpcll
binds to PRE (promoter for repressor establishment) and stimulates binding of RNA polymerase. The gpcIII
protects gpcll from degradation by host nucleases.
Fig: Choice between lysis and lysogeny.
Lambda repressor (gpcl) is rapidly synthesized (B), binds to OL and OR, and inhibits the synthesis of mRNA
and production of gpcll and gpcIII (proteins) (C). The repressor activates the promoter for repressor
maintenance (PRM) which induces cI gene to be transcribed continuously at a low rate. This process goes
on continuously and ensures the stable lysogeny when it is established (C).

During the course of time the gpcro also accumulates. It binds to OL and OR, turns of the transcription
repressor gene cl and represses PRM function (D). The repressor (gpcl) can block cro transcription.
Therefore, there is a race between the production of gpcl and gpcro proteins.
The detail of this competition is not yet clear but the environmental factors influence the result of this race
for choice of the two cycles. If the repressor wins the competitions, the circular DNA is inserted into the E.
coli genome. The amount of gpcro and the outcome of competition with gpcl decide the establishment of
lysogenic or lytic pathways.

1. Host DNA gyrase puts negative supercoils in the circular chromosome, causing A-T-rich regions to
unwind and drive transcription.
2.

1. Structural proteins and phage genomes self-assemble into new phage particles.
2. Products of the genes S,R, Rz and Rz1 cause cell lysis. S is a holin, a small membrane protein that, at
a time determined by the sequence of the protein, suddenly makes holes in the membrane. R is
an endolysin, an enzyme that escapes through the S holes and cleaves the cell wall. Rz and Rz1 are
membrane proteins that form a complex that somehow destroys the outer membrane, after the
endolysin has degraded the cell wall. For wild-type lambda, lysis occurs at about 50 minutes after
the start of infection and releases around 100 virions.

1. For the first few replication cycles, the lambda genome undergoes θ replication (circle-to-circle).
Soon, the phage switches to a rolling circle replication. This does not release single copies of the
phage genome but rather one long molecule with many copies of the genome: a concatemer.
2. These concatemers are cleaved at their cos sites as they are packaged. Packaging cannot occur from
circular phage DNA, only from concatomeric DNA.
Lambda phage exists as a phage but also integrates into the E. coli chromosome at the attB site to form a
prophage. The integration reaction occurs when integrase makes staggered cuts in the center of the
phage attP site and in the center of the bacterial site attB. The ends then connect so that the phage DNA is
integrated, but notice that the sequences are different than the original. These are
called attL and attR after integration. This reaction can be reversed, but since the two sites are different
after integration, another enzyme called Xis, or excisionase, removes the inserted DNA and relegates the
broken DNA.

