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BACTERIOPHAGE S AND VIRUSES
BACTERIOPHAGES AND VIRUSES
Study of viruses is called VIRIOLOGY. Study of viruses as a causative agent of infections began in late 19 th century.
In 1882, Adolf Mayer (1843–1942) described a condition of tobacco plants, which he called "mosaic disease".
In 1892, Dmitry Ivanovsky showed that the sap from a diseased tobacco plant remained infectious to healthy
tobacco plants despite having been filtered. Dmitry Ivanovsky used the filter to study what is now known as the
tobacco mosaic virus. His experiments showed that crushed leaf extracts from infected tobacco plants remain
infectious after filtration. Ivanovsky suggested the infection might be caused by a toxin produced by bacteria.
In 1898, the Dutch microbiologist Martinus Beijerinck repeated the experiments and proved that the filtered
solution contained a new form of infectious agent. He called it a contagium vivum fluidum (soluble living germ)
and re-introduced the word virus.
Bacteriohages were discovered in the early 20th century, by the English bacteriologist Frederick Twort (1877–
1950).
Félix d'Herelle (1873–1949) was a mainly self-taught French-Canadian microbiologist. In 1917 he discovered that
"an invisible antagonist", when added to bacteria on agar, would produce areas of dead bacteria.The antagonist,
now known to be a bacteriophage could pass through a Chamberland filter. He accurately diluted a suspension of
these viruses and discovered that the highest dilutions (lowest virus concentrations), 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 viruses in the original suspension. He realised that he had discovered a
new form of virus and later coined the term "bacteriophage". Between 1918 and 1921 d'Herelle discovered
different types of bacteriophages that could infect several other species of bacteria including Vibrio cholera.
In 1935 Wendell Meredith Stanley (1904–1971), who proved that infectious agents were particles.
Friedrich Loeffler (1852–1915) and Paul Frosch (1860–1928) discovered the cause of foot-and-mouth disease
was virus.
Ernst Ruska (1906–1988) and Max Knoll (1887–1969), showed that virus particles, especially bacteriophages,
were shown to have a complex structure.
In 1939, Stanley and Max Lauffer (1914) separated the virus into protein and RNA parts.
In 1933 Schlsinger was first determine the composition of a virus.
In 1955 ,Rosalind Franklin discovered the full DNA structure of the virus .
In 1952 Hershey and Chase studied and demonstrated that genetic material is DNA and infection I due to the
penetration of DNA into cells.
In 1965, Howard Temin (1934–1994) described the first retrovirus.
In 1983 Luc Montagnier and his team at the Pasteur Institute in France, first isolated the retrovirus now called
HIV.
Viruses are distinguished by three properties:
1. They are infectious agents of diseases.
2. They are quite small and hence are invisible in the light microscope and able to pas through the filters that
retain most bacteria and
3. They do not proliferate in the culture media designed to support growth of bacteria.
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BACTERIOPHAGE S AND VIRUSES
Nature of Viruses
1. Size. The size range of viruses is from about 20 to 300 nm. On the whole, viruses are much smaller than
bacteria. Most animal viruses and all plant viruses and phages are invisible under the light microscope.
2. Simple structure. Viruses have very simple structures. The simplest viruses are nucleoprotein particles
consisting of genetic material (DNA or RNA) surrounded by a protein capsid. In this respect they differ from
typical cells which arc made up) of proteins, carbohydrates, lipids and nuc1eicacids.
The more complex viruses contain lipids and carbohydrates in addition to proteins and nucleic acids, e. g. the
enveloped viruses.
These viruses are surrounded by a membranous envelope which is derived from the host cell. It protects the
virus and also serves for transmission from one host to another. The envelope consists of a lipid bilayer and
proteins with special functions.
The membrane proteins are of two types, glycoproteins and matrix: proteins. Glycoproteins have a hydrophobic
end fixed in the lipid bilayer and a hydrophilic glycosylated end which protrudes into the medium.
The spikes on the outer surface of the virions consist of glycoproteins. In the some animal (orthomyxoviruses,
paramyxoviruses and rhabdoviruses) viruses, there is an unglycosylated matrix protein layer on the inner surface
of the envelope. This layer appears to connect the envelope with the capsid.
The envelope and capsid proteins are specified by viral genes. The lipid and carbohydrate of the glycoprotein are
derived from the host cell. Since some viruses can be grown in different cell types, they often have different lipid
and carbohydrate moieties.
3. Absence of cellular structure. Viruses do not have any cytoplasm, and thus cytoplasmic organelles like
mitochondria, Golgi complexes, lysosomes, ribosomes, etc., are absent.
They do not have any limiting cell membrane. They utilize the ribosomes of the host cell for protein synthesis
during reproduction.
4. No independent metabolism.
Viruses cannot multiply outside a living cell. No virus has been cultivated in a cell-free medium. Viruses do not
have an independent metabolism. They are metabolically inactive outside the host cell because they do not
posses enzyme systems and protein synthesis machinery.
Viral nucleic acid replicates by utilizing the protein synthesis machinery of the host. It codes for the synthesis of a
limited number of viral proteins, including the subunits or capsomeres of the capsid, the tail protein and some
enzymes concerned with the synthesis or the release of virions.
