Virology

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 Contents
Chapter title Page

1 Introduction……………………………………………. 3

2 Historical Introduction………………………………… 5

3 The nature of the virus………………………………… 19

4 Cultivation of viruses………………………………….. 26

5 Purification of viruses…………………………………. 39

6 Chemistry of viruses…………………………………... 66

7 Bacteriophages………………………………………… 84

8 References……………………………………………... 106

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INTRODUCTION

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 A virus is a very small ultra-filterable and ultra-microscopic
infectious agent that replicates only inside the living cells of other
organisms (obligate intracellular parasite). Viruses are pathogenic that
can infect all types of life forms, from animals and plants to
microorganisms, including bacteria and archaea causing diseases for
them (Koonin et al., 2006).
Virus particles consist of two or three parts:-
i) The genetic material made from either DNA or RNA, long
molecules that carry genetic information, may be linear or closed,
double stranded or single stranded.
ii) A protein coat that protects these genes called "capsid" formed by a
number of morphological units called "capsomers" each one
composed of smaller building units called protomers that compose
of amino acids.
iii) In some cases an envelope of lipids that surrounds the protein coat
when they are outside a cell.
They are morphologically differing may be:-
 Spheroid (most animal viruses).
 Elongated (most plant viruses).
 Spheroid and elongated (bacteriophage).
 Complex.
The symmetry of viruses may be helical forms in elongated
viruses and icosahedral forms in spheroid and complex structures.
They replicate though host cell take over and complete their life
cycle inside the host cell.
The infection may be reproductive, non-reproductive or
integrated.
They cannot be classified under any kingdoms and need a separate one
because:-
 They are a cellular entities (lake cytosol).
 Present in crystalline form.
 Lake machinery system (no organelles).
 Cannot be cultivated on synthetic media.
 The only entity that contain double stranded RNA.
However viruses need advanced tools for examination,
investigation, cultivation and purification, they had been discovered
even before modern times.

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Historical Introduction

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Ancient civilization

 Viruses have been recorded as disease symptoms.

1) Pharaonic civilization:-
 Poliomyelitis disease has been recorded by Ancient Egyptians as
paintings on the wall of temples and carvings, this paintings and
carvings such as children with canes at young age or as healthy people
with withered limbs (Daniel and Robbins, 1997).
 Small box disease is found in the Egyptian mummies of people who
died some 3000 years ago (Fenner et al., 1988).
2) Indian and Chinese civilization:-
 Some form of inoculation was developed in India or China before the
16th century (Lombard et al., 2007).
 The Earliest documented example of vaccination is a vaccine for small
box disease, where vaccination with powdered scabs from people
infected with small box was used to protect against the disease in India
and China in the 17th century ( Lund et al., 2005).

Fig.1 Poliomyelitis in Ancient Egypt

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Dark centuries

 Appearance of epidemic diseases such as:

1) Yellow fever :-
 Phylogenetic analyses indicate that the virus originated from East or
Central Africa, with transmission between primates and humans, and
spread from there to West Africa (Bryant et al., 2007).
 The virus was probably brought to the western hemisphere and the
Americas by slave trade ships from Africa (Haddow, 2012).
2) Foot and mouth of cattle (FMD) :-
 In 1546 Hieronymous Fracastorius described an epidemic that occurs
in cattle (Brooksby, 1958).
3) Common cold:-
 The disease has been with humanity since antiquity (Eccles and
Weber, 2009).
 The name "cold" came into use in the 16th century, due to the similarity
between its symptoms and those of exposure to cold weather.
 These diseases cause death of millions of people and animals.

Carlos Closus (1575)

 Tulip break virus (TBV):-

 The earliest recorded plant virus.
 First described in 1575 by Carlos Closus. He observed appearance of
different bands with different colours on the petals of Tulip flowers and
he called this phenomena “Tulip break".
 As early as 1637 Dutch growers were able to produced new broken
varieties through bulb grafting, by combining "broken" bulbs infected
with the virus with healthy bulbs that produced uniformly colored
flowers.

Fig.2 (TBV)

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19th Century

1) Louis Pasteur:-
 Father of Microbiology.
 He denied the "spontaneous generation theory" that means, certain
forms such as fleas could arise from inanimate matter such as dust and
established the "germ theory" that means, any infectious diseases are
caused by germs.
 Pasteur demonstrated that the skin of grapes was the natural source of
yeasts, and that sterilized grapes and grape juice never fermented. He
drew grape juice from under the skin with sterilized needles, and also
covered grapes with sterilized cloth. Both experiments could not
produce wine in sterilized containers. His findings and ideas were
against the prevailing notion of spontaneous generation (Farley and
Geison, 1974; Roll-Hansen, 1979 and Magner, 2002).
 To prove his correctness, Pasteur exposed boiled broths to air in swan-
neck flasks that contained a filter to prevent all particles from passing
through to the growth medium, and even in flasks with no filter at all,
with air being admitted via a long tortuous tube that would not allow
dust particles to pass. Nothing grew in the broths unless the flasks were
broken open, showing that the living organisms that grew in such
broths came from outside, as spores on dust, rather than spontaneously
generated within the broth. This was one of the last and most important
experiments disproving the theory of spontaneous generation
(Schwartz, 2001and Ligon, 2002).

Fig.3 Louis Pasteur experiment

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2) Robert Kouch:-
 Father of Pathology.
 He is known for his role in identifying the specific causative agents of
tuberculosis, cholera, and anthrax and for giving experimental support
for the concept of infectious disease.
 He put Kouch's postulates as general guidelines to identify pathogens
(Walker et al., 2006).
 Couch’s postulates are a series of four generalized principles linking
specific microorganisms to specific diseases that remain today the
"gold standard" in medical microbiology (Brock, 1999).
 Kouch's postulates:
1. The microorganism must be found in abundance in all organisms
suffering from the disease, but should not be found in healthy
organisms.
2. The microorganism must be isolated from a diseased organism and
grown in pure culture.

3. The cultured microorganism should cause disease when introduced

into a healthy organism.
4. The microorganism must be re-isolated from the inoculated,
diseased experimental host and identified as being identical to the
original specific causative agent.
3) Adolf Mayer (1882-1886) :-
 He was a German agricultural chemist who played an important role in
the discovery of tobacco mosaic virus (TMV) and viruses in general, so
considered the father of virology (Zaitlin and Milton, 1998).
 Symptoms of TMV :-
 Light green coloration between the veins of young leaves followed
quickly by the development of a "mosaic" or mottled pattern of light
and dark green areas in the leaves. Rugosity may also be seen where
the infected plant leaves display small localized random wrinkles.
These symptoms develop quickly and are more pronounced on younger
leaves. Its infection does not result in plant death, but if infection
occurs early in the season, plants are stunted. Lower leaves are
subjected to "mosaic burn" especially during periods of hot and dry
weather. In these cases, large dead areas develop in the leaves.

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 Mayer experiments:-
a. Mechanical inoculation of infectious sap to a healthy leaf
Symptoms appear.
b. Filtration of the infectious sap by using a pad of filter papers
No symptoms appear.
c. Thermal treatment of the infectious sap at different temperatures:

30◦C Sever symptoms

50◦C Few symptoms
70◦C Little symptoms
100◦C No symptoms

d. Treatment of infectious sap by organic solvent that separated into

precipitate containing the causative agent and a clear sap, taking the
clear sap and inoculating into a healthy leaf No symptoms
appear.
 Mayer results:-
a. The causative agent of mosaic can be transmitted by mechanical
inoculation.
b. The infectious sap loses its effect after filtration.
c. The ability of the causative agent to cause the disease is decreased
by raising the temperature.
d. The causative agent is precipitated by organic solvent.
 Mayer conclusion:-
 The causative agent of mosaic disease is bacteria.
 Mayer mistakes:-
i. Using a pad of filter paper.
ii. Making his experiments only one time.
4) Dmitri Iwanowski (1892) :-
 He gave the first evidence for the existence of a non-bacterial
infectious agent, showing that infected sap remained infectious even
after filtering through chamberland filter (a filter invented by Charles
Chamberland, had pores smaller than bacteria)( Zaitlin and Milton,
1998).
 He called these agents filterable agents and supposed that they are
smaller than bacteria.

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 Fig.4 Filterable agent

5) Martinus Beijerinck (1897) :-

 He agree with Iwanowski in that the causative agent is filterable but he
suggested that this agent is fluid in nature and this fluid is a toxic living
substances secreted by bacteria. He called this agent cantagium vivum
fluidum and introduced the word "virus" (Sussman et al., 1998).
 He could not culture the filterable infectious agent, however he
concluded that the agent can replicate and multiply in living plants
(Zaitlin and Milton, 1998).

20th century

1) Adelchi Negri (1903) :-

 He discovered the eponymous Negri bodies defined
as cytoplasmatic inclusion bodies located in the purkinje cells of
the cerebellum in cases of rabies in animals and humans.
 These inclusion bodies appeared only in the infected cells.

Fig.5 Inclusion Bodies

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2) Friedrich Loeffler and Paul Frosch :-
 They found evidence that the cause of foot-and-mouth disease in
livestock was an infectious particle smaller than any bacteria. This was
the first clue to the nature of viruses, genetic entities that lie
somewhere in the grey area between living and non-living states.
 The foot and mouth disease virus was the first animal virus described.
 They succeeded in producing the first protective serum against foot and
mouth disease, which however was not used for cost reasons.

3) Peyton Rous (1907) :-

 He was involved in the discovery of the role of viruses in the
transmission of certain types of cancer. He made his seminal
observation, that a malignant tumor (specifically, a sarcoma) growing
on a domestic chicken could be transferred to another fowl simply by
exposing the healthy bird to a cell-free filtrate .This finding that, cancer
could be transmitted by a virus and this virus is oncogenic virus or
retrovirus (Rous, 1910).
4) Frederick Twort (1915) :-
 At the time, smallpox vaccines had to be made in the skin of calves and
were almost always contaminated with the bacterial
genus Staphylococcus. Twort plated some of the smallpox vaccines
on nutrient agar slants and obtained large bacterial colonies of several
colours. Upon closer examination of the colonies with a magnifying
glass, he found minute glassy areas that would not grow when
subcultured. He quickly realized these glassy areas were the result of
the destruction of the bacterial cells and was able to pick from some of
these areas and transmit this from one staphylococci colony to another
(Ackermann, 2009).
 He believed this agent must be one of the following:
a) A stage in the life cycle of the bacteria.
b) An enzyme produced by the bacteria themselves.
c) A virus that grew on and destroyed the bacteria.
5) Félix d'Herelle (1917) :-
 He discovered an invisible antagonistic agent, when added to bacteria
dysentery bacillus on agar medium, would produce areas of dead
bacteria, this antagonist could pass through a Chamberland filter.

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 He accurately diluted a suspension of this agent and discovered that the
highest dilutions, rather than killing all the bacteria, formed discrete
areas of dead organisms. Counting these areas and multiplying by the
dilution factor allowed him to calculate the number of these agents in
the original suspension.
 He concluded that:
 These agents are viruses that infect bacteria; he called them
bacteriophage bacteria-eater.
 "The antagonistic agent can never be cultivated in media in the absence
of the dysentery bacillus. It does not attack heat-killed dysentery
bacilli, but is cultivated perfectly in a suspension of washed cells in
physiological saline. This indicates that the anti-dysentery agent is an
obligate bacteriophage.

Fig.6 Bacteriophage by Electron Microscope

6) Wendell Stanley (1935) :-
 He was the 1st scientist purify virus.
 He crystallizes TMV using ammonium sulphate in a process known as
"salting out" and shows that it remains infectious and it has properties
of both living and non-living matter. However, he does not recognize
that the infectious material is nucleic acid and not protein (Zaitlin and
Milton, 1998).

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7) Norman Pirie and Frederick Bawden (1938):-
 Pirie and Bawden attempted to isolate the potato virus a virus difficult
to purify and in 1936 reported the association of its infectivity with
material sensitive to various proteinases. Their enthusiasm was
heightened by the 1935 report of Wendell M. Stanley.
 They studied the tobacco mosaic virus, demonstrating that the virus
contained ribonucleic acid.
8) Electron Microscope (1940):-
 The "inclusion bodies" were seen in many virus-infected cells
using optical microscopes but these aggregates of virus particles were
still too small to reveal any detailed structure until the invention of the
electron microscope in 1931 by the German engineers Ernst Ruska
and Max Knoll.
 The first practical electron microscope was constructed in 1939.
 The first examined virus was TMV, it was rod shaped particle that
confirmed the particulate nature of viruses.

