VIRUS

INTRODUCTION:

Viruses are the smallest known infective agents and are perhaps the simplest form of life known. Viruses do not
possess a cellular organization and they do not fall strictly into the category of unicellular microorganisms. Even the
simplest of microorganisms are cells enclosed within a cell wall, containing both types of nucleic acid (DNA and
RNA), synthesizing their own macromolecular constituents.

MAIN PROPERTIES OF VIRUSES

1. Viruses do not have a cellular organization.
2. They contain only one type of nucleic acid, either DNA or RNA but never both.
3. They are obligate intracellular parasites.
4. They lack the enzymes necessary for protein and nucleic acid synthesis and are dependent for replication on the
synthetic machinery of host cells.
5. They multiply by a complex process and not by binary fission.
6. They are unaffected by antibacterial antibiotics.

MORPHOLOGY OF VIRUSES
Size
Viruses are much smaller than bacteria. The extracellular infectious virus particle is called the virion. It was their
small size and ‘filterability’ (ability to pass through filters that can hold back bacteria) that led to their recognition as
a separate class of infectious agents. Hence they were for a time known as ‘filterable viruses’. They were called
‘ultramicroscopic’ as they were too small to be seen under the light microscope. Some of the larger viruses, such as
poxviruses can be seen under the light microscope when suitably stained. The virus particles seen in this manner are
known as ‘elementary bodies’.
The unit for measurement of virion size is nanometers (nm). Viruses vary widely in size from 20 nm to 300 nm. The
largest among them is pox virus (300 nm) and is as large as the smallest bacteria (mycoplasma). The smallest viruses
are the parvovirus (about 20 nm).
Shape of the Virus
The overall shape of the virus particle varies in different groups of viruses. Most of the animal viruses are roughly
spherical, some are irregular and pleomorphic. Poxviruses are brick-shaped, rabies virus is bullet-shaped, tobacco
mosaic virus is rod-shaped. Bacteriophages have a complex morphology. The extracellular infectious virus particle is
known as virion.
STRUCTURE AND CHEMICAL COMPOSITION OF THE VIRUSES

A. Viral Capsid
B. Virus Symmetry
C. Viral Envelope
D. Viral Nucleic Acids

A. Viral Capsid
Viruses consist of nucleic acid core surrounded by a protein coat called capsid. The capsid with the enclosed nucleic
acid is known as nucleocapsid. The capsid is composed of a large number of capsomers which form its
morphological units. The chemical units of the capsid are polypeptide molecules which are arranged symmetrically
to form molecules to form an impenetrable shell around the nucleic acid core (Fig. 53.1).
Functions of Capsid
i. Protection: It protects the viral genome from physical destruction and enzymatic inactivation by nucleases in
biological material.
ii. Binding sites: It provides binding sites which enable the virus to attach to specific receptor sites on the host cell.
iii. It facilitates the assembly and packaging of viral genetic information.
iv. Vehicle of transmission: It serves as a vehicle of transmission from one host to another.
v. Antigenic: It is antigenic and specific for each virus type.
vi. Host’s defence: It is of paramount importance in the host’s defence to virus infection.
vii. It provides the structural symmetry to the virus particle.

B. Virus Symmetry

Viral architecture can be grouped into three types based on the arrangement of morphologic subunits:
(1) Icosahedral symmetry (2) Helical symmetry (3) Complex structures.

1. Icosahedral Symmetry
An icosahedral ( icosa, meaning 20 in Greek) is a polygon with 12 vertices or corners and 20 faces or sides. Each
facet is in the shape of an equilateral triangle. Two types of capsomers constitute the icosahedral capsid. They are the
pentagonal capsomers at the vertices (pentons) and the hexagonal capsomers making up the facets (hexons). There
are always 12 pentons but the number of hexons varies with the virus group, e.g., adenoviruses.
2. Helical Symmetry
The nucleic acid and the capsomers are wound together in the form of a helix or spiral.
Examples: Single-stranded RNA viruses such as influenza (Fig. 53.2), the parainfluenza viruses, and rabies.

3. Complex Symmetry
Viruses (e.g. poxviruses) which do not show either icosahedral or helical symmetry due to complexity of their
structure are referred to have complex symmetry.