5. Nucleic acids. Viruses have only one nucleic acid, either DNA or RNA. Typical cells have both DNA and RNA.
Genomes of certain RNA viruses can be transcribed into complementary DNA strands in the infected host cells, e.
g. Rous Sarcoma Virus (RSV). Such RNA viruses are therefore also called RNA-DNA viruses.
6. Crystallization. Many of the smaller viruses can be crystallized, and thus behave like chemicals.
7. No growth and division. Viruses do not have the power of growth and division. A fully formed virus does not
increase in, size by addition of new molecules. The virus itself cannot divide.
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BACTERIOPHAGE S AND VIRUSES
Only its genetic material (RNA or DNA) is capable of reproduction and that too only in a host cell.
It will thus be seen that viruses do not show all the characteristics of typical living organisms. They, however,
possess two fundamental characteristics of living systems. Firstly, they contain nucleic acid as their genetic
material.
The nucleic acid contains instructions for the structure and function of the virus. Secondly, they can reproduce
themselves, even if only by using the host cells synthesis machinery.
The debate as to whether viruses are living or non-living is actually superfluous. A decision on this matter would
ultimately depend upon the criteria adopted to distinguish between living and non-living.
Definition of Virus: Luria in 1967 gave a composite definition of virus. “ Viruses are entities whose genome is an
element of nucleic acid, either DNA or RNA, which reproduces inside living cells and uses their synthetic
machinery to direct the synthesis of specialized particles, the virions, which contain viral genome and transfer
it to other cells.”
MORPHOLOGY AND STRUCTURE OF BACTERIOPHAGES
The bacteriophages are commonly called 'phages'. The phages possess dsDNA, ssDNA, dsRNA or ssRNA as
genetic material. Three common forms (viz., tailed, cubic, and filamentous) of bacteriophages are known.
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BACTERIOPHAGE S AND VIRUSES
MORPHOLOGICAL GROUPS OF BACTERIOPHAGES :On the basis of EM studies, Bradley (1967) has
described the following six morphological types of bacteriophages.
TYPE
A
MORPHOLOGY hexagonal
head,
a
rigid tail
with
contractile
sheath and
tail fibers
B
a
hexagonal
head but
lacks
contractile
sheath. Its
tail
is
flexible
and mayor
may have
tail fibe
NUCLEIC ACID ds DNA
AND NO. OF
STRANDS
EXAMPLE
T2, T4,
D
head which
is made up
of
capsomers
but lacks
tail,
E
F
head made Filamentous
up of small phage
capsomers
but
contains no
tail
G
Pleomorp
hic,
no
capsid
dsDNA
C
hexagonal
head and
a
tail
shorter
than head.
Tail lacks
contractie
sheath
and
mayor
may not
have tail
fiber,
dsDNA
ssDNA
ssRNA
ssDNA
dsDNA
T1, T5
T3, T7.
φX174
F2, MS2
fd, fl)
MV-L2
Source: peoi.org
BINAL STRUCTURE OF BACTERIOPHAGE (T-EVEN PHAGES )
The T-even phage is characterized by the presence of a hexagonal head about 900 Å wide. It consists of dsDNA
molecule protected by a protein coat made up of numerous facets. The DNA molecule, measuring about 52,000
Å in length, is coiled and packed inside the head. The head is attached with a cylindrical tail consisting of a hollow
core surrounded by protein sheath. The hollow central core measures about 80-100 Å in diameter and is
considered continuous from the head to the end of the tail forming a channel through which the nucleic acid
moves into invade the host cell being infected.
The protein sheath is spirally coiled and consists of 24 annular rings, which often forms a tube is connected to a
thin disc-like structure called collar at the base of the head and to a hexagonal end plate at the end of the tail.
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BACTERIOPHAGE S AND VIRUSES
The protein sheath of the tail is capable of contracting in the longitudinal direction. At the six corners of the
hexagonal plate there are small spikes to which very long fibers called tail-fibers are connected. The tail fibers are
the organs of attachment to the wall of the bacterial cell.
Structure of T-even Bacteriophage (Diagrammatic). A. External
Structure, B. Internal Structure, and C.
End
Plate
(Enlarged).
1. Head
2. Protein Sheath
3. Coiled DNA
4. Collar
5. Central Core
6. Protein Sheath
(Helical)
7. Tail
8. End Plate
9. Tail Fibres
10. Hexagonal Plate
11. Spike
LYTIC (VIRULENT) AND TEMPERATE (NON VIRULENT) BACTERIOPHAGES:
Bacteriophages may have a lytic cycle or a lysogenic cycle, and a few viruses are capable of carrying out both.
With lytic phages such as the T4 phage,
bacterial cells are broken open (lysed) and
destroyed after immediate replication of
the virion. As soon as the cell is destroyed,
the phage progeny can find new hosts to
infect.
Picture source:(mcdevittapbio.wikispaces.com)
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. The virus
remains dormant until host conditions
deteriorate, perhaps due to depletion of
nutrients, and 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.
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BACTERIOPHAGE S AND VIRUSES
CLASSIFICATION OF BACTERIOPHAGES
Bacteriophages are assigned designations or code symbols by investigators as the taxonomic development within
the bacteriophages is slow and difficult. Coliphages are extensively studied. They infect the non motile strains of
E.coli. They are designated T1 to T7. The bacterial virus subcommittee has now recommended names ending in
Viridae.