Fig.7 Electron Microscope, 1940

9) George Hirst (1941) :-

 Was an American virologist , particularly known for inventing the"
hemagglutination assay", a simple method for quantifying viruses and

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 adapting it into the "hemagglutination inhibition assay", which
measures virus-specific antibodies in serum.
 In 1941, Hirst discovered that adding influenza virus particles to red
blood cells caused them to agglutinate or stick together forming a
lattice, a phenomenon called "hemagglutination".
 Hemagglutination provided a convenient method of diagnosing
influenza in the laboratory, which had previously been performed by
cultivating the virus in ferrets. Hirst developed this reaction into the
"hemagglutination assay", which allows the amount of virus in the
sample to be measured (Schlesinger and Granoff, 1994). This
technique is rapid, accurate and convenient, and later proved to be
applicable to many other viruses (Joklik, 1999 and Racaniello, 2009).
 He soon realized that the" hemagglutination assay" could easily be
adapted to measure the levels of antibody specific to the virus strain in
human serum: any antibodies present bind to the influenza virus
particles, prevent them from crosslinking red blood cells and so inhibit
hemagglutination (Schlesinger and Granoff, 1994 and Racaniello,
2009).
10) Alfred Hershey and Martha Chase (1952) :-
 Hershey–Chase experiments were a series of experiments conducted in
1952 by Alfred Hershey and Martha Chase that helped to confirm that
DNA is the genetic material (Hershey and Chase, 1952).
 Viruses were known to be composed of a protein shell and DNA, so
they chose to uniquely label each with a different elemental isotope.
This allowed each to be observed and analyzed separately. Since
phosphorus is contained in DNA but not amino acids, radioactive
phosphorus-32 was used to label the DNA contained in the T2 phage.
Radioactive sulfur-35 was used to label the protein sections of the T2
phage, because sulfur is contained in amino acids but not DNA.
 Hershey and Chase inserted the radioactive elements into the
bacteriophages by adding the isotopes to separate media within which
bacteria were allowed to grow for 4 hours before bacteriophage
introduction. When the bacteriophages infected the bacteria, the
progeny contained the radioactive isotopes in their structures. This
procedure was performed once for the sulfur-labeled phages and once
for phosphorus-labeled phages. The labeled progeny were then allowed

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 to infect unlabeled bacteria. The phage coats remained on the outside
of the bacteria, while genetic material entered.
 Centrifugation allowed for the separation of the phage coats from the
bacteria. These bacteria were lysed to release phage progeny. The
progeny of the phages that were originally labeled with P-32 remained
labeled, while the progeny of the phages originally labeled with S-35
were unlabeled. Thus, the Hershey–Chase experiment helped confirm
that DNA, not protein, is the genetic material (Hershey and Chase,
1952).

Fig.8 Hershey–Chase experiment

11) James Watson and Francis Crick (1953):-

 In 1953, James Watson and Francis Crick suggested what is now
accepted as the first correct double-helix model of DNA structure
(Watson and Crick, 1953).
 Their double-helix, molecular model of DNA was then based on a
single X-ray diffraction image taken by Rosalind Franklin and
Raymond Gosling in May 1952, as well as the information that the

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 DNA bases are paired also obtained through private communications
from Erwin Chargaff in the previous years.

Fig.9 Watson and Crick DNA model Fig.10 Franklin X ray crystallography

12) Heinz Conrat and Robley Williams (1956):-

 They made the following experiments:

a. Inoculation of TMV protein coat into healthy leaves….No symptoms
appear.
b. Inoculations of TMV nucleic acid into healthy leaves….Symptoms
appear.
c. Put viral protein andnucleic acid in a tube containing pyrophosphate as
a catalyst, PH =7.2 and at 37 ͦ C and left for 60 min. Then examined
the tube.
 They found that, particles similar to TMV particles are formed. When
these particles inoculated into healthy leaves….Symptoms appear.
 They concluded that:
i. Only nucleic acid is responsible for carrying genetic information,
replication and infection.
ii. The viruses have the ability of "self assembly" and can cause
infection after assembly.
13) Lowff (1966 -1969):-
 He tried to classify and put them in a special kingdom known as
viratae.

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14) Viruses as a tool of genetic engineering (1976), why?
 Viruses can be used to transmit genes of special traits from one
organism to another as :-
 Viral nucleic acid is simple and carrying few genes.
 Viral nucleic acid can integrate with host cell nucleic acid.
 Viruses are obligate intracellular parasites and their nucleic acid
penetrates the host cells.
 Viruses are acellular entities.

15) Acquired Immunodeficiency Syndrome (AIDS) (1984):-

 The causative virus is Human Immunodeficiency Virus (HIV).

 Spherical virus (Govern et al, 2002).
 With a diameter of about 120 nm, around 60 times smaller than a red
blood cell, yet large for a virus.
 Composed of two copies of positive single-stranded RNA that codes
for the virus's nine genes enclosed by a conical capsid composed of
2,000 copies of the viral protein p24.

16) Poweil (1986):-

 He obtained the first transgenic plant which can resist diseases.
17) Molla (1991):-
 He succeeded to create virus such as poliovirus and other viruses which
used in biological wars (Conrat idea).
18) Ebola (1995):-
 The first science talked about epidemic viral disease which causes
infection and explosion of the brain.
 He called this disease "Ebola".

21st century

 Emergence and Discovery of:-

 New types of viruses.
 New ways for creation of viruses.
 Entities smaller than viruses.
 Molecular Virology.

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The nature of the virus

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1-Introduction

Viruses were first discovered after the development of a porcelain

filter, called the Chamberland-Pasteur filter, which could remove all
bacteria visible in the microscope from any liquid sample. In 1886,
Adolph Meyer demonstrated that a disease of tobacco plants, tobacco
mosaic disease, could be transferred from a diseased plant to a healthy one
via liquid plant extracts. In 1892, Dmitri Ivanowski showed that this
disease could be transmitted in this way even after the Chamberland-
Pasteur filter had removed all viable bacteria from the extract. Still, it was
many years before it was proven that these "filterable" infectious agents
were not simply very small bacteria, but were a new type of tiny, disease-
causing particle.

1.1. Definitions of viruses

 Viruses are obligate parasitic pathogen, their dimension less than

200m-micron (Bawden, 1950).
 A particle whose atoms are arranged in a more or less constant
relationship to each other (Crick and Watson, 1956).
 Submicroscopic infectious entities that multiply only intracellularly
and are potentially pathogenic (Bawden, 1964).
 Viruses could be surely set as apart from other microorganisms on
the bases of five discriminative features:-
 Presence of a single type of nucleic acid,
 Reproduction from the genetic material only
 Incapacity to grow and divide
 Absence Lippman”s system
 Absence of genetic information required for synthesis of
cellular systems (absolute parasite) (Lowff and Tournier,
1967)

 Viruses are viruses because they are viruses ( Lowff )

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1.2. General characters of viruses

1.2.1. Size

 Viruses are minute in nature: they are very small in size, where they
are the second smallest entities in our planet (10-350 nm).
 Viruses are ultra-filterable agents: as they pass through bacterial
filters.
 Viruses are ultra-microscopic agents: as they could not be seen by
naked eye or by light microscope but could be examined by electron
microscope.

1.2.2. Nature

 Viruses are acellular entities: they are simple in structure as they lack
cytoplasm.
 Viruses may be present in crystalline form outside ( as pure crystal)
or inside (as inclusion body) their hosts.

Pure crystal Inclusion body

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1.2.3. Nutrition

 Viruses are obligate intracellular parasite.

 Viruses are metabolically inert: as they lack lippman system,
precursors and machinery system.
 Viruses are pathogenic: all viruses are obligate parasites causing
diseases for their host.
 Viruses cannot be cultivated on synthetic media.

1.2.4. Shape

Viruses are morphologically differ, they may be spheroid (as most

animal viruses), elongated (as most plant viruses), spheroid and elongated
(as most bacterial viruses) and complex.

Fig.11 virus shapes

1.2.5. Structure

 Viruses have the genetic material (core)

enclosed in protein capsid.

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 So, viruses chemically are nucleoprotein. Some infectious agents even
simpler than viruses based on structure:-

 Viroids, smaller and simpler than even viruses, consist of tiny molecules
of naked circular RNA that infect plants.
 Prion: A group of proteinaceous infectious agent (lacking nucleic acid)
with high molecular weight and cause diseases to animals only.

 Viral genetic material is only one type of nucleic acid: nucleic acid
in viruses may be DNA (single or double stranded) or RNA (single
or double stranded). So, based on nucleic acid viruses are classified
into 3 groups: RNA, DNA and RNA< >DNA.

In RNA< >DNA both RNA&DNA cannot be present inside the molecule at

the same time, i.e. there is one type, but when the RNA viral injection of the
breadwinner cell turns to DNA and vice versa, i.e. it is reflexive viruses
(carcinogen viruses or reterovirus e.g HIV).

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 Viral protein nature: viral protein coat is formed of a number of units
called capsomers, each capsomer consists of smaller building blocks
called protomers which is composed of amino acids.

capsomer

protomer

capsid

Core (nucleic acid)

 Viruses are repeated identical units with known symmetry:

helical or icosahedral.

Helical Icosahedral

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1.2.6. Replication

 Viruses replicate through host cell take over

when the viral nucleic acid enters the host cell, it controls all
processes in the cell and then cause host cell hydrolysis.
 Viruses complete its life cycle only inside host cell.
 Viral reproduction may be reproductive, non-reproductive or
integrated.

 Viruses are geometrically fixed: they could not growth.

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Cultivation of viruses

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1. Importance of cultivation

Is to propagate the viral particle in its specific host and increase viral
concentration through support viral replication.

2. Cultivation of viruses

Viruses can be cultivated within suitable hosts, such as a living cell.

To study bacteriophages, for example, bacteria are grown in a suitable
growth medium; then bacteriophages are added. The bacteriophages
multiply within the bacteria and increase their numbers substantially.

3. Cultivation of animal viruses

Viruses are obligate intracellular parasites. They depend totally on

their host cells for their existence. Their total host dependence makes it
extremely difficult to get good insight of them natural conditions, because
the internal characteristics of the host cells are likely to interfere with the
observations. Due to these reasons, it has been found desirable that viruses
are cultivated or grown in the laboratory itself.
It has two advantages:
 This enables to get sufficient amount of virus particles at any given
time.
 This would mean lesser degree of contamination from the host cell
material, ensuring a purer virus sample.

Since viruses are host dependent, it is not possible to cultivate them

solely in presence of organic or inorganic nutrient medium. They can be
grown only if living cells and tissues are used as culture medium. These
tissues and cells would act as the host for the virus in laboratory
conditions. For this purpose, the relevant cells or tissues must be cultivated
first.

3.1. Methods of cultivation of animal viruses

There are three methods for cultivation of animal viruses:

1- Organized tissue method.

2- Cell culture method.
3- Laboratory animal’s method.

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 3.1.1. Organized tissue method

This method is also called Chick-Embryo technique or

Embryonated Hen Egg or Wood and Good Pasture. The animal viruses
can be successfully cultivated using chick-embryo technique. In this
method fertile hen eggs are selected. Eggs must not be more than 12
days old. To prepare the egg for virus cultivation, the shell surface is
first disinfected with iodine and penetrated with a small sterile drill.
After inoculation, the drill hole is sealed with gelatin and the egg is then
incubated. Viruses may be able to region. For convenience, the myxoma
virus grows well on the chorioallantoic membrane, whereas the mumps
virus prefers the allantoic cavity. The infection may produce a local
tissue lesion known as pock, whose appearance often is characteristic of
the virus.

Fig.12 Organized tissue method

The embryonated offers several sites for the cultivation of viruses:

A. Chorioallantoic membrane (CAM):
CAM is inoculated mainly for growing poxvirus. Herpes simplex
virus is also grown. Virus replication produces visible lesions, grey white
area in transparent Cam. Each pock is derived from a single virion. Pocks
produced by different virus have different morphology. Under optimal
conditions, each infectious virus particle can form one pock. Pock
counting, therefore can be used for the assay of pock forming virus such as
vaccinia.

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B. Allantoic cavity:
Inoculation into the allantoic cavity provides a rich yield of influenza
and some paramyxoviruses. Allantoic inoculation is employed for growing
the influenza virus for vaccine production. Other allantoic vaccines
include Yellow fever (17D strain), and rabies vaccines. Duck eggs are
bigger and have a longer incubation period then hen’s egg. They therefore
provide a better yield of rabies virus and were used for the preparation of
the inactivated non-neural rabies vaccines.
C. Amniotic cavity:
The amniotic sac is mainly inoculated for primary isolation of influenza
a virus and the mumps virus.
D. Yolk sac:
It is inoculated for the cultivation of some viruses as well as for some
bacteria like Chlamydiae and Rickettsiae.

There are two techniques for inoculation of virus inside embryonated

egg the first is by injection in which virus is inoculated directly into the
embryo and the second is by spreading in which virus is sprayed on the
exposed membrane(chorioallantoic membrane).

Fig.13 Embryonated egg

3.1.2. Cell culture method

Cell culture is the process by which cells are grown under controlled
conditions, generally outside of their natural environment. In practice, the
term "cell culture" now refers to the culturing of cells derived from multi-

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cellular eukaryotes, especially animal cells. However, there are also
cultures of plants, fungi, and microbes, including viruses, bacteria and
protists. The historical development and methods of cell culture are
closely interrelated to those of tissue culture and organ culture.

Fig.14 Cell culture method

In this method, specific host cells from susceptible animal are

cultivated and grown on a specific media under laboratory conditions to be
used as a media for virus cultivation.