C. Viral Envelope
Virions may be enveloped or nonenveloped (naked).
Enveloped Virus
The envelope or outer covering of
virus containing lipid is derived
from the plasma membrane
of the host cell during their
release by budding from the cell
surface. The envelope is
glycoprotein in nature. The lipid is
largely of host cell origin
while the protein is virus-
encoded. Enveloped viruses are
susceptible to the action of lipid
solvents such as ether, chloroform
and detergents, whereas most
viruses existing as naked
capsids are more likely to be
resistant to them.
Peplomers
In mature virus particle, the
glycoproteins often appear as
projecting spikes on the outer
surface of the envelope. These are
known as peplomers (from
peplos, meaning envelope).
A virus may have more than one
type of peplomers, e.g., the
influenza virus carries two kinds
of peplomers, the
hemagglutinin which is a triangular
spike and the
neuraminidase which is a
mushroom-shaped structure.
Envelope confer chemical,
antigenic and biological properties
on viruses.
Functions of Peplomers
i. Mediate attachment: Many peplomers mediate attachment of the virus to the host-cell receptors to initiate the
entrance of the virion into the cell.
ii. Attach to receptors: Some viral glycoproteins also attach to receptors on red blood cells, causing these cells
to agglutinate (hemagglutination).
iii. Enzymatic activity: Other glycoproteins possess enzymatic activity like neuraminidase which cleave
neuraminic acid from host cell glycoproteins.
iv. Major antigens: Glycoproteins are also major antigens for protective immunity.

D. Viral Nucleic Acids

Viruses contain a single kind of nucleic acid—either DNA or RNA—that encodes the genetic information necessary
for replication of the virus. The genome may be single-stranded or double-stranded, circular or linear, and segmented
or nonsegmented. The type of nucleic acid, its strandedness, and its size are major characteristics used for classifying
viruses into families.

VIRAL REPLICATION

The genetic information necessary for viral replication is contained in the viral nucleic acid but lacking bio synthetic
enzymes, the virus depends on the synthetic machinery of the host cell for replication. The viral multiplication cycle
can be divided into six sequential phases, though the phases may sometimes be overlapping: 1. Adsorption or
attachment, 2. Penetration, 3. Uncoating, 4. Biosynthesis, 5. Maturation, and 6. Release.

1. Adsorption or Attachment
Virions come in contact with cells by random collision but adsorption or attachment is specific and is mediated by
the binding of virion surface structures, known as ligands, to receptors on cell surface. In case of influenza virus, a
surface glycoprotein (the hemagglutinin) binds specifically to sialic acid residue of glycoprotein receptor sites on the
surface of respiratory epithelium. In case of human immunodeficiency virus-1 (HIV1), surface glycoprotein gp
120 acts as a ligand. It binds to the CD4 60kDa glycoprotein on the surface of mature T lympho cytes. Similarly,
rabies virus binds to the acetylcholine receptor found on neural cells.

2. Penetration
After binding, the virus particle is taken up inside the cell. In some systems, this is accomplished by receptor-
mediated endocytosis (viropexis), with uptake of the ingested virus particles within endosomes. Most nonenveloped
viruses enter the cell by receptor-mediated endocytosis or by viropexis. Enveloped viruses fuse their membranes
with cellular membranes to deliver the nucleocapsid or genome directly into the cytoplasm.

3. Uncoating
This is the process of stripping the virus of its outer layers and capsid so that the nucleic acid is released into the cell.
With most viruses, uncoating is effected by the action of lysosomal enzymes of the host cell. The genome of DNA
viruses, except for poxviruses, must be delivered to the nucleus, whereas most RNA viruses remain in the cytoplasm.

4. Biosynthesis
This phase includes synthesis not merely of the viral nucleic acid and capsid protein but also of enzymes nec essary
in the various stages of viral synthesis, assembly and release. In addition, certain ‘regulator proteins’ are also
synthesized which serve to shut down the normal cellular metabolism and direct the sequential production of viral
components. The site of viral synthesize depends on the type of virus. In general, most DNA viruses synthesize their
nucleic acid in the host cell nucleus. The exceptions are the poxviruses, which synthesize all their components in the
host cell cytoplasm. Most RNA viruses synthesize all their components in the cytoplasm, except for
orthomyxoviruses, some paramyxoviruses and retroviruzes which are synthesized partly in the nucleus. Viral protein
is synthesised only in the cytoplasm.