On the basis of presence of single or double strands of genetic material, the bacteriophages are categorized as
under:
1. The ssDNA Bacteriophages
(i) Icosahedral phages = φ ´X174, St-1, φR, (ii) Helical (filamentous)
(a) The Ft group: They are F specific phages and absorb to the tip of F type sex pilus, e.g., E.coli phages (fd,).
(b) If group: They are absorbed to I-type sex pilus specified by R factors e.g., If1.
(c) The third group is specific to strains carrying RF1 sex factor.
2. The dsDNA Phages
(vii) The phage of Haemophilus e.g., HPl.
Following are the examples of dsDNA phages:
(viii) The phage of Pseudomonas e.g., PM2.
(i) T-odd phage of E.coli e.g. T1, T3, T5, T7
3. The ssRNA phages
(ii) T-even phage of E.coli e.g.T2, T4, T6
Examples of the ssRNA bacteriophages are as below:
(iii) The other E.coli phages e.g., P1, P2, Mu, φ80.
(i) Group I : E. coli. phages such as f2, MS2, M12,
(iv) The phages of Bacillus subtilis e.g., PBSI, PBSX,
R17, fr, etc.
PBSI, SPOI, SPO2.
(ii) Group II : The QP phages.
(v) The phage of Shigella a e.g., P2
(vi) The phage of Salmonella e.g., PI, P22.
4. The dsRNA phages
Example: The φ6 bacteriophage.
28
BACTERIOPHAGE S AND VIRUSES
24
FAMILIES OF BACTERIOPHAGES (Representative):
NON ENVELOPED
dsDNA
Myoviridae:T2
ENVELOPED
dsDNA
Plasmoviridae:MV-L2
Styloviridae:P2
NON ENVELOPED
ssDNA
ssRNA
Microviridae:ØX174 Leviviridae:MS2
ENVELOPED
dsRNA
Cystoviridae: Ø6
DESIGN AND CONSTRUCTION OF VIRUSES
The intact virus unit or infectious particle is called the virion. Each virion consists of a nucleic acid core surrounded
by a protein coat (capsid) to from the nucleocapsid.
The nucleocapsid may be naked or may be surrounded by a loose membranous envelope. It is composed of a
number of subunits called capsomeres.
The capsid protects the nucleic acid core against the action of nucleases.
Viruses occur in three main shapes, spherical (actually polyhedral), helical (cylindrical or rod like) and complex.
Polyhedral and helical viruses may have naked capsid, or the caps ids may be covered by envelopes.
Viral capsids are built from large number of small ‘morphological units’ or capsomeres that are attached each other
by non covalent bonds. Electron microscopy shows that capsomeres are disposed in regular geometrical figures to
form capsid of either cubical or helical symmetry. At first sight the capsomeres often seem to be spherical but on
close scrutiny at very high resolving powers they are frequently found to be hollow and pyramidal in shape and to
consist of aggregate s of even small structural units of differing polypeptide chains.
A capsomeres may contain only one structural unit .E.g. the capsomeres of the TMV are known as ‘monomers’
because they contain only one structural unit; a poly peptide chain of a molecular weight 20-30 daltons.
BACTERIOPHAGE S AND VIRUSES
25
Capsomeres of poliovirus are oligomeres and contain four different polypeptides. The fitting of capsomeres and
their components together construct the capsid that is precisely accurate and symmetrical because an impenetrable
shield has to be formed to protect the nucleic acid from damage by enzymes actions and destructive mechanisms of
host.
HELICAL SYMMETRY:
The helical capsids consist of monomers arranged in helix around a single rotational axis. The monomers curve into
a helix because they are thicker at one end than the other.
The size and shape of the monomers determines the shape of the virus. Helical capsids may be naked (e. g. the
tobacco mosaic virus) or surrounded by an envelope (e.g. the influenza virus).
Tobacco Mosaic Virus - TMV
In 1936 Stanley isolated the tobacco mosaic virus in crystalline state from the sap of infected tobacco plants.
The virus is rod shaped, about 300nm long and 15-18 nm in diameter.
X-ray diffraction studies have shown that the virus consists of a protein
tube with a lumen of 20A enclosing a single strand of helically coiled RNA.
The tube is made up of a number of identical subunits (monomers) of
protein arranged in a helical manner.
Studies have shown that there are 49 subunits of protein for three turns of
the helix, thus giving a total of 2,130 subunits for the rod.
Each subunit has a molecular weight of 17,500, and consists of a single
polypeptide chain made up of 158 amino acids whose sequence has been
established.
The RNA is a single stranded molecule coiled into a helix BOA in diameter.
It follows the pitch of the protein helix. Each turn of the RNA helix contains
about 49 nucleotides, and has a pitch of 23°. The RNA is infective by itself,
although much less so than the intact virus.
Picture source:pathmicro.med.sc.edu
This is because unprotected RNA is subjected to the action of enzymes
(nucleases), and is thus destroyed. The protein functions as a protective
tube around the RNA.
Picture Source: bahankuliahkesehatan.blogspot.com
BACTERIOPHAGE S AND VIRUSES
26
CUBICAL SYMMETRY: (ICOSAHEDRAL) SYMMETRY
Crick and Watson have shown that the polyhedral capsids can have
three possible types of symmetry, viz. tetrahedral, octahedral and
icosahedral.