The tissue is first removed from the organism concerned. This tissue
is then broken down into its constituent cells by utilizing suitable physical
means. Homogenization in a homogenizer is common method utilized.
The complete tissue is then converted into many small pieces. The tissue
fragments are washed with salt solutions. Sterile physiological saline or
other types of solution like saline or other types of solution like hank’s
solution or eagle’s solution are used. The pieces are converted into their
constituent cells by a process called dispersion of the cells from tissue. It
is done by breaking down the proteinaceous cementing material (i.e.
Haluronic acid), joining the cells with the help of proteolytic enzymes like
trypsin and mechanical shaking. This step is called as trypsinization.
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 The washed tissue fragments are then placed in a flask with sterile
trypsin solution at 4°c for about 18 hours. During this period, the tissue
fragments area gradually dispersed into their cellular components.
Presence of chemicals like EDTA helps in dispersion of cells. The cells
are then centrifuged and resuspended in washing medium. It is done
repeatedly. The washed suspended cells are then cultivated in a suitable
growth medium. The essential constituents of growth medium are
physiological amounts of essential amino acids, and vitamins, salts and
glucose and a buffering system generally consisting of bicarbonate in
equilibrium with atmosphere containing about 5% calf or fetal calf serum.
Antibiotics are added to prevent bacterial contaminants and phenol red as
indicator. Such media will allow most cell types to multiply with a
division time of 24-48 hrs.
Cultivation is done after adjustment of the number of cells per unit
volume. The required number of cells is suspended in the growth medium
taken in a tube or flask. The entire culture is then incubated at 36°c for
72hrs. The cells in culture multiply and cover the bottom of the glass
container with a thin but continuous layer, which is often one cell thick.
Such cell layers are called as monolayer.
Sometimes the dispersed cells are not allowed to settle down at the
bottom of the container. Rather they are kept floating by shaking the flasks
continuously on the mechanical shaker. This type of culture is called as
suspension culture. A vigorously growing monolayer or suspension
culture is then inoculated with the types of viruses to which it is
susceptible. The inoculation is done by mixing or spreading the viral
suspension with the cultivated host cells. The virus particle infects the host
cells in due course. They multiply in number within the host cell and
eventually come out by destroying the host cell. They are thus liberated
into the surrounding medium and infect the neighboring cells. The cell
culture looks disintegrated. The initially formed virus particles soon lead
to the production of many more viruses. These areas appear to be
completely disintegrated and take shapes of white patches called as
plaques.
3.1.3. laboratory animal’s method
Suitable living mammals (such as sheep or calves or rabbits) are
selected for cultivation of viruses. The selected animals should be healthy
31
and free from any communicable diseases. The specific virus is introduced
into the healthy animals by direct injection. The site of administration
varies according to the type of virus is allowed to grow in the living
animal. At the end of incubation period, the animals are slaughtered and
washed thoroughly and viruses are obtained from them.
The injection may be intracerebral, intramuscular, intraperitoneal,
intravenous and intranasal.
Advantages:
 Very simple, can obtain virus at any time by bleeding the animal.
 Cheap.
Disadvantages:
 Virus may be transmitted to human.
 Death to animal.
3.1.4. Types of cell culture
There are three types of tissue culture; organ culture, explant culture and
cell culture.
 Organ cultures are mainly done for highly specialized parasites of
certain organs e.g. tracheal ring culture is done for isolation of
coronavirus.
 Explant culture is rarely done.
 Cell culture is mostly used for identification and cultivation of
viruses.

 Cell culture is the process by which cells are grown under

controlled conditions.
 Cells are grown in vitro on glass or a treated plastic surface in a
suitable growth medium.
 At first growth medium, usually balanced salt solution containing
13 amino acids, sugar, proteins, salts, calf serum, buffer, antibiotics
and phenol red are taken and the host tissue or cell is inoculated.
 On incubation the cell divide and spread out on the glass surface to
form a confluent monolayer.

32
[Link]. primary cell culture

A culture derived directly from a tissue of the host and grown on

suitable media and characterized by heterogeneous, limited cell division,
short duration (not exceed 3days), easy and simple.

Fig.15 Primary cell culture

[Link]. Diploid cell culture

Subsequent culture is derived from primary cell culture and

characterized by homogenous, static, high number of cell division and
long duration period (10 days).

Fig.16. Diploid cell culture

33
[Link]. Suspension cell culture

A culture in it, the dispersed cells are not allowed to settle down, but
they are kept floating by continuous shaking of the flasks and
characterized by no monolayer sheet and number of cell division is high.

[Link]. Permanent cell culture

A culture separated from single separated cell and characterized by

indefinite number of cell division and very long duration time.

Fig.17 Permanent cell culture

4. Cultivation of plant viruses

Tissue culture techniques are today widely used in studies on the

mechanism of virus replication and pathogenesis, i.e., the biochemical
processes of virus multiplication in the infected cells and the different
interactions between the virus components and the host organism.
However, contrary to animal and bacterial virology, the plant virology
section suffered for a long time from the lack of established tissue culture
systems, which were suitable for biochemical or molecular biological
investigations. Several attempts were made in the past to introduce plant
callus cultures into plant virology or to use separated cells from virus-
infected plants. However, all these studies contributed only a little to the
understanding of functional aspects in virus multiplication. Only the recent
work of H. H. Murakishi and his colleagues, who offered several
promising possibilities for overcoming the limitations of cell culture
systems for biochemical studies on plant viruses, revealed some new

34
perspectives. On the other hand, plant tissue culture methods could be
helpful in solving special problems such as the “hypersensitivity” of
plants, which is a phenomenon primarily related to intact tissue. Finally,
plant tissue culture is of fundamental value in the production of virus-free
plants through meristem tip and callus cultures, and for the mass
propagation of many cultured plants.

There are several methods of cultivation of viruses such as:

1- Protoplast and callus method.

2- Mechanical method.
3- Biological method.

4.1. Methods of cultivation of plant viruses

4.1.1. Protoplast and callus method

This method is similar to cell culture, in which selection of plant host

and specific organ in the specific host then cut into small segments by
tissue grinder in a process called homogenization then by buffer with
centrifugation for three times then applying lysozymes enzymes to
separate plant cells into solitaries then centrifuge and growing on suitable
media (contains plant hormones, buffer, carbon source and water).
The result is callus if media is solid and protoplast if media is liquid, in
case of callus, the virus is inoculated by mechanical inoculation by using
sterilized needle and put drops on virus but in case of protoplast, the virus
is inoculated directly and then incubated in suitable conditions (aerated
and lighted incubator) for a suitable time.

Fig.18 Protoplast and callus method

35
4.1.2. Mechanical method

Leaves are mechanically inoculated by rubbing with a mixture of

viruses and an abrasive such as carborundum. When the cell wall is broken
by the abrasive, the viruses directly contact the plasma membrane and
infect the exposed host cells. (The role of the abrasive is frequently filled
by insects that suck of crush plant leaves and thus transmit viruses.) A
localized necrotic lesion often develops due to the rapid death of cells in
the infected area. Even when lesions do not arise, the infected plant may
show symptoms such as change in pigmentation or leaf shape. Some plant
viruses can be transmitted only if a diseased part is grafted onto a healthy
plant.

4.1.3. Biological method

In this method, virus is transmitted from the infected plants into

healthy plants by living transport system. This occurs by incubating the
insect with the infected plant to feed on it and then the insect is transferred
and incubated with healthy plant to infect it in specific box.

5. cultivation of bacterial viruses

Bacterial viruses of bacteriophages (phages for short) are cultivated

in either broth or agar cultures of young, actively growing bacterial cells.
Several host cells are destroyed that turbid bacterial cultures may clear
rapidly because of cell lysis.

Fig.19 Cultivation of bacterial virus

36
 Cultures are prepared by mixing the bacteriophage sample with
cool, liquid agar and a suitable bacterial culture medium. The mixture is
quickly poured into a petri dish containing a bottom layer of sterile agar.
After hardening, bacteria in the layer of top agar grow and reproduce,
forming a continuous, opaque layer or lawn. Wherever a virion comes to
rest in the top agar, the virus infects an adjacent cell and reproduces.
Finally, bacterial lysis generates a plaque or clearing in the lawn.

Fig.20 Plaque assay method

6. Cultivation of insect viruses

The most suitable method for cultivation is on their larvae.

Before considering an insect virus as potentially usable in microbiological
control, one must consider not only its identification and its
standardization but also the method of production. The purity and quality
of virus preparations are primarily a function of its method of production.
Among the methods available, virus production by multiplication of high
insects on artificial media, currently offers the best way to get a quality
product.

Insect viruses can be identified by their physical, morphological,

chemical as well as by serological tests and vis-a-vis host specificity.
However, none of these methods are adequate in isolation. In the current

37
state of knowledge, the joint use of the last two tests provides the best
features for the specific identification of an insect virus.

The standardization of a virus should be based both on its ability to

produce disease and on the enumeration of the inclusion bodies. The
variation found in the measurement of viral activity may be a consequence
of the presence of released virus particles, the number of viral particles in
the inclusion bodies, the size and the chemical constitution thereof or the
presence of second biotypes. Various techniques usually based on studies
of lethal dose and mortality of time can be used to estimate the insecticidal
activity of the virus preparations. Whatever the technical utilize, it is
necessary to determine as precisely as possible, the titration method and
parameters to estimate and present the results statistically.

Summary

As the viruses do not reproduce independent of living host cells, they

cannot be cultured in the same as bacteria and eukaryotic microorganisms.
they can be cultivated through different hosts as cultivation of animal
viruses, cultivation of plant viruses, cultivation of bacterial viruses and
cultivation of insect cultivation.

Cultivation of viruses is an important to propagate the viral particle in

its specific host and increase viral concentration through support viral
replication.

Tissue culture is a useful method for cultivating clinical samples

suspected of harboring a virus.

38
Purification of viruses

39
1. Importance of purification:

1. Biological study of viral particles (infectivity).

2. Molecular characterization of virus (Type), shape of nucleic acid
and protein.
3. Academic studies of viruses (shape and chemical structure).
4. Serological characters (antigen-antibodies reactions).
5. Vaccines preparation of selective virus.

Ever since the pioneering discovery of the first step of virus

purification by alcohol precipitation of tobacco mosaic virus (TMV) by
Beijernick, 1898, purification of plant viruses have developed along
different lines. A variety of ways have been shown exploiting the
differences in the particles of viruses and host’s normal cell constituents.
Thus, purification of any virus involves at least three distinct steps, viz. (i)
extraction and clarification, (ii) removal of non-viral contaminants and
(iii) concentration of the virus.

Purification of the viruses is imperative for detailed knowledge of

the virus and the fact can be illustrated by the simple example that the
nature of plant viruses remained obscure despite several decades of study
on viruses until Stanley, 1935 purified TMV. Further, virus purification
aids in a number of chemicals, physical and biochemical investigations.
As is now known, different viruses vary widely and hence during the last
four decades, many efforts were directed in the purification of different
types of viruses.

Still it is easy only to purify the viruses which are stable and are
presents in high concentration in their hosts. However, a number of
procedures, developed for clarification of different viruses are viz.
differential centrifugation, filtration of viruses, heating and freezing,
emulsification with organic solvents like butanol, ethanol, chloroform, and
acetone.

Partially clarified extracts are further purified by gel filtration,

salting, isoelectric point precipitation, and electrophoresis, different types
of chromatography, ultra-centrifugation, and density gradient
centrifugation. But for different categories of viruses, other than the
commonest type i.e. single-component nucleoproteins, different types of

41
purification procedures have to be used as follows Multi component
nucleoproteins: viruses in this group, such as cowpea mosaic virus are
indicated to have multipartite genome of which two or more are essential
for biological activity.

The virus can be initially purified by the common method for other
single components have to be separated either by using difference in their
sedimentation coefficients or densities. Similarly, purification of defective
or incomplete viruses, such as satellite viruses, TMV mutants, potato
spindle tuber viroid (PSTV. or enveloped viruses (Rhabdo virus), needs
gentle methods to avoid any damage to their biological activity.

Since purification aims at elimination of all hosts constituents and

retaining the viral infectivity, selection of the procedure becomes
imperative which will take minimum time, produce the maximum virus
(with infectivity) without altering the morphology of the virions. Different
step in this regard are being referred here as under: Selection of the source
host: It should be a host which can be readily grown, kept free from other
virus contaminations and can yield maximum virus/gm tissue without
posing the problem of less manageable host constituents.

Extraction from the host tissue: Very often systemically than

locally infected leaves are the best tissues. Extraction is better, if the
leaves are frozen at least for 10-12 hrs and washed before sap extraction,
by blending infected leaves with buffer (s) to extract the maximum virus
even from fiber. Use of a cold room or to purify the viruses at 4-5oC is
effective as unstable viruses get degraded at high temperatures. Most of
the plants have slightly acidic sap but contributes and spinach has slightly
alkaline sap while some ornamental/medicinal plants have sap with a pH
range of 3-4 or 9-11. However, most viruses have the stability and
solubility up to pH 7 while their isoelectric point lies around pH 4.

This is why viruses like alfalfa mosaic, potato X, Y or tobacco

mosaic can be extracted better and more at pH 7 than pH 5-6. On the other
hand, viruses with isoelectric point near pH 7.0 (viz. satellite viruses of
TMV, TRSV, Bromo mosaic virus) are stable at pH5 than pH7.

41
 Further, isometric virions are easily freed from host plant constituents
like proteins and ribosomes by simply grinding the plant tissue with
acetate buffer at pH 4.5-5.0 (0.1M).

However, for elongated viruses, use of high molarities buffers at pH

almost about neutral (6.57.5) viz. citrate (pH 6.5); phosphate (pH 7.0) or
borate (pH 7.5) which chelate Mg2+ to degrade ribosomes; aggregate
flexuous rods, respectively. At the same time unstable viruses like tomato
spotted wilt virus (TSWV) can be safely purified with 0.1M phosphate
buffer + 0.1M sulphite.

Nevertheless, extraction of viruses at high morality does not mean

that they are equally good for virus purification for because the virus
particles may be more stable in dilute buffers. Scott, 1963 observed that
CMV extracted from plants in 0.5 citrate buffer (pH 6.5) but maximum
purification occurs with 0.005M borate buffer (pH 0.9). Similarly, a
number of additives can help either in disaggregation of elongated virions
like that of PVY, Henbane mosaic virus (HbMV) which often gets
denatured on aggregation. Detergent like Tween-80 either prevents or
even reversed the effect of Igepon t-73 another dispersing agent.