5. Maturation
Assembly of the various viral components into virions occurs shortly after the replication of the viral nucleic acid
and may take place in either the nucleus (herpes and adenoviruses) or cytoplasm (picorna and poxviruses). In case of
enveloped viruses, the envelopes are derived
from the host cell nuclear membrane (herpes virus) and from plasma membrane when the assembly occurs in the
cytoplasm of the host cell (orthomyxoviruses and paramyxoviruses).

6. Release
Viruses can be released from cells after lysis of the cell, by exocytosis, or by budding from the plasma membrane.
Viruses that exist as naked nucleocapsids may be released by the lysis of the host cell (polioviruses) or they may be
extruded by a process which may be called reverse phagocytosis. Release of many enveloped viruses occurs after
budding from the plasma membrane without killing the cell.

CULTIVATION OF VIRUSES
Because viruses are obligate intracellular parasites, their growth requires susceptible host cells capable of replicating
them. They cannot be grown on any inanimate culture medium. Three methods are employed for the cultivation of
viruses

A. Animal inoculation
B. Embryonated eggs
C. Cell culture.

A. Animal Inoculation
Uses of Animal Inoculation
i. Primary isolation of certain viruses
ii. For the study of pathogenesis, immune response and epidemiology of viral diseases
iii. For the study of oncogenesis.
1. Monkeys
Monkeys were used for the isolation of the poliovirus but find only limited application in virology due to their cost
and risk to handlers.
2. Mice
The use of white mice, pioneered by Theiler (1903) extended the scope of animal inoculation greatly. Infant
(suckling) mice are very susceptible to coxsackie and arboviruses, many of which do not grow in any other system.
Mice may be inoculated by several routes— intracerebral, subcutaneous, intraperitoneal or intranasal. The growth of
the virus in inoculated animals may be indicated by death, disease or visible lesions. The viruses are identified by
testing for neutralization of their pathogenicity for animals, by standard antiviral sera.
B. EmbrYonated Eggs

The embryonated hen’s egg was first used for the cultivation of viruses by Goodpasture (1931) and the method was
further developed by Burnet. The embryonated egg (8-11 day old) are inoculated by several routes for the cultivation
of viruses such as chorioallantoic membrane (CAM), allantoic cavity, amniotic cavity and yolk sac (Fig. 53.4). After
being inoculated, eggs are incubated for 2-9 days.
1. Chorioallantoic Membrane (CAM)
Inoculation on to chorioallantoic membrane (CAM) can be used to cultivate herpesvirus, smallpox virus (variola),
vaccinia, myxoma virus, Rous sarcoma virus and eastern equine encephalitis virus.
Inoculation on the chorioallantoic membrane (CAM) produces visible lesions (pocks). Each infectious virus particle
can form one pock under optimal conditions. Pock counting, therefore, can be used for the assay of pock-forming
viruses, such as variola or vaccinia. Different viruses have different pock morphology.
2. Allantoic Cavity
Allantoic inoculation is employed for growing the influenza virus for vaccine production. Other chick embryo
vaccines are yellow fever (17D strain) and rabies (Flury strain) vaccines. Duck eggs are bigger and have a longer
incubation period than hen’s eggs. Therefore, they provide a better yield of rabies virus and were used for the
preparation of the inactivated, non-neural rabies vaccine.Inoculation into the amniotic sac is employed for the
primary isolation of the influenza virus.
4. Yolk Sac
Yolk sac inoculation is used for the cultivation of some viruses, chlamydiae, Coxiella burnetti and rickettsiae.

C. Tissue Culture
Three types of tissue cultures are available
1. Organ Culture
Small bits of organs can be maintained in vitro for days and weeks, preserving their original architecture and
function. Organ cultures are useful for the isolation of some viruses which appear to be highly specialized par asites
of certain organs. For example, the tracheal ring organ culture is employed for the isolation of coronavirus, a
respiratory pathogen.
2. Explant Culture
Fragments of minced tissue can be grown as ‘explant’ embedded in plasma clots and was originally known as ‘tissue
culture’. This method is now seldom employed in virology. Adenoid tissue explant cultures were used for the
isolation of adenoviruses.
3. Cell Cultures
This is the type of culture routinely employed for growing viruses. Tissues are dissociated into the component cells
by the action of proteolytic enzymes such as trypsin and mechanical shaking. The cells are washed, counted and
suspended in a growth medium. Such media will enable most cell types to multiply with a division time of 24-48
hours. The cell suspension is dispensed to the glass surface and on incubation, divide to form a confluent monolayer
sheet of cells covering the surface within about a week. Cell culture tubes may be incubated in a sloped horizontal
position, either as ‘stationary culture’ or may be rolled in special ‘roller drums’ to provide better aeration. Some
fastidious viruses grow only in such roller cultures.