It has been shown that an icosahedron is the most efficient shape for
the packing and bonding of the subunits of a near spherical virus
Therefore viruses are icosahedral rather than tetrahedral of octahedral.
A large number of intermolecular bonds can be formed in this type of
structure, and it therefore has low free energy. An icosahedron is a
regular polyhedron with 20 faces formed by equilateral triangles, and
12 intersecting points or corners. An axis entering at one of these
vertices and passing through the centre of the figure enables the
icosahedron to be rotate through five new positions in each of which
the same appearance is presented. If the axis enters through the center
of any one of the equilateral triangle only three identical positions can
be obtained and if the point of entry is through the center of any of the
edges of the triangular facets there can be two such positions. Thus an
icosahedrons is said to have 5.3.2.rotational symmetry.
As mentioned previously, each capsid consists of many capsomeres.
Each capsomere is composed of few monomers which form polygonal
rings, each with a central space of up to 40 A.
The monomers are the structural units, and are made up of one or
more polypeptide chains.
There are two types of capsomeres, pentameres and hexameres. The
pentamere or pentagonal capsomere is made up of 5 monomers. The
hexamere or hexagonal capsomere consists of 6 monomers.
Picture source: Prescott’s Microbiology
The monomers are held together by bonds, each monomer having
bonds with two neighbouring monomers. The capsomeres are also
held together by bonds.
These bonds appear to be weaker than the bonds between the
monomers, because in some viruses the capsid breaks down into
capsomeres during purification.According to the rules of
crystallography, only a certain number of capsomeres can be present
in an icosahedral capsid.The minimum number of capsomeres can
theoretically be 12, followed by 32,42,72,92,162,252,362,492,642 and
812. Of these capsomeres, 12 are pentameres occupying the 12
corners, while the rest are hexameres.
The twenty triangular facets of the icasahedron can be further
subdivided into smaller triangles and resulting solid is then called an
icosadeltahedron.
BACTERIOPHAGE S AND VIRUSES
27
The simple formula 10T+2 gives the total number of capsomeres in the capsid. T=small triangle numbers. E.g. for
Herpes simplex virus T=16 so this virus has 10 x 16 =160+2=162(10T+2) capsomeres. Another method to calculate
the total numbers of capsomeres is to use the formula 10(n-1)2 +2 where ‘n’ is the number of capsomeres seen by
the electron microscope to be situated along the edge of one equilateral triangle. E.g. for Herpes simplex virus ‘n’ is
5, therefore 10(5-1)2+2=162.
The actual number of capsomeres found in different viruses are: φX174, 12; turnip yellow mosaic virus and
poliovirus, 32; and papilloma virus, 72; reoviruses, 92; herpesviruses, 162; adenoviruses, 252, and tipula iridescent
virus,812.
φX174. The bacteriophage φXI74 contains 12 capsomeres. It has been suggested that each capsomere is actually a
cluster of five units. Therefore the capsid is probably made up of 60 identical units.
Enveloped Viruses
Some icosahedral and helical animal viruses are surrounded by a membranous envelope l00-150A thick. An external
envelope is also present in some plant viruses and bacteriophage.
Enveloped Constituents - Proteins
Viral envelopes contain host cell proteins as well as, proteins specified by the virus. In arboviruses, rhabdoviruses,
and, myxoviruses, there is overwhelming evidence that all envelope proteins are coded by viral genomes.
The membranes of all classes of enveloped viruses contain glycoproteins. This protein is a glycoprotein. In the
Sindbis virus and the Semliki Forest Virus the protein contains a relatively high proportion of hydrophobic amino
acids, indicating that it is associated with the envelope lipids. Rhabdoviruses have one glycoprotein in. their
envelopes, paramyxoviruses two glycoproteins and influenza viruses (orthomyxoviruses) four different
glycoproteins.
The herpesviruses and the leukoviruses also have glycoproteins in their envelopes. The, spikes on the outer surface
of virions are glycoproteins.
Carbohydrates
Viral envelopes contain a significant amount of carbohydrates. Galactose, man nose, glucose, fucose, glucosamine
and galactosamine have been found in the influenza virus, the parainfluenza virus SV5 and in the Sindbis virus.
The total carbohydrate content and the proportions of hexoses and hexosamines are very similar in these viruses.
Carbohydrates in enveloped viruses are not only found as glycoproteins but also as glycolipids. In arboviruses and
myxoviruses it appears that at least a part of the carbohydrate structure is specified by the host cell.
At least some carbohydrate can arise by host modification. In the vaccinia viruses there is evidence that the
carbohydrates of the viral glycoproteins might be virus specific, and that these viruses have their own glycosylating
enzymes.
Lipids Present in Viral Envelope
It is generally accepted that the lipids in virus envelopes are derived from the host cell. This is shown by the facts
that:
(i) viruses rarely have lipids not found in host cells,
(ii) when viruses are grown in different host cells, they show differences in their lipid patterns, and
BACTERIOPHAGE S AND VIRUSES
28
(iii) radioactively labelled cellular lipids are incorporated into virions.
The lipids of viruses budded from preformed cellular membranes are early of host cell origin.
In viruses assembled without continuity with host cell membranes, the evidence of cellular is not so clear cut. In the
vaccinia viruses lipid biosynthesis in infected host cells is not basically altered. The virus does not have any unusual
or novel lipids. Unusual lipids are, however, found in some viruses.