Urea 0.5M in the extraction buffer has been found equally effective
in extraction of potyviruses (Damirgah and Shephard, 1970). Chelating
agents like EDTA and sodium diethyl dithiocarbamate or use of reducing
agents like thioglycolic acid or nicotine help virus extraction by chelating
copper and/or inhibiting polyphenoloxidase and thereby virus inactivation
due to O-quinones. After extraction, the virus in sap can be freed from
plant material by a number of physical or chemical techniques.

42
2. Purification Outlines

2.1. Preparation of harvest

Means the viral cultivation (increase the number of viral particles

inside the host cells or highly concentration of virus). I.e. cultivate the
virus in its specific host , replicate , then extract the infectious sap by
degrading (homogenate ) the host cells (convert it to liquid form) either by
:

1. Mechanical methods (by maceration or blinding).

2. Chemical Methods: use of chemical substance that remove wall
such as SDS, hypochlorite, any detergent or enzymes (pectinase).
3. Physical Methods (Freezing and Defreezing ) or (Thawing and
Dethawing), Put tissue in (-20 c) ,leave for 2-3 days ,then take it out
2-3 days ,then freezing again for 2-3 days .
4. Sonication: use ultrasonic to degrade wall of the host cell.

3. Methods of Purification
3.1. Ultrafiltration

This operation is based upon the fact that, generally speaking, virus
particles have a smaller size dimension than any other microorganism.
Therefore, to separate viruses from amongst a mixture of variously sized
particles, one has simply to use filtration device capable of retaining the
larger particles while allowing the smaller virus particles to pass through.

43
For instance, let us suppose a viral suspension present in a bacterial
medium is to be isolated by filtration.

All one has to do is to allow the fluid suspension to pass through

suitable filter capable of allowing only the virus particles to pass through,
while keeping back the larger particles. Seitz type filter or porcelain G-5
filter may be conveniently used. The clear filtrate thus obtained should
contain solely viruses. In order to retain these virus particles the viral
suspension may further be passed through ultrafilters made of cellulose
acetate or collodion membranes (Elford 1939).

The very small pore size of these filters enables them to retain the
comparatively larger particles of the virus. Such membrane filters with
graded porosity, like Millipore filters (manufactured by the Millipore
Filters Corporation U.S.A.) have accurately determined pore size. These
can, therefore, be used to segregate particles having different sizes.

Particles larger than a particular pore size get wedged in the small
pores of these filters. Thus the smaller ones get through but may again be
retained by filters having still smaller pore size .The filtered particles of a
particular size can be brought in fluid suspension by washing the filters in
reverse direction. In recent years, diatomaceous earth has been increasing
in use as a filtering aid. Charcoal and bentonite have also been
successfully used. More sophisticated gel filtration techniques using
Agar- agar or sephadex allow selective filtration of virus particles.

The filtration of viruses, no matter how small be the pore size, is not
fool-proof against possible presence of other organisms. This is because
there are a few other types of microorganisms which have size ranges
comparable to that of larger viruses. The Rickettsia, a pathogenic group of
microorganism and mycoplasma tales, a type of bacteria are two examples
of such organisms. Therefore, the technique of ultrafiltration is likely to be
useless in case the virus suspension contains these or similar
microorganisms as well.

44
3.2. Centrifugation

Depending on the sedimentation rate, majority of the viruses can be

separated from their host constituents by choosing and appropriate speed
and period of centrifugation or even by adjusting the density of vehicle or
suspending medium except for lipid – enveloped virus (e.g. Lettuce
necrotic yellow virus) or unstable virus like TSWV. Generally a short low
speed centrifugation to clarify the extract is followed by a long high speed
centrifugation to sediment the virus from clarified extract.

For greater degree of purification, mostly for the stable viruses, a

number of alternating low and high speed centrifugation cycles may
denature the unstable viruses or deplete the smaller or rarer components of
a virus with multipartite genome. After clarification of the extracted plant
sap, the third and most important step is to separate or isolate the virus in a
concentrated from.

Centrifugation, in the present day centrifuges, allows handling large

quantities even at high gravitational fields of 100,000 xg or more to
quickly sediment even smallest virions by leaving the soluble plant
proteins and other smaller molecules in suspensions.

45
3.2.1. Differential Centrifugation

The simplest form of separation by centrifugation is differential

centrifugation, sometimes called differential pelleting. Particles of
different densities or sizes in a suspension will sediment at different rates,
with the larger and denser particles sedimenting faster. These
sedimentation rates can be increased by using centrifugal force. A
suspension of cells subjected to a series of increasing centrifugal force
cycles will yield a series of pellets containing cells of decreasing
sedimentation rate.

Differential Centrifugation: Particles of different densities or size will

sediment at different rates with the largest and most dense particles
sedimenting the fastest followed by less dense and smaller particles.

46
 Differential pelleting is commonly used for harvesting cells or
producing crude subcellular fractions from tissue homogenate. For
example, a rat liver homogenate containing nuclei, mitochondria,
lysosomes, and membrane vesicles that is centrifuged at low speed for a
short time will pellet mainly the larger and more dense nuclei.

Subsequent centrifugation at a higher centrifugal force will pellet

particles of the next lower order of size (e.g., mitochondria) and so on. It is
unusual to use more than four differential centrifugation cycles for a
normal tissue homogenate.

Due to the heterogeneity in biological particles, differential

centrifugation suffers from contamination and poor recoveries.
Contamination by different particle types can be addressed by
resuspension and repeating the centrifugation steps (i.e., washing the
pellet).

3.2.2. Rate-Zonal Centrifugation

In rate-zonal centrifugation the problem of cross-contamination of

particles of different sedimentation rates may be avoided by layering the
sample as a narrow zone on top of a density gradient. In this way the faster
sedimenting particles are not contaminated by the slower particles as
occurs in differential centrifugation. However, the narrow load zone limits
the volume of sample (typically 10%) that can be accommodated on the
density gradient. The gradient stabilizes the bands and provides a medium
of increasing density and viscosity.

47
 Rate-Zonal Centrifugation Sample is layered as a narrow zone
on the top of a density gradient (2B). Under centrifugal force,
particles move at different rates depending on their mass (2C).

The speed at which particles sediment depends primarily on their size

and mass instead of density. As the particles in the band move down
through the density medium, zones containing particles of similar size
form as the faster sedimenting particles move ahead of the slower ones.
Because the density of the particles is greater than the density of the
gradient, all the particles will eventually form a pellet if centrifuged long
enough.

3.2.3. Isopycnic Centrifugation

In isopycnic separation, also called buoyant or equilibrium separation,

particles are separated solely on the basis of their density. Particle size
only affects the rate at which particles move until their density is the same
as the surrounding gradient medium. The density of the gradient medium
must be greater than the density of the particles to be separated. By this

48
method, the particles will never sediment to the bottom of the tube, no
matter how long the centrifugation time.

Isopycnic Centrifugation Starting with a uniform

mixture of sample and density gradient (3A) under
centrifugal force, particles move until their density is
the same as the surrounding medium (3B).

Upon centrifugation particles of specific density sediment until they

reach the point where their density is the same as the gradient media (i.e.,
the equilibrium position). The gradient is then said to be isopycnic and the
particles are separated according to their buoyancy.
Since the density of biological particles is sensitive to the osmotic
pressure of the gradient, isopycnic separation may vary significantly
depending on the gradient medium used. Although a continuous gradient
may be more suited for analytical purposes, preparative techniques
commonly use a discontinuous gradient in which the particles band at the
interface between the density gradient layers.
This makes harvesting certain biological particles (e.g.,
lymphocytes) easier. The cellular mixture is centrifuged through a steep
density gradient that contains a high concentration of sucrose, or more
often, cesium chloride (CsCl). In these gradients, the molecules being
studied have a density somewhere in between the highest and lowest
densities of sucrose or CsCl generated in the gradient.
The components of a sample begin to move down this gradient in
the same way as they do in a rate-zonal density gradient. When a

49
component of the mixture reaches a point where the density of the solution
is equal to its own density, it stops moving further and forms a distinct
band. The position of the band in the tube is characteristic of the buoyancy
of that component. Buoyancy or buoyant density of a substance is its
tendency to float in a medium, which in this case is the density gradient.
Hence soluble proteins which have similar density (p=1.3 g cm-3 in sucrose
solution) cannot usually be separated by this method, whereas subcellular
organelles (e.g. Golgi apparatus p=1.11 g cm-3, mitochondria p=1.19 g cm-
3
and peroxisomes p=1.23 g cm-3in sucrose solution can be effectively
separate.

3.2.4. Suitable Density Gradient Medium Selection

The primary function of density gradient centrifugation is to separate

particles, either on the basis of their buoyancy density or their rate of
sedimentation. For rate-zonal separations, the function of the gradient is to
provide a gradient of viscosity which improves particle resolution while
stabilizing the column from convection currents. For isopycnic
separations, the important feature is that the maximum density of the
gradient media is higher than that of the particles. An ideal density
gradient media has the following properties:

 Sufficient solubility to produce the range of densities required

 Does not form solutions of high viscosity in the desired density
range
 Is not hyperosmotic or hypoosmotic when the particles to be
separated are osmotically sensitive
51
  Solutions of the gradient should be adjustable to the pH and the
ionic strengths that are compatible with the particles being separated
 Does not affect the biological activity of the sample
 Nontoxic and not metabolized by cells
 Does not interfere with assay procedures or react with the centrifuge
tubes
 Exhibits a property that can be used as a measure of concentration
 Easily removed from the purified product
 Autoclavable
 Reasonable cost

No single compound can satisfy all of the above criteria. Therefore a

wide range of gradient media are used for the different types of samples.
Most media are capable of producing the range of densities required and
being easily removed from the particles of interest.

The effect of osmolality on biological particles requires special

consideration. The osmolality of most mammalian fluids is 290-300
mOsm). This is the osmolality of balanced salt solutions (e.g., 0.85-0.9%
NaCl) and most common media. High osmolality solutions not only
remove water from the interior of membrane bound particles, they also
remove water bound to macromolecules like DNA.

Loss of water from cells will reduce their size and increase their
density, thereby affecting their buoyancy and rate of sedimentation. The
osmotic effect on cells and macromolecules may be reversible, though it is
a possible source of error that should be avoided.

Over the years, a variety of different compounds have been

developed as density gradient media in order to enhance the separation
process and to overcome osmolality and viscosity problems. There are five
main classes of density gradient medium:

 Polyhydric (sugar) alcohols

 Polysaccharides
 Inorganic salts
 Iodinated compounds
 Colloidal silica

51
3.3. Precipitation

The precipitation technique is often used for stable viruses like

TMV, PVX and also for want of sophisticated equipment like
ultracentrifuge. The two most common precipitants are alcohol and
ammonium sulphate. Alcohol results in an instantaneous coagulation of
the plant protein and/or the virus being used.

In the case of host proteins being precipitated, the supernatant

containing the virus is centrifuged at a low speed and separated. However,
not all viruses can tolerate high levels of the alcohol or they may even be
precipitated by dilute alcohol along with host’s constituents due to pH of
plant sap favorable for it (Markham, 1959). Normally, 33% saturation of
ammonium sulphate is enough for precipitating the viruses.

Precipitants: In suspensions the viruses are soluble because of their

electrical of these characters are damped by addition of some compounds,
they will precipitate reversibly particularly at their isoelectric points. Apart
from ethanol or ammonium sulphate, already mentioned above, diethyl
ether, n-butanol, acetone, carbon tetrachloride, chloroform and
fluorocarbon Freon 113 can also be used to separate a variety of viruses.

Use of organic solvents as an initial step has advantages in removal

of many contaminants from the virus suspension and a mixture of equal
volumes of buffer, n-butanol and chloroform, and infected plant tissues
gives considerably concentrated infectious nucleoprotein normally without
much contaminant (Steere, 1956). Polyethylene glycol (PEG) also
precipitates a number of viruses depending on its own concentration or its
buffer vehicle, e.g. TMV can be precipitated in the same amount by 4%
PEG in 0.1M NaCl or 2% PEG in 0.3 NaCl (Hebert, 1963).

3.4. Adsorption

Adsorbants: They can adsorb proteins from suspensions. Hydrated

calcium phosphate or activated charcoal and celite can selectively adsorb
plant proteins from the virus containing extracts.

Benthonite can also be used similarly but under controlled

conditions so that it adsorbs ribosomes and plant proteins but not virus
52
particles either by its saturation with sodium and not magnesium (Dunn
and Ditchborn, 1965). Still, Bentonite in any form at about pH 6 adsorbs
enzymic ribonuclease and is hence used for either extracting viral-RNA or
inoculating plants with naked RNA.
a) Ion exchange :
This method is more advanced than
gel filtration, we use negatively charged
beaded chemical substance (Dowex).In this
method dowex lose electron (-ve charges) so
the negatively charged impurities and virus
particles (-ve) are adsorbed on the dowex
particles to equalize the lost [Link]
(+ve) impurities not adsorbed on dowex
.virus particles could be separated from –ve
impurities br washing the dowex column
with specific buffer (eluent) which have
high affinity to virus particles.

b)Heamadsorption :
This method is suitable for purification of animal viruses only
1. Prepare of mixture of partially purified virus + RBCs.
2. Incubate at 4 c for 1 hr. (the virus particles will adsorbed on specific
sites on RBCs).
3. Scattering pattering will formed then centrifugate at 5000 rpm for
10 min.
4. Pellet is virus adsorbed on RBCs (the pellet will be RBCs and the
supernatant will be the virus particles).
5. Centrifuge at 5000 rpm for 10 min then take the pellet and
resuspend in saline solution and incubate at 37 c for 2 hrs, when we
incubate the mixture at 37 c ,this to activate N- acetyl nuraminidase
(enzyme present with viruses or RBCs.) which decay N-acetyl
nuraminic acid on RBCs and separate virus from RBCs.).