Classification of Cell Cultures

Cell cultures are classified into three types based on their origin, chromosomal characters and the number of
generations through which they can be maintained (Table 53.4).
1. Primary Cell Cultures
These are normal cells freshly taken from the body and cultured. They are capable of only limited growth in culture
(5-10 passages) and cannot be maintained in serial culture. Primary cell cultures are useful for the isolation of viruses
and their cultivation for vaccine production. Important examples of primary cell cultures are monkey kidney; human
embryonic kidney, human amnion and chick embryo cell cultures.
2. Diploid (Semi-continuous) Cell Strains
These are cells of a single type that retain the original diploid chromosome number and karyotype during serial
subcultivation for a limited number of times. They undergo ‘senescence’ after about fifty serial passages. Diploid
cells developed from human fibroblasts are susceptible to a wide range of human viruses.
They are useful for isolation of some fastidious pathogens and for the production of viral vaccines, e.g. rabies
vaccine is produced by cultivation of the fixed rabies virus in WI-38 human embryonic lung cell strain.
3. Continuous Cell Lines
These are the most widely used for diagnostic work. These are cells of a single type, usually derived from cancer
cells, that are capable of continuous serial cultivation indefinitely. They have been derived from diploid cell lines or
from malignant tissues. They invariably have altered and irregular number of chromosomes. Such cells often
produce tumors if injected into susceptible animals.
Standard cell lines derived from human cancers, such as HeLa, Hep-2 and KB cell lines have been used in lab-
oratories throughout the world for many years. These cell lines may be maintained by serial subcultivation or stored
in the cold (–70°C) for use when necessary. cell lines are now permitted to be used for vaccine man ufacture, for
example, Verocell for rabies vaccine

DETECTION OF VIRUS GROWTH IN CELL CULTURE

Virus growth in cell cultures can be detected by the following methods:

1. CYTOPATHIC EFFECT
Many viruses cause morphological changes in cultured cells in which they grow. These changes can be readily
observed by microscopic examination of the cultures and are known as ‘cytopathic effects’ (CPE) and the viruses
causing CPE are called ‘cytopathogenic viruses’.

2. ELECTRON MICROSCOPY
3. IMMUNOFLUOROSCENCE
4. DETECTION OF VIRAL NUCLEIC ACIDS

Note:
VIROIDS
Viroids are small infectious agents which are circular single stranded RNAs without a protein coat. They cause
disease of plants. Viroids are agents that do not fit the definition of classic viruses.
PRIONS
Prions (proteinaceous infectious particles) are infectious particles composed solely of protein with no detectable
nucleic acid. Unlike viruses, the agents are resistant to a wide range of chemical and physical treatments. They are
highly resistant to inactivation by heat, formaldehyde, and ultraviolet light that inactivate viruses. The prion protein
is encoded by a single cellular gene

CLASSIFICATION OF VIRUS BASED ON HOST

1. VIRUS WHICH AFFECT PLANT. Ex: tobacco mosaic virus

2. VIRUS WHICH AFFECT ANIMAL/HUMAN ex: pox virus
3. VIRUS WHICH AFFECT BACTERIA ex: bacteriophage

CLASSIFICATION OF
VIRUS BASED ON
GENETIC MATERIAL

1. RNA VIRUS
2. DNA VIRUS
 BACTERIOPHAGE:

INTRODUCTION:

Viruses that infect bacteria are called bacteriophages, or simply phages..