The different classes of lipids present in viral envelopes are as follows:
(a) Phospolipids: e.g. sphingomyelin, phosphatidyl choline phosphatidyl ethanolamine, phosphatidyl serine and
phosphatidyl inositol. Of these the former three are predominant, and the other two are usually present in smaller
amounts.
(b) Cholesterol : It is significantly higher than in the host cell. The molar ratio cholesterol: phospholipid is about I in
viral envelopes and 0.2 in host cells
(c) Fatty acids: The phospholipids consist predominantly of saturated and unsaturated acids, with chain lengths of
16, 18 and 20 C atoms. Virus fatty acids contain higher amounts of saturated fatty acids than whole cells.
(d) Glycolipids: Glycosphingolipids consist of sphingosine, fatty acids and carbohydrates. Arboviruses and
rhabdoviruses also posses gangliosides.
Types of Viral Nucleic Acids
Viral nucleic acids show considerable diversity. Viruses may contain DNA or RNA which may be single or .double
stranded, linear or circular. Some may have plus polarity while others may have plus polarity.
With respect to the number of strands, four types of nucleic acids are found in viruses:
Single stranded DNA (ssDNA)
Double stranded DNA (dsDNA)
Single stranded RNA (ssRNA) and
Double stranded RNA (dsRNA).
Terminal Redundancy of Some Viruses - The DNA of Some contains repeated nucleotide sequences at its terminus.
This is viruses known as terminal redundancy. Thus in the T-even phages bout 5% of the total molecule is repeated
at the ends.
Structural Viral Proteins (Nucleocapsid Proteins)
The capsids of viruses are made up entirely of proteins.
The capsid proteins enclose the nucleic acid and protect it from nucleases in biologic fluids.
The capsid also promotes attachment to susceptible cells. The virus cannot have too many genes to specify different
protein types. Hence it is made up of many identical protein units or protomers.
Helical capsids usually consist of a single protein type. Thus the TMV consists of a single RNA molecule coated by a
single type of polypeptide. Icosahedral capsids may have one or several types of proteins. Adenoviruses contain at
least 14 protein types.
The bacteriophage T4 contains some 30 different polypeptide chains.
The protomers are arranged in a definite architecture in toe capsid. This permits bonding between suitable chemical
groups on their surfaces.
Internal OR Core ProteinsThese are proteins associated with the nucleic acid of the virion, e. g. proteins V and VII of
adenoviruses and the nucleoproteins of vesicular stomatitis virus (VSV) and the influenza virus.
Viral Enzymes
Several virion specific enzymes have been found in animal viruses, most of these activities being confined to
enveloped viruses.
Thus dsRNA viruses contain enzymes for the synthesis of viral mRNA, including the addition of a 'cap'.
BACTERIOPHAGE S AND VIRUSES
29
Such enzymatic activities have not been detected in plant or bacterial viruses, except for the dsRNA viruses of
plants.
Enzyme
Product OR Function
Virus
Enzymes affecting interaction of NANA split off from surface polysaccharides.
host cell surface with virions
Degradation of surface. Modification of lipid
Neuraminidase.
bilayer.
Endoglycosidase.
Fusion factor.
Ortho
paramyxovirus.
E.
coli
Paramyxovirus.
DNAmRNA
transcription Transcribes ss mRNA.
enzymes
Transcribes ss mRNA.
DNA-dependent RNA polymerase. Transcribes (+) strand ss mRNA.
dsRNA
transcriptase
.
ssRNA transcriptase.
Poxvirus, Phages
SPO2.
dsRNA viruses.
ssRNA (+) viruses.
Enzymes adding specific terminal
groups to viral mRNA
Nucleotide
Phosphohydrolase.
Guanylyl
transferase.
RNA
methylases.
Poly (A) polymerase.
Viruses
synthesizing
mRNA in virions
(e. g. poxviruses and
reoviruses).
Converts 5'-ppp to 5'-PP. Adds guanylyl
residue to 5'-pp in mRNA.
Methylates 5' end guanyl residues in mRNA.
Synthesizes 3' end poly (A) tail in mRNA.
and
phages.
N4,
RNA- DNA transcription enzymes DNA-RNA hybrids; dsDNA.
Reverse transcriptase. RNase H Degrades RNA in RNA-DNA hybrids.
(with above).
Closes ss breaks in dsRNA.
Polynucleotide ligase.
Retroviruses.
-do-do-
Nucleic acid replication or Synthesizes dsDNA.
processing enzymes
Break DNA strands and crosslinks.
DNA-dependent DNA polymerase. mRNA processing.
DNases
(exoand
endo-).
Endoribonucleases.
Hepatitis B.
Pox-,
retroadenoviruses.
Ss mRNA viruses (e. g:
poxviruses).
Other
enzymes
Protein Phosphorylate proteins.,Aminoacylate tDNA. Retro-,
orthomyxokinasestRNA aminoaovlases.
paramyxo,herpesand
adenovirusesRetroviruses
PLANT VIRUSES
Plant viruses
Most plant viruses have been found in angiosperms (flowering plants). Relatively few viruses are known in
gymnosperms, ferns, fungi or algae. Plant viruses are of great economic Importance, since they cause plant diseases
in a variety of crops.