53
3.5. Solvent Extraction

Purification of viral preparations may also be achieved by what is

known as partition between solvents. It is well known that a substance
could be soluble in more than one solvent. Usually the solubility of the
material in these solvents (the amount that can be dissolved in a fixed
volume of the solvent) also differs. In other words, a substance is more
soluble in one solvent than in the other.

If we wish to purify a virus preparation containing some impurities,

then the suspension is first mixed with one of the solvents. Since the
suspension is in water, the entire mixture is then shaken vigorously and
allowed to stand. If the impurities are more soluble in the second solvent
than they are in water, they gradually pass into it.

Virus particles remain in the water medium. In this manner,

impurities are eventually removed and the viral preparation becomes purer
and purer. Since the two solvents are immiscible, the separation of pure
suspension and the impurities is almost total. Organic solvents like
fluorocarbon and dextran are often used as the second solvent.

54
3.6. Other Methods

a. Electrophoresis
Owing to the quality of virus particles to have a charge, except on
their isoelectric point, they can migrate in an electrical field and the
elctrophoretic mobility of various viruses or even strains of a virus are
different. Although this has been little used to purify plant viruses, it has a
promise for doing so in a liquid column stabilized by sucrose density
gradient (Van Regenmortel, 1966 and Brakke, 1995).

Two phase liquid systems: Solutions of organic polymers viz.

dextran and polythene glycol (PEG) or dextran and methylcellulose which
separate on standing into two phases can also help in separating the virus
suspensions by concentrating them in one phase. This method, however,
does not excel the salting out or precipitation of viruses.

b. Restricted diffusion
It can also be helpful in purification of small sized virus particles. In
this either dialysis through viscose cellulose tubes to concentrate the virus
by allowing exosmosis of the water, sugars and salts.

c. Serological methods
Antigen –Antibody reaction.
d. H.P.L.C
Depend on the molecular weight.

e. By enzymatic reactions

By using enzymes which can degrade impurities such pigments.

55
4. Criteria of Purification

When a sample has been purified in this manner the question that
comes up is about its purity. This question has two aspects. The purity or
the homogeneity status has to be considered from the (ii) biological point
of view. Chemically, a preparation may be regarded as the purest when it
is available in crystallized state. Even in suspension, an extremely
purified sample shows certain characteristics of its own. For instance, a
purified suspension of tobacco mosaic virus shows a very brilliant shine.
In most cases an unusual brightness is visible.

Physical criteria like size, shape, solubility and molecular weight

are also helpful in establishing the purity status. In recent years,
electrophoresis and serological data have been found useful in
determining the homogeneity of a sample. However, even in a state of
high apparent purity, foreign material like protein molecules of the host
may still be present. Absolute purity is, therefore, rarely, if at all,
achieved.

Biological purity of virus preparations has also to be ensured.

Biological purity relates to the capacity of the preparations to infect a
host. If this capacity is lost during the preparation of the sample, it would
biologically be impure. This capacity should, therefore, be tasted at every
step to see if it is still there.

With successive stages of purification, the infectivity tends to get

diminished with relation to number of observed particles. If proper
precaution is maintained throughout the operation, this loss can be
substantially checked. However, Absolute constancy in relation between
infectivity and number of particles at every range is rarely achieved
(Knight, 1979).

4.1. The Purity of Viral Preparation Can be Determined By

1. spectrophotometer
2. Isoradiation
3. Infectivity
4. Serological
5. Electron Microscope

56
1. Spectrophotometer

The idea of spectrophotometer depends on that when a

monochromatic light is incident on a sample of any solution, a part of the
light will absorbed (due to solvent and particles in the solvent) and
another part will transmit, and then the amount of absorbed part can be
measured by galvanometer. The amount of absorbed light (as a result of
solution particles) could be calculated from the relation:

Absorbance (Optical Density) =O.D of sample – O.D of blank

The amount of absorbed light is directly proportional to the

concentration of particles in this solution. Spectrophotometer can be used
for determination of:

A-Yield

Used to determine concentration of virus. Yield means the amount

of virus particles obtained from purification, it calculated from the
relation:

A260*dilution factor (D.F)

Virus concentration = -----------------------------------

Extinction coefficient (E.C)

Virus concentration is expressed as mg/ml

A260=absorbance for nucleic acid

E.C=constant for each viral family

D.F=Dilution factor
57
B- Protien / nucleic acid ratio

A280 absorbance of protein

------------------------------------------

A260 absorbance of nucleic acid

This ratio is constant for each virus.

EX: suppose A280/A260 of TMV=1.3 if this ratio was found to be

1.6 in any other sample of the same virus. This mean that there is an extra
protein (may be from the viral host cells) present in the sample, or there is
loss in viral nucleic acid during purification.

C- Scan

We used scan spectrophotometer which is computerized to measure

the optical density of purified virus over a range of wave lengths (220-320
nm).For purified virus only one peak should be appear at wave length 260
nm.

58
2. Isoradiation

After Purification

By replacing the sulfer atom of viral protein coat by isotopic sulfer

35
S or replacing the phosphorus atom of viral nucleic acid by isotopic
phosphorus P32 and then measure the amount of radiation for viral protein
or viral nucleic acid and compare to standard value for the same virus.
After purification measure the radiation in the purified sample by using
Geiger apparatus.

Before Purification

By replacing the carbon atom of some host cell by isotopic carbon

14
C and add these cells to the infectious viral sap that will be purified, then
carry out the purification process.

If there is no radiation, the sample is completely purified. If the

radiation is detected, low radiation means that the purification process is
not sufficient. High radiation means that this method of purification
should be excluded.

59
3. Infectivity

a) Local lesions assay

By inoculating the purified virus mechanically into a differential

host (on which the symptoms will appear as local lesions) and determine
the number of local lesions appeared after 21 day. The concentration of
the purified virus could be determined by using the standard curve for
this virus (Relation between different concentrations of standard virus
and number of local lesions appeared).

61
 b) Plaque assay

As the same of local lesions but here we count the number of

plaques appear. This method is suitable for bacterial viruses.

4. Serological (antigen and antibody reaction)

In serological reaction there must be specificity between the antigen

and the antibody. The degree of interaction between antigen and antibody
can be taken as an indicator for virus purity (Where the more purified
virus is more reacted with specific antibodies).

61
Ouchterlony test

In this test a plate of water agar is prepared and several wells are
made in the dish, then in the central well we put the specific antibody
and in the other wells we put the antigen (virus purified by several
methods) and leave the dish for 12 hrs. The antigen and the antibody will
diffuse through the agar and reacted with each other (precipitation test),
the reaction zone will appear as a band. The width of the band appears
can be considered as indicator for virus purity (the wider band indicates
more purified virus).

5-Electron Microscopy

In this method the purified virus is examined directly by electron

microscope.

62
4.2. Maintenance of purified viruses

It is considered important that viruses, once isolated from their host

environs, be maintained and preserved as infective agents. This is
necessary for various reasons. Specific physico-chemical characterization
of viruses would require that the viral specimens be pure as well as
biologically active or infective (Similarly for using).

Viruses as diagnostic reagents or for inactivating or attenuating

viruses for production of vaccines, it is essential that all the properties of
viruses are preserved after their initial separation.

Virus particles are highly sensitive to fluctuations in environmental

conditions. This is because the outer coat or the capsid is made up of
protein units which are affected adversely by conditions such as high
temperature, change in pH or the ionic concentration of the medium. As a
result the three dimensional unique features of the capsid proteins and
their component, alters resulting into Lon of infectivity.

Viruses surface proteins are thermolabile at temperatures above 55

o
C and denature within minutes between 55 oC and 60 oC. Thus the
particles are no longer capable of attachment to the host cell surface or to
uncoat normally. Event at much lower ambient temperatures (25 oC) decay
in inactivity occurs though the rate of decay is low. Therefore, viral
preparations must be stored at very low temperatures. While 4 oC is found
suitable for preserving the particles for a day or two , much lower
temperature such as of frozen CO2 (dry ice) _ 70 oC and more preferably
liquid nitrogen, -196 oC, are required for long term preservation .

63
 Fenner et al., (1987) have noted that as a rule of thumb, the half-
life of infectivity of most viruses can be measured in seconds at 60 oC,
minutes at 37 oC, hours at 20 oC, days at 4 oC, years at -70 oC or lower.
The enveloped viruses are more heat labile than non-enveloped viruses,
though they tend to loose infectivity if kept at low temperatures.

Normal practice in laboratories is to freeze the viral suspensions

very rapidly in a medium containing protective proteins, followed by
storage at _70 oC _ 196 oC. Dehydration of frozen viral suspensions under
vacuum (lyophilization) followed by preservation of the dried powder at
_
20 oC is also recommended. It is desirable that stored viral preparations
are checked for their level of activity from time to time.

4.3. The purification procedures for common viruses TMV and

CMV.

TMV can be successfully filtered through a celite filter pad for

clarification of the sap (Nagaich, 1965) and purified with acid ammonium
sulphate for its precipitation followed by separation at long centrifugation
and followed by separation at long centrifugation and re suspended in
0.1M phosphate buffer (Phatak and Verma, 1967). Because of a high
concentration in the source host and stability, TMV can also be purified by
subjecting the sap from infected leaves to differential centrifugation
(Nagaich, 1965).

CMV, a common aphid-borne virus and rather difficult to handle

for purification, can be successfully purified by extracting the infected
tobacco leaves with a mixture of chloroform (1g: 1ml) and 0.5M pH 6.5
citrate or phosphate buffer containing 0.1% thioglycolic acid. The
emulsion is broken down by centrifugation at 12000 xg for 10 min and
dialyzing the aqueous phase against 0.005 M borate buffer pH 9 for 20-24
hrs.
The dialysate is centrifuged at 54000 xg for 15 min and then the
clarified dialysate centrifuged for 150min. at 78,000 xg. For purified
preparations, the pellet may be re suspended in 0.005 M borate buffer (pH
9) and again clarified at 5400 xg for 15 min and if required once gain
centrifuged at 105,000 xg for 90 min. to obtain a final pellet to be

64
suspended in the above said borate buffer (Tomlinson et al., 1959 and
Scott, 1963).

Leberman (1966) employed coacervation or two phase liquid

system one of which is poor in colloid and the other is rich in it. The
extracted sap is clarified by centrifugation at 12,000 xg for 30 min. and
then for each 100 ml are added 134 g of sodium dextran sulphate in a 20%
solution, 29 g of PEG in a 30% solution and 5 g of 5M NaCl.

The resultant mixture is held at 4oCin separating funnels for

overnight and lower + interphase are collected and centrifuged for
pelleting the virus. DS can be precipitated out by adding 0.2ml of 3 M KCl
per gram of original amount of DS solution. Shanker et al. (1969) also
successfully purified CMV by using butanol for clarification followed by
2-3 cycles of differential centrifugation.

Summary

Purification of virus was and is fundamental to the determination of

essential chemical and morphologic features of viruses. More importantly,
qualities of viruses that are significant in their interactions with cellular
constituents are brought out by reflection on the principles and techniques
that have been applied in the purification of viruses.

Purification of virus is important because the only feasible defense

against animal viruses at present is vaccination, and this procedure has
hazards which are reduced by use of purified viruses.

Purification of virus means that, to obtain the viral particles in a

single form out of their host cell debris or to remove all impurities
(different organelles of the host) in the sample. It is essential that the
viruses area available in a pure form to achieve this they must be isolated
first and then purified.

The principal techniques used for this purpose are ultra-filtration,

ultra-centrifugation and chemical precipitation .In recent years
chromatographic, electrophoretic and solvent extraction techniques have
also been increasingly used.

65
Chemistry of viruses

66
 1. Introduction

The particulate nature of viruses was partially established by Max

Schlesinger in 1933, the chemistry of viruses was sought to be elaborated
in further detail.

Schlesinger isolated a large bacteriophage, WLL, Infecting the

bacterium Escherichia Coli, by differential ultra-centrifugation. On
chemical investigation, he found proteins and phosphoric acid to be
associated with these particles.

He suggested the presence of nucleic acid in them. His conclusion

suggested that these bacteria phage particles were nucleoproteins. The
chemical identity of a virus could be determined only after tobacco mosaic
virus (TMV) was crystallized by Stanley in 1935. Later, Bawden and
Pirrie were the first to establish the nucleoprotein nature of three strains
of TMV. They also showed that the nucleic acid and component was
ribonucleic acid (RNA) (Bawden and pirie 1937).

During the last four decades. Enormous amount of data have been
collected on various aspects of the chemistry of different classes of
viruses.

2. Chemical Composition of Viruses

Chemically, viruses are

nucleoproteins. A majority of viruses
consist of two components. One is a
single usually linear molecule of
either deoxyribonucleic acid (DNA)
or ribonucleic acid (RNA).

The other is an outer proteinaceous covering or sheath surrounding

the inner nucleic acid molecule. However, some of the larger animal and
bacterial viruses are containing lipids (Franklin, 1962) and carbohydrates
(Compans and Choppin 1973).The lipids are also associated with a few
plant viruses (Best, 1968).

67
A causal of the chemical composition of different categories of viruses
shows that:-

1- The composition varies, both qualitatively and quantitatively from

virus to virus.

2- Bacterial and animal viruses are mostly DNA viruses whereas

plant viruses are in most cases RNA viruses. However there are exceptions
in both the cases. For example, Coliphage F2 and influenza virus are RNA
viruses and cauliflower mosaic and golden yellow mosaic viruses are
DNA containing viruses.