Phages can readily be isolated from a wide range of environments such as feces, sewage and other natural sources of
mixed bacterial growth.
Discovery of bacteriophage:
Twort (19l5) described a degenerative change in staphylococcal colonies isolated from calf lymph, which could be
transmitted serially by application of culture filtrates from the original growth. d’Herelle (1917) observed that the
filtrates of feces cultures from dysentery patients induced transmissible lysis of a broth culture of a dysentery
bacillus. He suggested that the lytic agent was a virus and gave it the name bacteriophage (phage: to eat). Phages
occur widely in nature in close association with bacteria.
ROLE OF BACTERIOPHAGES
1. They play an important role in the transmission of genetic information from one bacterium to another by the
process of transduction.
2. They also play a role in the evolution of bacterial types and in the transmission of some virulence characters.
3. Phages may indeed be effective in treating bacterial infections, including those caused by antibiotic-resistant
bacteria.
4. Phages have been used as cloning vectors in genetic manipulations.
5. They may have a role in the control of bacterial populations in natural waters.

STRUCTURE OF BACTERIOPHAGE:
Most phages are tadpole-shaped, possess a hexagonal
head and a cylindrical tail.
Head
The head consists of a tightly packed core of nucleic
acid (double stranded DNA) surrounded by a protein
coat or capsid. The size of th e head varies in
different phages from 28 nm to 100 nm. The size of
the head varies in different phages from 28 nm to 100
nm. The head of phage T4 has a diameter of 65 nm
and is 100 nm long.

Tail
The tail is cylindrical and is composed of a central
hollow core or tube, a contractile sheath surrounding
the core and a terminal base plate which has attached to it prongs or tail fibers (usually six in number) or both that
bind to specific receptor sites on the bacterial surface.

LIFE CYCLE OF BACTERIOPHAGE:

Phages exhibit two different types of life cycle. In the virulent or lytic cycle, intracellular multiplication of the phage
culminates in the lysis of the host bacterium and the release of progeny virions. In the temprate or lysogenic cycle the
phage DNA becomes integrated with the bacterial genome, replicating synchronously with it, causing no harm to the
host cell

Lytic Cycle
Replication of a virulent phage can be considered in the following stages adsorption, penetration, synthesis of phage
components, assembly, maturation and release of progeny phage particles
i. Adsorption
By random collision, phage particles attach to virus-specific receptors on the host cell by its tail. Adsorption is a
specific process and depends on the presence of complementary chemical groups on the receptor sites on the
bacterial surface and on the terminal base plate of the phage. Adsorption is a very rapid process under optimal
conditions, being complete within minutes. Any component on the bacterial surface can serve as receptor for some
phage.

ii. Penetration
After adsorption, most phages inject their nucleic acid into the bacterial cytoplasm and leave their protein cap sid
outside. The process of penetration resembles injection through a syringe. Following adsorption, six tail pins make
contact with the host cell surface and firmly attach the phage plate to it. The contractile tail sheath then contracts
forcing the hollow interior tail tube into the bacterial cell wall. The phage DNA then passes through the hollow
interior tail tube. The empty head and tail of the phage remain outside the bacterium as the shell or ‘ghost’ after
penetration. Penetration may be facilitated by the presence on the phage tail of lysozyme which produces a hole on
the bacterial wall for the entry of the phage core.

iii. Synthesis of Phage Nucleic Acid and Proteins

The synthesis of the phage components is initiated immediately after penetration of the phage nucleic acid.
The first products to be synthesized (called early proteins) are the enzymes necessary for the building of the complex
molecules peculiar to the phage. Subsequently, late proteins appear, which include the protein subunits of the phage
head and tail. During this period, the synthesis of bacterial protein, DNA and RNA ceases and the cell is forced to
make viral constituents.

iv. Assembly and Maturation

The phage structural proteins and nucleic acid are assembled in definite pathways to form the mature progeny phage
particle. Phage DNA, head protein and tail protein are synthesized separately in the bacterial cell. The DNA is
condensed into a compact polyhedron and ‘packaged’ into the head and, finally, the tail structures are added. This
assembly of the phage components into the mature infective phage particle is known as maturation.

v. Release
Many phages lyse their host cells at the end of the intracellular phase. Release of the mature progeny phages
typically occurs by lysis of the bacterial cell. The bacterial cell wall is weakened and it assumes a spherical shape
during the replication of the phage. Phage enzymes act on the weakened cell wall causing it to burst or lyse resulting
in the release of mature daughter phages.

Eclipse Phase
The interval between the entry of the phage nucleic acid into the bacterial cell and the appearance of the first
infectious intracellular phage particle is known as the eclipse phase. It represents the time required for the synthesis
of the phage components and their assembly into mature phage particles.