BACTERIOPHAGE S AND VIRUSES
30
Virion Morphology
The essentials of capsid morphology are similar to the other viruses as outlined earlier since they do not differ
significantly in construction from
their animal virus and phage
relatives. Many have either rigid
or flexible helical capsids (tobacco
mosaic
virus).
Others
are
icosahedral or have modified the
icosahedral pattern with the
addition of extra capsomers
(turnipyellow mosaic virus). Most
capsids seem composed of one
type of protein; no specialized
attachment proteins have been
detected. Almost all plant viruses
are RNA viruses, either single
stranded or double stranded.
Caulimoviruses and geminiviruses
with
their
DNA
genomes
areexceptions to this rule.
ANIMAL VIRUSES
Morphology is probably the most
important characteristic in virus
classification. Animal viruses can
be
studied
with
the
transmissionelectron microscope
while still in the host cell or after
release. The nature of virus nucleic
acids is also extremely important.
Nucleic acid properties such as the
general type (DNA or RNA),
strandedness,
size,
and
segmentation are all useful.
Genetic relatedness can be
estimated by techniques such as
nucleic acid hybridization, nucleic
acid and protein sequencing, and
by determining the ability to undergo recombination.
BACTERIOPHAGE S AND VIRUSES
GENERAL PROPERTIES OF MAJOR GROUPS OF ANIMAL VIRUS
DNA VIRUSES
Parvovirus
NO.OF
STRAND
S
1
Papovirus
2
Icosahedron
N
Spherical
40-55
Adenovirus
2
Icosahedron
N
Spherical
80
Linear dsDNA
Herpes virus
2
Icosahedron
E
Roughly spherical
100
Linear dsDNA
Pox virus
2
Complex
E
Brick shaped
300x200x100
Linear dsDNA
Baculovirus
2
Polyhedral
E
Rod shaped
300x40
Reovrus
2
Icosahedron
N
RNA VIRUSES
Spherical
80
Orbivirus
Picarnovirus
2
1
Icosahedron
Icosahedron
N
N
Spherical
Spherical
60
20-30
Togavirus
1
Icosahedron
E
Spherical
40-70
Retrovirus
1
Icosahedron
E
Roughly spherical
100
Orthomyxoviru
s
1
Helical
E
Roughly spherical
80-120
Minus strand
RNA
Paramyxovirus
1
Helical
E
Pleomorphic
100-300
Minus strand
RNA
Rhabdovirus
1
Helical
E
Bullet shaped
175x70
Minus strand
RNA
GROUP
N=NAKED, E=ENVELOPED
SYMMETRY
N/E
SHAPE
Icosahedron
N
STRUCTURE
SIZE IN NM
NUCLEIC ACID
Spherical
20
Linear ssDNA
Circular
dsDNA
Circular
dsDNA
RNA
Segmented
10-13
molecules
RNA
Plus Strand
RNA
Plus Strand
RNA
Plus strand
RNA
31
BACTERIOPHAGE S AND VIRUSES
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Cyanophages : Morpology and Growth Cycle
These are the phages that attack cyanobacteria. Cyanophages were first discovered by Safferman and Morris from
a waste stabilization pond of Indiana Universit. The first cyanophage studied by Safferman and Morris was the
cyanophage attacking Lyngbya. Plectonema and Phormidium.. They named the virus as LPP-I using the first letter of
the three genera. Thereafter, several serological strains of LPP were isolated from different parts of world and
named LPP-I, LPP-2, LPP-3, LPP-4 and LPP-5. Besides LPP groups of cyanophages, a large number of other
cyanophages such as SM-I, AS-I, N-I, C-I, AR-I and AI etc. have been reported in recent years.
Diagram of Cyanophages
Waste stabilization ponds, eutrophic lakes and polluted water support the
luxurient growth of cyanobacteria. These can be obnoxious bloom in water
reservoirs like lakes and result in fish mortaility. Therefore, the cyanophages can
playa significant role in control of blooms. So far the problems with them that they
are specific to genus and difficult to isolate.
Morphology of Cyanophages
LPP group of cyanophages resemble T3 and T7 bacteriophages as they possess
icosahedral head (580 Å diam.) and short (20 x 15 nm) tail. N-I cyanophages
resemble T2 and T4 phage because their head (550 Å diam.) is icosahedral but the
tail is long (110 x 10 nm). SM-I cyanophages have tailless icosahedral head (880 Å
diam.) whereas As viruses posses hexagonal head (900 A diam.) and long tail (243 x
22 nm). Like R-even phages, the tail may be contractile or non-contractile. AS-I group has the largest cyanophages.
Cyanophages resemble T-even bacteriophages in their growth cycle.
Mycoviruses (Mycophages): Morphology and Replication
M. Hollings of Glasshouse Crop Research Institute, USA for the first time gave experimental evidence of viruses in
cultivated mushroom Agaricus bisporus causing die-back disease in 1962, The most characteristic and consistent
features of mushroom virus diseases are the loss of crop and the degeneration of mycelium in the compost. Several
terms have been proposed to denote such viruses, viz., fungal viruses, mycophages, ds-RNA plasmids, mycoviruses
and virus-like particles (VLPs); the last two terms have been frequently used by the microbiologists.