2.1. Nucleic Acids

The nucleic acid is the active disease, specific and host specific
infective part of a virus. It should be noted that both the types of cellular
nucleic acids, the DNA and the RNA are not present together in any one
virus. This trait distinguishes viruses from the known living organisms all
of which possess both the types of nucleic acids. Viruses possessing DNA
are referred to as deoxy viruses, while those possessing RNA as ribo-
viruses.

2.1.1. Types of Nucleic Acids

The nucleic acid of most viruses is linear straight chain structures.

Some, however, such as those from DNA tumor viruses and a few bacteria
phages like Pseudomonas phage PM2 (Biswas and Amita 1996) and
Rhodospirillum phage RQGP (Pembetron and Tucker 1977) contain
cyclic or circular structure. These are known to be supercoiled or twisted
like aball of thread (plate IV). The straight chain or cyclic structures could

68
be single stranded. There could be eight categories of viruses, depending
upon nucleic acid types. These are:-

1. Viruses with double stranded DNA (SS).

2. Viruses with single stranded DNA (S).
3. Viruses with double stranded RNA (SS).
4. Viruses with single stranded RNA (S).
5. Viruses with double stranded cyclic DNA.
6. Viruses with single stranded cyclic DNA.
7. Viruses with double stranded cyclic RNA.
8. Viruses with single stranded cyclic RNA.

2. 1. 2. Nucleic acid contents per particle

The nucleic acid content per particle varies from virus to virus.
Larger the size of the virus particle, larger is the contents of nucleic acid
and vice-versa.

For instance, Vaccinia virus having a length of 250nm has 160x106

Daltons of DNA per particle. Creator amount of nucleic acid is necessary
for the synthesis of more complex and larger structural components of the
larger viruses.

The nucleic acid content of a virus can be calculated from the

molecular weight of a virus and its nucleic acid percentage. For instance,
the molecular weight of tobacco mosaic virus (TMV) has been calculated
to be 40x106 Daltons. Since it contains about 5 percent RNA, its content

69
can be calculated to be roughly 2x106 Daltons. The content per particle
can be confirmed by calculating the molecular weight of RNA isolated
from TMV particles.

A direct correspondence between the molecular weight calculated on

the basis of percentage nucleic acid content and the one calculated from
isolated RNA gives an idea about the number of nucleic acid molecules. A
one to one correspondence means that there is a single, linear or circular,
molecule. For example, the molecular weight of a isolated RNA (TMV) is
approximately 2x106, calculated on the basis of percentage RNA and
particle molecular weight, it comes to about the same. This indicates that
there is one molecular of RNA per particle.

2. 1. 3. Primary Structures of Viral Nucleic Acids

Nucleic acid is long chain molecules made up of nucleotides. The

nucleotides are named according to the Purina and Pyramiding base they
contain.

For RNA, the nucleotides are the denylic, guanilic, uridylic and
cytidlic acids. For DNA, these are deoxyadenylic, deoxy guanilic, deoxy
cytidilic and thymidilic acids.

These nucleotides are made up of three simple components phosphate

group, nitrogen bases and pentose sugar:-

 phosphate group
In both DNA & RNA

71
  Pentose sugar
- Ribose (RNA)
- Deoxyribose (DNA)

 Nitrogen bases

The bases are adenine, guanine, cytosine and thymine in DNA and
the first three and uracil instead of thymine in RNA.

Adenine and guanine are the purine bases and cytosine, thymine and
uracil are the pyrimidine bases. In double stranded nucleic acid, the two
strands are held together by hydrogen bonds between purine and
pyrimidine bases. There is a specific association between the purine and
the pyrimidine bases in that adenine (A).

From an association with thymine (T) and guanine (G) associates

with cytosine (C). In case of double stranded RNA molecules, as in
reovirus, the association is between adenine and uracil (U).

71
 The primary structure of nucleic acid relates to the proportion and
arrangement of these various nucleotides in a specific manner. An
important step in characterizing the primary structure of nucleic acid
would be to determine:-

(1) The proportion of pureness and pyrimidines present.

(2) The sequence of arrangement of the bases or of the nucleotides
they are part of.

72
 2.1.4. Determination of Base Ratios and Nucleotide proportion

Both chemical and physical methods have been used to determine the
base ratios of viral nucleic acids.

Molar base or nucleotide ratios can be determined by hydrolyzing the

viral nucleic acid under controlled conditions. The nucleotides can be
identified and separated quantitatively by chromatography paper,
column1.

An analysis of the molar base rations of the viral nucleic acids reveals
that:-

(1) There is a great variation in the base ratios of nucleic acid, both
RNA and DNA, obtained from different viruses. This is clearly
reflected in AT/GC or AU/GC ratios.
(2) In double stranded nucleic acid molecules there is direct one to
one correspondence between the purine and the pyrimidine
bases.
The nucleic acid from all the DNA containing viruses except Φx174
which has single stranded DNA, wound tumor Andrea viruses are double
stranded RNA, a higher proportion of uracil than other bases is found
influenza virus.

(3) Similar viruses may have almost identical base ratios. For
example; shope popilloma and polyma virus and simian virus
40 all of which are oncogenic and possess nucleic acid with
similar base ratios.
(4) Dissimilar viruses may also have almost identical base ratio,
for example; coliphage T4 and Tipula iridescent virus, both
having similar GC content.
2.1.5. Homology Analyses by hybridization

Is method giving information about nucleic acid sequences is based

on hybridization of nucleic acid molecules? The procedure is as follows.

The two strands of a double stranded nucleic acid are associated

together mainly by hydrogen bonds between complementary bases (A: T
or U and G: C) of the two strands. The two strands could be separated by

73
heating which break up the hydrogen bonds. Separation could be heating
by chemical means.

Separation of nucleic acid strand is called its denaturation. When this

is an achieved by heating, the process is also referred to as molecular
melting.

When heat separated nucleic acid strands are allowed to cool

gradually, they tend to re-associate. This phenomenon is called
renaturation or annealing of strands. If denatured strands of a second
species of nucleic acid are added to those of originally denatured one
during the annealing process, strands of the second nucleic acid may
compete with the original ones in reforming the double strands structure.
This would occur only if substantial nucleotide sequences of both the
species are the same or very similar. Such similarity or homology would
lead to formation of hybrids between complementary strands of the two
nucleic acids. Therefore, the procedure is known as hybridization.

The strands of the second nucleic acid species are similarly

denatured and broken or sheared into smaller fragments. The second
species is made radioactively. The broken radioactive strands are then
made to anneal with the immobilized original strands. After a suitable
period of incubation the excess of unassociated nucleic acid fragments are
washed off. The immobilized DNA of the original species are then
analyzed for radioactivity.

Hybridization experiments essentially give us information regarding

the homology existing between two species of nucleic acids. This
information has been used in:-

(1) Evaluation of the degree of similarity of nucleic acid from

different or similar types of viruses.
(2) Investigation of the amount of replicating viral nucleic acid
present at different times after infection.
(3) Estimation of the viral genome incorporated into host nucleic acid.
(4) Determination of the presence or absence of virus specific
messenger RNA, and

74
 (5) Discrimination between types of the RNA present at various
stages after infection (Knight, 1974).
2.1.6. Secondary Structure of Viral Nucleic Acids

The nucleic acid component of viruses is generally a single linear

molecule. A few, like the Simian Virus 40 and polyoma virus have
circular molecules. The structure i.e. the three dimentional configuration
of these molecules assumes importance since these single molecules have
to be specifically accommodated in the mature virus particles.

The circular single stranded nucleic acid molecules as in colipha

Φx174 remain in a twisted or a supercoiled state. Various physical and
chemical techoiques, have been used in the elucidation of the secondary
structure of nucleic acid. Considerable work has been done to elucidate
the secondary structure of the TMV – RNA. It has been shown that the
alignment of the RNA molecule within the TMV particle is different from
isolated TMV – RNA molecule (Gierer 1960).

Studies on some other RNA viruses reveal that in some of them, like
the reovirus, the RNA genome is fragmented into ten segments (Bwas and
Amita 1996).

The secondary structure of viral DNAs seem to be more stable in the

sense that they don't show much change when isolated from intact
particles. The form is double helix certain characteristics enable linear
viral DNAs to adopt a circular configuration during replication. For
example, the linear dsDNA of some herpes viruses contain repeat
sequences at the ends of the molecule when the linear ds molecules are
partially desisted from their 5'ends by an exonuclease, the exposed ends
are found to be complementary to each other. Also on heating followed by
gradual cooling the ds DNA linear molecules reanneal (join together) as
circular ds DNA. The genomes of RNA viruses also show variation in
their secondary structure. For example; the ss RNA of arenaviruses have
sticky ends. Hence, there molecules occur as cyclic structures. These
viruses also have regions where base pairing occurs. Such double stranded
portions serve as signals for controlling translation transcription or for
packaging into capsids.

75
2.2. Proteins

Proteins are one of the basic biochemical units constituting a cell

which are composed of twenty odd amino acids linked together covalently
by the group – HN – C – O -, which is formed by the basic group – NH2,
of one amino acid with the carboxylic group – COOH of the next amino
acid. Thus is formed a peptide chain which naturally has a free H2N group
at one end and a free – COOH group at the other. This chain represents the
primary structure of the proteins. In proteins containing more than one
chain there may be interchange covalent linkage through disulphide bond
(- S – S -) between sulphur containing amino acids like cysteine.

In some cases, the peptide chain is

not wholly straight. In parts it shows
helical configuration. The helical
configuration, also called the alpha
helix, is formed due to hydrogen
bonding between – CO and group of
amino acids situated at regular intervals
– the combined straight and helical
configuration represent the secondary
structure of a protein.

76
 The peptide chain is often folded. The folded structure represents the
tertiary configuration of there may be more than one peptide chain or
subunit forming a protein structure. The number and spatial relationship of
these subunits represents the quaternary structure of the proteins. The
tertiary and quaternary structure can be determined by X-ray
crystallographic studies (Kendrew 1959).

Generally, proteins form the outer component of virus particles. In

effect, it is the outer proteins part or the coat, sometimes also called the
sheath which gives the virus particles their characteristic shape. In most
cases the protein coats are not unitary structures, rather are composed of a
varying number of identical subunits. These subunits are the capsomers
which assemble to from the capside. Each capsomere is composed of one
to six polypeptide molecules, depending upon the virus. In case, many of
the same type (homo polymers) or sometimes of different types (hetero
polymers).

The coat has antigenic properties and is mainly protective toward the
central nucleic acid against host cell nucleases. Conjugate proteins such as
glycoproteins make up the polemers present on the surface of the viral
envelope.

2.2.1. Primary Structure of Viral Proteins

The protein component in viruses has to be separated from the non-

protein constituents.

Under controlled conditions, exposure of the virus particles to mild

alkali or detergents leads to partial to total release of the particle proteins.

77
 One problem associated with the preparation of viral proteins is the
establishment of their homogeneity.

Homogeneity by is essential, otherwise no proper clue to their

primary structure can be obtained. While selective precipitation, such as at
the isoelectric point have been attempted with some success, more
sophisticated techniques like electrophoresis, gel filtration and density
gradient centrifugation are also in use.

In general, all of the methods in use to prepare viral proteins are

capable of causing various degrees of degenerative changes. These
changes are generally at the level of quaternary configuration of the
protein. Some of these changes are reversible. Some, however, are not.

In order to distinguish between native and denatured viral proteins,

Anderer (1959) suggested a list of criteria.

Taking TMV protein as the model is noted that a native viral protein
should be noted that a native viral protein should be:

(1) Soluble in aqueous solution.

(2) Aggregate into the parent virus like rods at certain specific PH
levels.
(3) With appropriate viral nucleic acid should able to reconstitute
into infectious particle and
(4) Show resemblance to the intact virus particles, in the amount of
the specific viral antibody it binds.
2. 2. 2. Amino Acid Compositions of Viral Proteins

The amino acid residues of purified viral proteins are usually released
by hydrolysis of the protein under controlled conditions. This is achieved
by taking 205 mg of protein in one m1 of 6 NHCl in a sealed glass tube
and heating it at 110oCfor 36hours under vacuum. By this method a
majority of the constituent a mina acids are almost quantitavely liberated.
However, while most of the amino acids are little affected by such
treatment, a few like threonine, tryptophan and cysteine are destroyed to a
considerable extent.

78
 The amino acids thus liberated can be qualitatively determined by
paper chromatography using the convention technique (Biswas and
Amita 1996). Quantitative analysis can be efficiently done using
automatic amino acid analyses. These analyses are built on the principles
of ion exchange chromatography as utilized for amino acid analyzers by
Biswas and Amita (1996).

The analyzers records the amino acids present in the protein

hydrolysate both qualitatively and [Link] viral coat proteins
are constituted of the common protein amino acids.

2. 3. Carbohydrates

Carbohydrates found in viruses can be categorized into two groups

depending on their location within the virus particle. In the main category
would fall the carbohydrates associated with viral nucleic acids, namely,
the ribose and the deoxyribose sugars. Found in all the viruses.

The other category called the non-nucleic acid carbohydrates. These

are found mostly associated with animal viruses but are reported from
bacterial and plant viruses.

The non-nucleic acid carbohydrate may be simple sugar like glucose

or gentiobiose found in T- coliphages in which they are linked to the
hydroxyl methyl cytoseine residue. They may also be found associated
with proteins (glycoproteins) or lipids (glycolipids) as in the animal and
plant viruses.

Analysis of glycoprotein and glycolipid compounds reveals that the

carbohydrates are fructose, galactose, glucesamine and mannose. The
protein and carbohydrate in glycoproteins are linked by for motion of
bonds between the carbohydrates chain and asparagine, Serine and
thereonine residues of proteins.