Lysogeny and prophage formation is a characteristic of tailed bacteriophages. Regulatory genes encoding
the CI repressor, CII activator, and CIII protein are essential for induction and maintenance of lysogeny. The
gene cI encodes a repressor molecule (CI) that maintains lysogeny, shuts down lysis by preventing
transcription from the early promoters PR and PL, and provides immunity to the bacterial host from
infections by other related phages. It was among the first genes to be identified that auto-regulates its own
synthesis. The CI repressor forms concatemers that bind to operator regions controlled by the PL and
PR promoters causing a loop formation in the DNA between these two promoters and turning off
transcription of the N and cIII genes (from the PL promoter) as well as the cro and cII genes (from the
PR promoter). It weakly activates the PRM promoter that continues expression of the cI and rex genes (that
render the host cell immune to phage infections). As a result, the synthesis of CI continues even in the
absence of CII and CIII proteins. At high concentrations of CI, transcription of cI gene from the PRM promoter
is reduced until an optimum level is achieved that represses lysis and infection by other phages. The Cro
protein is a weak repressor that can also bind to the PRM promoter, and reduces the transcription of cI gene.
The ability of CI repressor to shut off transcription of cro gene (essential for the lytic phase) and in turn, be
regulated by Cro protein (a product of the first gene of the PR operon) has been referred to as a genetic
switch modulating lysogeny or lysis. The CII activator is the major switch for lysogeny that turns on the
transcription of cI gene from the early PRE promoter and int gene (encoding an integrase) from the
PI promoter. CII also activates a third promoter PAQ that produces a small antisense RNA molecule from
the Q gene that keeps late (transcriptional) operator shut off, preventing onset of the lytic phase. Several
host factors influence activity of the CII activator; in addition, CIII protein is important for the stability of the
CII activator. It inhibits a host-encoded protease complex from degrading the CII protein. In bacterial cells
growing in a limited nutrient environment, protease activity is low, making the cells stable and the invading
phage more prone to a lysogenic lifestyle.
The phage genome integrates into the host DNA by site-specific recombination at the att sites (present in
both the virus and the bacterial host) mediated by a virus-encoded integrase. This viral protein is part of an
Integration Complex that also includes host-encoded proteins like IHF (Integration Host Factor) and FIS. The
IHF is multifunctional. In addition to facilitating the protein–DNA interaction during recombination
at att sites, it also influences levels of CII translation and cIII transcription. Prophages can be induced to
excise out of the bacterial genome by UV and chemicals like mitomycin C. During excision, CI repression is
abolished and the prophage is released from the site of integration. The phage DNA circularizes at
the cos sites and replicates within the host. Replication initially proceeds bi-directionally from a single point
of origin, making many copies of each strand. Subsequently, replication proceeds by a rolling circle mode
before the phage genomes are packed into virions. The phage genes O and P participate in DNA replication.
Host proteins like DNA helicases are co-opted for the process. This stage corresponds to the release from CI
repression and resumption of lysis. The protein Xis is a phage-encoded protein that reverses the process of
integration. The levels of both Xis and Int proteins influence the continuation of the lysogenic or lytic phase
of lambda life cycle. Several nonessential lambda phage genes include lysogenic conversion genes,
like bor and lom that are involved in host interactions, and exo, bet, and gam that aid in recombination
events. Other exclusion genes such as rex and sie prevent further infections by phages.
Resumption of the lytic phase is preceded by assembly of virions. The phage head and tail assemble
independently and then combine spontaneously to form each virion. The phage gene E encodes the MCP.
The genes Nu1 and A encode a lambda terminase holoenzyme that participates in packaging of the virion
with the host-encoded IHF protein that bends/folds the phage DNA into the virion. Once large numbers of
virus particles accumulate in the host cell, lysis occurs and virions are released. Membrane proteins
called holins, phage proteins Rz and Rz1 of the spanin complex and S105/S107 participate in the actual lysis
and degradation of the bacterial peptidoglycan cell wall. Infection of new E. coli host cells involves
recognition of the phage J protein by the LamB protein of E. coli. The LamB protein is an outer
membrane protein normally involved in maltose uptake. It is the main receptor for lambda phage
recognition.
Induction

Transcriptional state of the PRM and PR promoter regions during a lysogenic state vs induced, early lytic
state.
The classic induction of a lysogen involved irradiating the infected cells with UV light. Any situation where a
lysogen undergoes DNA damage or the SOS response of the host is otherwise stimulated leads to
induction.
1. The host cell, containing a dormant phage genome, experiences DNA damage due to a high stress
environment, and starts to undergo the SOS response.
2. RecA (a cellular protein) detects DNA damage and becomes activated. It is now RecA*, a highly
specific co-protease.
3. Normally RecA* binds LexA (a transcription repressor), activating LexA auto-protease activity, which
destroys LexA repressor, allowing production of DNA repair proteins. In lysogenic cells, this
response is hijacked, and RecA* stimulates cI autocleavage. This is because cI mimics the structure
of LexA at the autocleavage site.
4. Cleaved cI can no longer dimerise, and loses its affinity for DNA binding.
5. The PR and PL promoters are no longer repressed and switch on, and the cell returns to the lytic
sequence of expression events (note that cII is not stable in cells undergoing the SOS response).
There is however one notable difference.
Tobacco mosaic virus

TMV is a plant virus which infects a wide range of plants, especially tobacco and other members of the family
Solanaceae.

The infection causes characteristic patterns, such as "mosaic"-like mottling(spots) and discoloration on the leaves
(hence the name). TMV was the first virus ever to be discovered. Most plant viruses are RNA viruses, and of these,
plus-strand RNA viruses are most common. Tobacco mosaic virus (TMV; family Virgaviridae) is the best studied
plus-strand RNA plant virus. TMV virions are filamentous with coat proteins arranged in a helical pattern.