Since their discovery, mycoviruses have been reported from all major taxonomic groups of fungi, the number of
fungal genera ranging from about 50 to 60. Some important fungi containing mycoviruses are Agaricus bisporus (2550 nm), Alternaria tenius (30-40 nm), Aspergillus foetidus (40-42 nm), A. glaucus (25 nm), A. niger (40-42 nm),
Penicillium brevicompactum (40 nm) P. chrysogenum (35 nm).However, it is interesting to note that most of the
species of Penicillium and Aspergillus have been found to be attacked by mycoviruses while the latter are not found
so frequently in other fungal genera..
Morphology of Mycoviruses
Mycoviruses show morphologically variable forms, viz., bacilliform, rod-shaped, filamentous and herpes types. But
majority of the known mycoviruses are typically isodiametric ranging usually from 25 and 50 nm in diameter and
particle weight from 6-13 x 106 dalton. The most outstanding feature common to mycoviruses is possession of
double-stranded ribonucleic acid (dsRNA) usually segmented into 1-8 segments with a total molecular weight of 2
to 8.5 X 106 dalton. The dsRNA segments are separately enclosed into identical capsids. This feature of mycoviruses
differentiates them from plant and animal dsRNA viruses in which the genetic material segments are, usually, all
enclosed in a single virion.
Replication of Mycoviruses
Highly specific virus-coded RNA polymerases are necessary for effective in vivo transcription and replication of
dsRNA. Such polymerase has been reported in some dsRNA mycoviruses. It is thought that the polymerases remain
confined within the virion during the replicative cycle of mycoviruses.
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The mechanism of infection and transmission of mycoviruses is still obscure. They have been found in fungal spores
and it is believed that they are transmitted through the spores. The presence of viral-RNA in the fungal cells does
not appear to affect any cellular properties such as antibiotic production. For example, Penicillium notatum contains
a dsRNA mycovirus, but penicillin production by the fungus is not affected at all. In recent years the dsRNA
mycoviruses have attracted the attention of scientists since they have ability to induce interferon production in
animal cells. Also, they do not appear to the animal cells be toxic unlike other chemicals that induce interferon
production.
Insect Viruses
Members of at least seven virus families (Baculoviridae, Iridoviridae,Poxviridae, Reoviridae, Parvoviridae,
Picornaviridae,and Rhabdoviridae) are known to infect insects and reproduce or even use them as the primary host
.Of these, probably the three most important are the Baculoviridae, Reoviridae,
and Iridoviridae .
The Iridoviridae are icosahedral viruses with lipid in their capsids and a linear double-stranded DNA genome. They
are responsible for the iridescent virus diseases of the crane fly and some beetles. The group’s name comes from
the observation that larvae of infected insects can have an iridescent coloration due to the presence of crystallized
virions in their fat bodies. Many insect virus infections are accompanied by the formation of inclusion bodies within
the infected cells. Granulosis viruses form granular protein inclusions, usually in the cytoplasm. Nuclear
polyhedrosis and cytoplasmic polyhedrosis virus infections produce polyhedral inclusion bodies in the nucleus or
the cytoplasm of affected cells. Although all three types of viruses generate inclusion bodies, they belong to two
distinctly different families. The cytoplasmic polyhedrosis viruses are reo-viruses; they are icosahedral with double
shells and have double-stranded RNA genomes. Nuclear polyhedrosis viruses and granulosis viruses are
baculoviruses—rod-shaped, enveloped viruses of helical symmetry and with double-stranded DNA.
The inclusion bodies, both polyhedral and granular, are protein in nature and enclose one or more virions. Insect
larvae are infected when they feed on leaves contaminated with inclusion bodies. Polyhedral bodies protect the
virions against heat, low pH, and many chemicals; the viruses can remain viable in the soil for years. However, when
exposed to alkaline insect gut contents, the inclusion bodies dissolve to liberate the virions, which then infect mid
gut cells. Some viruses remain in the mid gut while others spread throughout the insect. Just as with bacterial and
vertebrate viruses, insect viruses can persist in a latent state within the host for generations while producing no
disease symptoms. A reappearance of the disease may be induced by chemicals, thermal shock, or even a change in
the insect’s diet. Much of the current interest in insect viruses arises from their promise as biological control agents
for insect pests.
Many people hope that some of these viruses may partially replace the use of toxic chemical pesticides.
Baculoviruses have received the most attention for at least three reasons. First, they attack only invertebrates and
have considerable host specificity; this means that they should be fairly safe for non target organisms. Second,
because they are encased in protective inclusion bodies, these viruses have a good shelf life and better viability
when dispersed in the environment. Finally, they are well suited for commercial production since they often reach
extremely high concentrations in larval tissue (as high as 1010 viruses per larva).
The use of nuclearpolyhedrosis viruses for the control of the cotton bollworm, Douglas fir tussock moth, gypsy
moth, alfalfa looper, and Europeanpine sawfly has either been approved by the Environmental Protection Agency or
is being considered. The granulosis virus of the codling moth also is useful. Usually inclusion bodies are sprayed on
foliage consumed by the target insects. More sensitive viruses are administered by releasing infected insects to
spread the disease. As in the case of other pesticides, it is possible that resistance to these agents may develop in
the future.