2. 4. Lipids

Lipids have been found to be associated with a large number of

viruses representing all the major groups. However they are mostly found
associated with animal and bacterial viruses only a few plant viruses have
lipids as one of the chemical constituents where there are several kinds of
lipids extracted from viruses.

79
 Phospholipids of different types are found associated with some of
them. Cholesterol and triglycerides are very common. All the lipid
containing viruses are surrounded by a membranous envelope which is the
sole repository of the lipid components.

These viruses mature on the host cell membranes during replication

and are liberated by budding of the membrane itself. The viral lipids were
derived from the host cell membrane and were not viral.

There is significant difference, both qualitative and quantitative,

between the cell membranes of the hosts in which they were grown. For
example; it has been reported that simian virus 5 contains a phosphelipid
phosphatide; nostiol which is absent in the membranes on monkey kidney
cell in which the virus was grown (Biswas and Amita 1996).

The lipid are linked with protein formed called lipoprotein but when
linked with polysaccharides called glycolipid complexes.

81
 2. 5. Other chemical Components

In addition to nucleic acids, proteins and minute quantities of

carbohydrates and lipids, some other substances are found in highly
purified preparation of certain viruses for example; polyamines are found
in significant amount in both bacteria phages T2 and T4. The polyamines
reported are putrescine spermidine and spermino (Biswas and Amita
1996). Polyamines occur in herpes virus and influenza virus. Traces of
polyamines occur in plant viruses like turnip yellow mosaic virus and
broad been mottle virus. In organic metal cations have been found to be
associated with viral protein and nucleic acids.

3. Conclusion

Chemical composition of viruses is nucleic acids, proteins,

carbohydrates. Chemically, viruses are nucleoproteins. Viruses have one
type of nucleic acids either DNA or RNA. Proteins form the outer
component of virus particles. Carbohydrates found in viruses divided into
g two groups depending on their location within the virus particle.

4. Summary

1. Chemical composition of viruses.

Chemically, viruses are nucleoproteins. They consist of two components,

one is the single linear molecules of either DNA or RNA and the other is
an outer proteinaceous covering or sheath surrounding the inner nucleic
acid molecule.

1.1. Nucleic Acids

The nucleic acid moiety is the active disease, one type of nucleic acid
present in the virus either DNA or RNA.

1.1.1. Types of Nucleic Acids

1) Viruses have double strands or single strands of DNA.

2) Viruses have double or single strands of RNA.
3) Viruses have double or single cyclic strands of DNA.
4) Viruses have double or single cyclic strands of RNA.

81
 1.1.2. Nucleic acid contents per particle

The nucleic acid content per particle varies from virus to virus.
Generally, larger the size of the virus particle, larger is the contents of
nucleic acid and vice versa.

The nucleic acid content of a virus can be calculated from the

molecular weight of a virus and its nucleic acid percentage.

1. 1. 3. Primary Structures of Viral Nucleic Acids

Nucleic acid is long chain molecules made up of nucleotides, the

nucleotides are named according to the purine and pyramiding base they
contain. These nucleotides are made up of three simple components each
phosphate, a sugar (ribose or deoxy ribose) and a base (purine or
pyrimidine).

1. 1. 3. 1 Determination of Base Ratios and Nucleotide proportion

Chemical and physical methods have been used to determine the base
ratios of viral nucleic acid. Molar base or nucleotide ratios can be
determined by hydrolyzing the viral nucleic acid under controlled
conditions.

1. 1. 3. 2 Homology Analyses by hybridization

This method used for gaining information about nucleic acid

sequences.

The two strands of a double stranded nucleic acid are associated

together by hydrogen bonds between complementary bases of the two
strands. The two strands could be separated by heating which break up the
hydrogen bonds. When the heat separated strands allow calling, they tend
to re-associate.

1.1.4. Secondary Structure of Viral Nucleic Acids

The nucleic Acids component of viruses is generally a single linear

molecule. A few viruses have circular molecules.

The secondary structure of viral DNAs seem to be more stable in the

sense that they don't show much change when isolated from intact
particles. The form is double helix.

82
 2. 2 Proteins

Proteins are composed of twenty odd amine acids linked together in

various numbers and combinations. They are linked together covalently by
the group – HN – C – O – which is formed by the basic group – NH2, of
one amino acid with the carboxylic group COOH of the next amino acid.
Thus is formed a peptide chain.

2. 2. 1 Primary Structure of Viral Proteins

The protein component in viruses has to be separated from the non-

protein constituents.

Under controlled conditions, exposure of the virus particles to mild

alkali or detergents leads to partial to total release of the particle proteins.

2. 2. 2 Amino Acid Compositions of Viral Proteins

The amino Acids recorded can be qualitatively determined by paper

chromatography using the convention technique the viral coat proteins are
constituted of the common protein amino acids.

2. 3 Carbohydrates

Carbohydrates found in viruses can be divided into two groups

depending on their location within the virus particle. The carbohydrates
associated with viral nucleic acids namely, the ribose and deoxyribose
sugars. The other called the non-nucleic acid carbohydrates.

2. 4. Lipids

Lipids have been found to be associated with a large number of

viruses representing all the major groups. There are several kinds of lipids
extracted from viruses.

All the lipid containing viruses are surrounded by a membranes

envelope which is the sole repository of the lipid components.

2. 5. Other chemical Components

In addition to nucleic acids, proteins and minute quantities of

carbohydrates and lipids, some other substances are found for example;
polyamines which putrescine spermidine and spermine.

83
Bacteriophages

84
Introduction

A bacteriophage is any one of a number of viruses that infect

bacteria. They do this by injecting genetic material, which they carry
enclosed in an outer protein capsid. The genetic material can be ssRNA,
dsRNA, ssDNA, or dsDNA ('ss-' or 'ds-' prefix denotes single-strand or
double-strand) along with either circular or linear arrangement.
Bacteriophages are among the most common and diverse entities in
the biosphere (Mc Grath S and van Sinderen, 2007).The term is
commonly used in its shortened form, phage.

1. Discovery
Since ancient times, there have been documented reports of river
waters having the ability to cure infectious diseases, such as leprosy.
In 1896, Ernest Hanbury Hankin reported that something in the
waters of the Ganges and Yamuna rivers in India had marked antibacterial
action against cholera and could pass through a very fine porcelain filter.
In 1915, British bacteriologist Frederick Twort superintendent of
the Brown Institution of London discovered a small agent that infected and
killed bacteria. He believed that the agent must be one of the following:
 A stage in the life cycle of the bacteria.
 An enzyme produced by the bacteria themselves.
 A virus that grew on and destroyed the bacteria.
Independently, French-Canadian microbiologist Félix d'Hérelle
working at the Pasteur Institute in Paris, announced on September 3,
1917, that he had discovered an invisible, antagonistic microbe of the
dysentery bacillus. For d'Hérelle, there was no question as to the nature of
his discovery: In a flash I had understood: what caused my clear spots was
in called the virus a bacteriophage or bacteria-eater. He also recorded a
dramatic account of a man suffering from dysentery who was restored to
good health by the bacteriophages Félix d'Hérelle.
In 1923, the Eliava Institute was opened in Tbilisi, Georgia, to
research this new science and put it into practice.

85
 In 1969, Max Delbrück, Alfred Hershey and Salvador Luria were
awarded the Nobel Prize in Physiology and Medicine for their discoveries
of the replication of viruses and their genetic structure.

2. General characters of bacteriophages

a) Widely distributed
Bacteriophages present anywhere (water, soil, air…..) where its host
(bacteria) is present.
Phages are widely distributed in locations populated by bacterial
hosts, such as soil or the intestines of animals. One of the densest natural
sources for phages and other viruses is sea water, where up to 9×10 8
virions per milliliter have been found in microbial mats at the surface and
up to 70% of marine bacteria may be infected by phages. They have been
used for over 90 years as an alternative to antibiotics in the Eastern Europe
as well as in France. BBC Horizon, They is seen as a possible therapy
against multi-drug-resistant strains of many bacteria BBC article.
b) Size
Bacteriophages are the smallest viruses (10-350 nm) spheroid
phages such as group E and D not exceed 250 nm. T4 is among the largest
phages; it is approximately 200 nm long and 80-100 nm wide. Other
phages are smaller. Most phages range in size from 24-200 nm in length.
c) Cultivation
Bacterial viruses of bacteriophages (phages for short) are
cultivated in either broth or agar cultures of young, actively growing
bacterial cells. Several host cells are destroyed that turbid bacterial
cultures may clear rapidly because of cell lysis.

Cultures are prepared by mixing the bacteriophage sample with

cool, liquid agar and a suitable bacterial culture medium. The mixture is
quickly poured into a petri dish containing a bottom layer of sterile agar.
After hardening, bacteria in the layer of top agar grow and reproduce,
forming a continuous, opaque layer or lawn. Wherever a virion comes to
rest in the top agar, the virus infects an adjacent cell and reproduces.
Finally, bacterial lysis generates a plaque or clearing in the lawn.

86
 d) What is Plaques?

Plaques represent clearing the areas of bacterial growth due to lysis

by a bacteriophage. If lytic bacteriophage is absent bacteria grow
luxuriantly on culture medium. But if they are present, formation of clear
zones or plaques can be seen here and there on the bacterial lawn. Each
plaque corresponds to the site where a single bacteriophage particle acted
as an infectious unit and initiated its lytic reproductive cycle.

The spread of infectious bacteriophage from the initially infected

bacterial cell to the surrounding cells results in the lysis of many of the
bacterial cell and, therefore, a clear zone develops.

2. Composition and structure of bacteriophage

2.1. Composition

Although different bacteriophages may contain different materials

they all contain nucleic acid and protein. Depending upon the phage, the
nucleic acid can be either DNA or RNA but not both and it can exist in
various forms. The nucleic acids of phages often contain unusual or
modified bases. These modified bases protect phage nucleic acid from
nucleases that break down host nucleic acids during phage infection. The
size of the nucleic acid varies depending upon the phage. The simplest
phages only have enough nucleic acid to code for 3-5 average size gene
products while the more complex phages may code for over 100 gene
products.

87
 The number of different kinds of protein and the amount of each
kind of protein in the phage particle will vary depending upon the phage.
The simplest phage has many copies of only one or two different proteins
while more complex phages may have many different kinds. The proteins
function in infection and to protect the nucleic acid from nucleases in the
environment.

2.2. Structure

Like all viruses, bacteriophages are simple in their structure,

composed of nucleic acid surrounded by protein coat called capsid.

Generally bacteriophage composed of head, tail and basal plate.

a. Head

The head contains the double stranded DNA. How this DNA is
packed inside the head is not known.

The head has the shape of two halves of an icosahedron connected by

a short prism.

b. Tail

The tail is helical; the presence of two types of symmetry in the

protein coat is called banal symmetry and is found exclusively in
bacteriophages and cyanophages. The tail is made up of a cubical hollow
tube surrounded by a contractile sheath.

On one end it is joined to the head by a colar while at the other end
it has a hexagonal plate which has six short small fibers (tail pins) at every
corner and six tail fibers. While the long tail fibers are associated with
adsorption of the phage on the bacterial wall, the shot tail fibers hold the
phage fast to the host during sheath contraction and DNA injection.

During adsorption a pore is dissolved in the bacterial wall probably

by a phage enzyme and the DNA injected into the bacterial cell.

88
 d) Basal plate

Differ from phage to another, it contains fibers or no formed of

protein and are important for the attachment of phage to host cell.

2.3. Genome structure

Bacteriophage genomes are especially mosaic, the genome of any

one phage species appears to be composed of numerous individual
modules. These modules may be found in other phage species in different
arrangements. Mycobacteriophages (bacteriophages with mycobacterial
hosts) have provided excellent examples of this mosaicism. In these
mycobacteriophages, genetic assortment may be the result of repeated
instances of site-specific recombination and illegitimate recombination
(the result of phage genome acquisition of bacterial host genetic
sequences) (Morris et al., 2008).

3. Symmetry

89
4. Nucleic acid
The nucleic acid in bacteriophage may be DNA or RNA never the
two present in one virus particle. DNA may be simple or double stranded
and RNA may be single or double stranded or the nucleic as may be
circular.

Some phage may contain circular DNA outside the cell but inside
the host cell it converts to linear DNA. Nucleic acid of bacteriophage is
very long; it may reach 1000 time length of the particle itself, so it is much
coiled inside the capsid.

5. Morphology
Bacteriophage may be grouped into 6 morphological groups:

Group (A)

 Helical and icosahedral.

 The most complex type (spheroid and elongated) shaped.
 Have a hexagonal head with a tail with contractile sheath and base
has tail fibers.
 Its nucleic acid is DNA (ds DNA).
 e.g. T4 phage

Group (B)

 It has hexagonal head and its tail is long, flexible and lack
contractile sheath.
 Basal plate is absent or present.
 Its nucleic acid is DNA (ds DNA).
 e.g. ƛ Phage.

Group (C)

 It is has hexagonal head with a very short tail (shorter than head)
and lack contractile sheath.
 Basal plate present and contain fibers.
 Its nucleic acid is DNA (ds DNA).
 Ex: T7 phage

91
Group (D)

 Called spheroidal phages.

 It has a head only with icosahedral symmetry.
 The head composed of large capsomers.
 It lacks tail and contractile sheath and basal plate.
 Its nucleic acid is circular DNA (ss DNA).
 Ex: ɸX 174 phage.

Group (E)

 It is only spheroidal head, with icosahedral symmetry.