Each capsomer is made up of 158 amino acids. Rod shaped- 300nm x 180nm No. of Capsomers- 2130

The reproductive cycle of TMV consists of Five steps as

1. Entry into host cell

2. Uncoating
3. Intracellular Development
 4. Assembly (Maturation)
5. Release

RNA-dependent RNA polymerase ( RdRp)

methyltransferase (MT) and helicase (HEL)

Two proteins are produced, one smaller (about 126 kilodaltons; kD) and one larger (about 183 kD). The larger protein
is produced by readthrough of the stop codon at the end of the coding region for the 126 kD protein. The 183 kD
protein has RNA-dependent RNA polymerase activity and functions as both a transcriptase and a replicase. As the
replicase, it synthesizes negative-strand RNA using the plus-strand genome as the template.

The negative strand RNA serves as a template for synthesis of new genomes. It also serves as the template for
synthesis of subgenomic mRNAs that are translated into a variety of proteins needed by the virus.

TMV, usually enter the host through an abrasion or wound on the plant; biting insects are often involved in
transmission of the virus. Capsid is removed and nucleic acid is released into the cell cytoplasm. Nucleic acid of plant
viruses enters the host cell cytoplasm along with capsid. In the cytoplasm capsid is removed and nucleic acid is freed.

It requires assistance of host enzymes to remove capsids. Multiplication of plant viruses within their host depends on
the virus's ability to spread throughout the plant. Viruses can move long distances through the plant vasculature;
usually they travel in the phloem.

The spread of plant viruses in nonvascular tissue is hindered by the presence of tough cell walls. TMV thus spreads
slowly, about 1 mm per day or less, moving from cell to cell through the plasmodesmata. These slender cytoplasmic
strands extend through holes in adjacent cell walls and join plant cells by narrow bridges.

Viral "movement proteins" are required for transfer from cell to cell. TMV movement proteins accumulate in the
plasmodesmata, where they cause the plasmodesmata to increase in diameter. They are thought to coat the viral
RNA, causing the RNA to assume a linear conformation, rather than the helical conformation found in TMV virions.
This linear conformation allows it to fit through the plasmodesmata.

Although movement proteins seem to associate with microtubules, their role in movement of viral RNA through the
plasmodesmata is still being studied.

TMV contains SS Positive sense RNA as its genome. Replication of virion RNA thus involves synthesis of negative
strand RNA using positive strand RNA as a template. Thus, replication completes in 2 steps
 1) Synthesis of negative strand RNA using positive strand RNA as a template which forms doubles stranded
intermediate termed as “Replicative form (RF)”.

2) Synthesis of positive strand RNA using negative strand RNA as a template using virus coded RNA dependent RNA
polymerase.

After the coat protein and RNA genome of TMV have been synthesized, they spontaneously assemble into complete
TMV virions in a highly organized process. The protomers come together to form disks composed of two layers of
protomers arranged in a helical spiral.

Association of coat protein with TMV RNA begins at a specific assembly initiation site close to the 3' end of the
genome. The helical capsid grows by the addition of protomers, probably as disks, to the end of the rod. As the rod
lengthens, the RNA passes through a channel in its center and forms a loop at the growing end. In this way, the RNA
easily fits as a spiral into the interior of the helical capsid

Capsomers binds to each other to form disks. Each disk contains 2 layers each with17 subunits. Then helical capsid
grows by addition of disks to one end of rod.

Mechanism: At neutral pH carboxylic groups of adjacent capsomers have normal pK’s values. But at acidic pH (high
H+ conc.) repulsion between adjacent carboxylic group occurs due to increse in pk’s. This results in Lock “Washer
formation”.

Man plant viruses kills their hosts in which they multiply. They releases by autolysis of host cell which causes death
of cell.

Several cytological changes can take place in TMV infected cells. These include microscopically visible intracellular
inclusions that are similar to the replication complexes observed in animal cells infected with plus-strand viruses.

Hexagonal crystals of almost pure TMV virions sometimes develop in TMV-infected cells. In addition, host cell
chloroplasts become abnormal and often degenerate, while new chloroplast synthesis is inhibited
Figure: Self-assembly process of TMV. Model for the bidirectional self-assembly of nanotubular TMV particles, based
on the formation and consumption of different CP oligomers, i.e. "A-protein" and disks, in vitro. RNA is shown as a
black line. Assembly of TMV starts with insertion of the RNA origin of assembly (OAs)-loop into the central hole of a
protein disk, resulting in its conformational change into a helical “lockwasher” and the formation of an RNA “traveling
loop” (indicated with arrow) and integration of the adjacent RNA portion between the CP layers. Fast tube elongation
towards the 5’-tail of RNA is achieved by the serial addition of protein disks, while slow 3'-elongation occurs through
use of “A-protein”.