 The head composed of small capsomers.
 It lacks tail and contractile sheath.
 Its nucleic acid is RNA (ss).

Group (F)

 It called called filamentous fibrous phages.

 Its shape is flexible elongated rods.
 No head, contractile sheath and basal body, is elongated tail only.
 Its capsomers are arranged with helical symmetry.
 Its nucleic acid is DNA (ss).
 e.g. M13 phage
 Notes: A, B, C: unique to bacteriophage.
 D, E: distributed in animal viruses.
 F: distributed in plant viruses.

91
6. Replication

The process of multiplication in viruses is generally referred to as

replication. This is to distinguish it from the analogous process of
reproduction associated with the true living organisms.

Bacteriophages may have a lytic cycle or lysogenic cycle, and a few

viruses are capable of carrying out both. With lytic phages such as the T4
phages, bacterial cells are of the viron. As soon as the cell is destroyed, the
phage progeny can find new hosts to infect.

Lytic phages are more suitable for phage therapy. Some lytic phage
undergo a phenomenon known as lysis inhibition where completed phage
progeny will not immediately lyse out of the cell if there is a high
extracellular phage concentration. This mechanism is not identical to that
of temperate phage going dormant and is usually temporary.

In contrast, the lysogenic cycle does not result in immediate lysing

of the host cell. Those phages able to undergo lysogeny are known as
temperate phages. Their viral genome will integrate with host DNA and
replicate along with it fairly harmlessly, or may even become established
as a plasmid.

92
 The virus remains dormant until host conditions deteriorate. Perhaps
due to depletion of nutrients, then the endogenous phages (known as
prophages) become active.

At this point they initiate the reproductive cycle, resulting in lysis of the
host cell. As the lysogenic cycle allows the host cell to continue to survive
and reproduce, the virus is reproduced in all of the cell’s offspring.

An example of a bacteriophage known to follow the lysogenic cycle

and the lytic cycle is the phage lambda of E. coli (Mason et al., 2011).

Sometimes prophages may provide benefits to the host bacterium

while they are dormant by adding new function to the bacterial genome in
a phenomenon called lysogenic convert.

Multiplication in bacteriophages

Multiplication in bacteriophages has been chiefly elaborated by Dr

Max Delbruck and Dr [Link] and their associates in the United States and
by Professor Andre Lwoff’s school in France. These workers mainly
concentrated on the T- phages of the bacterium Escherichia coli,
particularly the T even phages.

We shall consider the multiplication process of virulent phages, also

called (lytic cycle).

Replication:

 Virulent ( lytic, permanent)

 A virulent ( lysogenic or temperate)

There are two types of replication cycles lytic and lysogenic

93
A. Lytic cycle of bacteriophage replication

The replicative process of virulent phages is called the lytic cycle

because at the end of it the host bacterial cell is lysed or destroyed. This
process can be divided into several distinct phages namely, Adsorption,
Penetration, the Eclipse, the Latent period, and Lysis Occurring in that
order.

A. a. Attachment and penetration

To enter a host cell, bacteriophages attach to specific receptors

on the surface of bacteria, including lipopolysaccharides, teichoic
acids, proteins, or even flagella. This specificity means that a
bacteriophage can infect only certain bacteria bearing receptors to
which they can bind which in turn determines the phages host range.
Host growth conditions also influence the ability of the phage to attach
and invade bacteria (Gabashvili et al., 1997).

As phage virions do not move independently, they must rely on

random encounters with the right receptors when in solution (Blood,
lymphatic, circulation, irrigation, soil, water, etc.)

Myovirus bacteriophages use a hypodermic syringe-like motion

to inject their genetic material into the cell. After making contact with
the appropriate receptor, the tail fibers flex to bring the base plate
closer to the surface of the cell this is known as reversible binding.
Once attached completely, irreversible binding is initiated and the tail,
(Prescott, 1993). Contracts, possibly with the help of ATP present in
the tail injecting genetic material through the bacterial membrane.

Podoviruses lack an elongated tail sheath similar to that of a

myovirus, so they instead use their small tooth-like tail fibers to
enzymatically degrade a portion of the cell membrane before inserting
its genetic material.

A. b. Synthesis of proteins and nucleic acid

Within minutes, bacterial ribosomes start translating viral

mRNA into protein. For RNA-based phages, RNA replicas are

94
synthesized early in the process. Proteins modify the bacterial RNA
polymerase so that it preferentially transcribes viral mRNA. The host’s
normal synthesis of proteins and nucleic acids is disrupted, and it is
forced to manufacture viral products instead. These products go on to
become part of new virions within the cell, helper proteins that help
assemble the new virions or proteins involved in cell lysis.

Walter Fiers (University of Ghent, Belgium) was the first to

establish the complete nucleotide sequence of a gene (1972) and of the
viral genome of bacteriophage MS2 (1976) (Fiers et al., 1976).

A. c. Virion assembly
In the case of T4 phage, the construction of new virus particles
involves the assistance of helper proteins.

The base plates are assembled first, with the tails being built
upon them afterwards.

The head capsids constructed separately will spontaneously

assemble with the tails. The DNA is packed efficiently within the
heads. The whole process takes about 15 minutes.

A. d. Release of virions

Phages may be released via cell lysis, by extrusion or in a few

cases by budding. Lysis by tailed phages is achieved by an enzyme
called endolysin, which attacks and breaks down the cell wall
peptidoglycan. An altogether different phage type, the filamentous
phages make the host cell continually secrete new virus particles.
Released virions are described as free and unless defective are capable
of infecting a new bacterium.

95
96
Bacteriophage infection can be easily monitored by plaque formation.
(In the lytic cycle, anyway):

Plaque forming Unit (p.f.u): is a measure of the number of particles

capable of forming plaques per unit volume (1,000 PFU/µl indicates
that there are 1,000 infectious virus particles in 1 µl of solution).

B. Lysogenic cycle of bacteriophage replication

This cycle occurs in several steps named adsorption, penetration,

integration, bacterial duplication, spontaneous induction, replication,
assembly and release.

B. a. Adsorption and penetration

To enter a host cell, bacteriophages attach to specific

receptors on the surface of bacteria, including lipopolysaccharides,
teichoic acids, proteins, or even flagella. This specificity means that
a bacteriophage can infect only certain bacteria bearing receptors to
which they can bind which in turn determines the phages host range.

97
 Host growth conditions also influence the ability of the phage to
attach and invade bacteria.

As phage virions do not move independently, they must rely

on random encounters with the right receptors when in solution
(blood, lymphatic, circulation, irrigation, soil, water, etc.)

Myovirus bacteriophages use a hypodermic syringe-like

motion to inject their genetic material into the cell, after making
contact with the appropriate receptor, the tail fibers flex to bring the
base plate closer to the surface of the cell this is known as reversible
binding. Once attached completely, irreversible binding is initiated
and the tail contracts, possibly with the help of ATP present in the
tail injecting genetic material through the bacterial membrane.

2-Integration:-

In this step the viral genome is integrated with the plasmid of the
bacterial cell (the plasmid is cleavage by restriction enzymes, then the
viral genome is integrated with the plasmid by ligases enzymes).

The integrated viral genome with the bacterial plasmid is called prophage.

In this stage all viral genes are suppressed except one gene called
repressor gene, this gene order the cell to form resistant protein which
prevent the lysis of the bacterial cell and prevent the replication of the
phage.

3- Bacterial duplication:-

The bacterial cell reproduces a sexually by normal binary fission

duplication of all constituents and separation into two new daughter cells
and so on.

During the bacterial duplications, the prophage also duplicated

many times and transferred to the daughter cells.

In this stage the viral genome is replicated but no progeny produced.

98
4-Spontaneousi induction:-

Mutation of temperate a virulent phage into virulent phage

In this stage the viral nucleic acid is separated from the bacterial
genome upon stimulation by a certain factor (radiation, extreme
temperature….) so the life cycle converted from lysogenic to lytic.

In this stage the repressor gene may be blocked and so the repressor
molecules not formed, or the cell formes protein act against suppressor
molecules.

5-Replication:-

Within minutes, bacterial ribosomes start translating viral mRNA into

protein. For RNA-based phages, RNA replicase is synthesized early in the
process. Proteins modify the bacterial RNA polymerase so that it
preferentially transcribes viral mRNA. The host’s normal synthesis of
proteins and nucleic acids is disrupted, and it is forced to manufacture
viral products instead. These products go on to become part of new virions
within the cell, helper proteins that help assemble the new virions,or
proteins involved in cell lysis.

Walter Fiers (University of Ghent, Belgium) was the first to establish

the complete nucleotide sequence of a gene (1972) and of the viral
genome of bacteriophage MS2 (1976).

6-Assembly:-

In the case of T4 phage, the construction of new virus particles

involves the assistance of helper proteins.

The base plates are assembled first, with the tails being built upon them
afterwards.

The head capsids constructed separarately, will spontaneously

assemble with the tails. The DNA is packed efficiently within the heads.

99
7-Release:-

Phages may be released via cell lysis, by extrusion or in a few cases

by budding. Lysis by tailed phages is achieved by an enzymecalled
endolysin, which attacks and breaks down the cell wall peptidoglycan. An
altogether different phage type, the filamentous phages make the host cell
continually secrete new virus particles. Released virions are described as
free and unless defective are capable of infecting a new bacterium.

111
Bacteriophage life- cycle:-

Mutation of temperate phage to virulent phage

• Resistant to repressor molecule.

• Loss repressor molecule production.

111
Lamda Phage, which affects E. coli, shows the conversion of replication.

112
 4- Importance of bacteriophages in fermentation processes

Any bacterial strain can be infected by virulent phages or harbour

one or more prophages. Therefore, bacteria-phage interactions are to be
regarded as fundamental properties of bacteria. In current industrial
fermentation processes phages can be advantageously employed for the
identification of bacterial production strains (phage typing). In some cases
phages are involved in the production of enzymes and special substances.

The fundamental importance of phages in any technical

fermentation process, however, is based on the peculiarities of their
obligately parasitic life cycle. The propagation of phages in fermentation
processes can cause complete (or at least partial) lysis of the production
strains and, consequently, serious disturbances in the production process
and considerable economic losses. The phage problem in the fermentation
industry has not yet been completely solved. For the protection of
technical processes against virulent phages five measures are discussed:
phage-protected sterile fermentation employment of alternative cultures,
employment of phage-resistant mutants, employment of phage inhibitors,
and employment of immobilized bacterial cells. The problem of the
protection of bacterial production strains from prophage induction is more
difficult and practically unsolved.

Two possibilities to minimize the process risk due to temperate

phages, the elimination of inducing factors during the fermentation
process, and the selection of production strains which are difficult to
induce, are discussed.

Bacteriophages in Medicine

Bacteriophages, or phages, are viruses of bacteria. Thus, by their

very nature, they can be considered as potential antibacterial agents. Over
the past decade or two, the idea of phage therapy, i.e. the use of lytic
bacteriophages for both the prophylaxis and the treatment of bacterial
infections, has gained special significance in view of a dramatic rise in the
prevalence of highly antibiotic-resistant bacterial strains paralleled by the
withdrawal of the pharmaceutical industry from research into new
antibiotics.
113
 As an alternative to "classic" phage therapy, in which whole viable
phage particles are used, one can also employ bacteriophage-encoded
lysis-inducing proteins, either as recombinant proteins or as lead structures
for the development of novel antibiotics. Two additional, rather less-
recognized potential medical applications of phages are the treatment of
viral infections and their use as immunizing agents in diagnosing and
monitoring patients with immunodeficiences. We also discuss very
interesting novel findings demonstrating the immunomodulatory activity
of bacteriophages, suggestive of a potential role of endogenous phages in
maintaining the homeostasis of the immune system.

In the environment

Metagenomics has allowed the in-water detection of bacteriophages

that was not possible previously. These investigations revealed phages are
much more abundant in the water column of both freshwater and marine
habitats than previously thought,[16] and they can cause significant
mortality of bacterioplankton.

Methods in phage community ecology have been developed to

assess phage-induced mortality of bacterioplankton and its role for food
web process and biogeochemical cycle to genetically fingerprint phage
communities or populations and estimate viral biodiversity by
metagenomics. The lysis of bacteria by phages releases organic carbon
that was previously particulate (cells) into dissolved forms, which makes
the carbon more available to other organisms. Phages are not only the
most abundant biological entities but also probably also the most diverse
ones.

The majority of the sequence data obtained from phage

communities have no equivalents in databases. These data and other
detailed analyses indicate phage-specific genes and ecological traits are
much more frequent than previously thought. To reveal the meaning of
this genetic and ecological versatility, studies have to be performed with
communities and at spatiotemporal scales relevant for microorganisms Mc
Grath S and van Sinderen D (2007).

Bacteriophages have also been used in hydrological tracing and

modelling in river systems, especially where surface water and
114
groundwater interactions occur. The use of phages is preferred to the more
conventional dye marker because they are significantly less absorbed
when passing through ground waters and they are readily detected at very
low concentrations Martin, (1988).

Phage typing

Phage typing is a method used for detecting single strains of

bacteria. It is used to trace the source of outbreaks of infections. The
viruses that infect bacteria are called bacteriophages ("phages" for short)
and some of these can only infect a single strain of bacteria. These phages
are used to identify different strains of bacteria within a single species.

A culture of the strain is grown in the agar and dried. A grid is

drawn on the base of the petri dish to mark out different regions.
Inoculation of each square of the grid is done by a different phage. The
phage drops are allowed to dry and are incubated: The susceptible phage
regions will show a circular clearing where the bacteria have been lysed,
and this is used in differentiation (Turbadkar, et al., 2007)

115
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116
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