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Viroids and Prions
Viroids: Discovery, Morphology, Replication, Transmission
Viroids are a novel class of subviral pathogens that are found to cause diseases on plants and are the smallest
known infectious agents. They are also known by the names metaviruses or pathogene and differ basically from
viruses in at least following features: (i) Virus-RNA is enclosed in a protein coat while the viroids lack any protein
coat and apparently exist as free-RNA, (ii) Viroid-RNA is of small size consisting of 246-375 nucleotides as compared
to 4-20 kb of virus-RNA, and (iii) Viroid RNA consists of only one molecular species only, while many virus-RNA exist
as more than one molecular species within the same capsid.
Discovery of Viroids
The first viroid was discovered by T.O. Diener in 1971 who found it to be the causative agent of Potato spindle tuber
disease (Diener, 1979), the disease previously considered to be caused by Potato spindle tuber virus.- Since then,
several other plant diseases are now known to be caused by viroids; some important ones are Chrysanthemum
chlorotic mottle disease, Chrysanthemum stunt disease, Citurs excortis disease,Coconut cadang-cadang disease,
Tomato bunchy top disease, Tomato apical stunt disease etc.
Morphology of Viroids
Viroids are small, circular, single-stranded RNA molecules ranging from 246 nucleotides (Coconut cadang-cadang
viroid) to 375 nucleotides (Citrus excortis viroid) in size. Their molecular weight is low and ranges from 85,000 to
1,30,000 daltons. The extracellular form of viroid is naked-RNA, there is no capsid of any kind. Even more
interestingly, the RNA molecule contains no protein encoding genes and, therefore, the viroid is totally dependent
on host function for its replication. Although the viroid is a single-stranded circular RNA molecule, there is such
considerable secondary structure possible that it resembles a short-stranded molecule with close ends
Diagram of viroid ss-RNA showing how single-stranded circular RNA forms a seemingly double-stranded structure
by intra-strand base pairing
Replication of Viroids
Viroids seem to be associated with the cell nuclei, particularly the chromatin, and possibly with the endomembrane
system of the host cell. There is evidence that viroids replicate by direct RNA copying in which all components
required for viroid-replication including the RNA polymerase are provided by the host.
The infecting viroid strand (marked +) enters a cell, moves into the nucleus and initiates the synthesis of minus (-)
strand (i.e., the complementary strand) by a rolling circle mechanism proposed earlier by Brown and Martin (1965)
for replication of certain viral RNAs.
The linear (-) strand of RNA then serves as a template (complementary) for replication of strand of (+) RNA. The (+)
RNA is subsequently cleaved by enzyme that release linear, unit length viroid (+) RNAs, and these circularize and
produce many copies of the original viroid RNA.
Transmission of Viroids
Viroids possibly cannot be transmitted as naked RNAs because of their susceptibility to nuclease enzyme. They,
however, are protected from this enzyme-attack by being localized within the nuclei of infected cells (Sanger, 1979).
Presumably, the viroids are transmitted in association with pieces of nuclei or chromatin and not as free RNA.
Their transmission from diseased to healthy plants takes place primarily by mechanical means, i.e., through sap
BACTERIOPHAGE S AND VIRUSES
35
carried on hands or tools during propagation or cultural practices, and by vegetative propagation. No specific insect
or other vectors of viroids are known.
Virusoids
Similar to viroids, the virusoids are small, low molecular weight, circular RNAs; they are always associated with a
larger RNA molecule of a virus. The virusoids were discovered by Randles). It is thought that some virusoids are
necessary for the replication of RNA of the virus with which they are associated, and may form part of the viral
genome (Robertson et aI., 1983). One virusoid has been found associated with Velvet tobacco mosaic virus. Other
virusoids have been found to be more like a satellite, i.e., extra RNA associated with virus capable only of replicating
in cells infected by the virus. It has also been found that virusoids produce such structures in infected cell suggest
that thereby that their replication cycles resemble those of the potato spindle tuber virusoid and other virusoids
(Branch and Robertson, 1984).
Prions : Structure, Chemical Nature, Replication
In 1970s S.B,. Prusiner, a bichemist at the University of California (USA), with his coworkers initiated the isolation
and identification of the infectious agent of scrapie. After exhaustive research for a decade, he in 1982 discovered
that the disease is caused by a proteinaceous infectious particle which he christened as prions. S.B. Prusiner has
been awarded Nobel Prize in 1997 for the discovery of prions.
Prions represent the other extreme from viroids. They are considered to be devoid of their own genetic material
(DNA or RNA) and consist of just a single or two or three protein molecules i.e., a prion is merely an infectious
protein. The discovery of prion, an infectious protein, has threatened the universally accepted concept that only the
genetic material (DNA, in some cases RNA) is infectious.
The prions, at present, are considered to be the causative agents of some of the diseases of animals and humans
such as Scrapie disease of sheeps and goats, Bovine spongiform encephalopathy in cattle (BSE or Mad cow
diseases);. Kuru, Creutifeldt Jacob disease (CJD), Gerstmann-Strausslar syndrome (GSS), Low Gehrig disease,
Parkinsons disease, Serite domentia and Multiple sclerosis in humans. In 1996, information available from England
indicates that the prion causing Bovine spongiform encephalopathy (BSE) in cattle might infect humans, resulting in
a variant of Creutzfeldt Jacob disease (CJD), called variant CJD or vCJD.
Structure of Prions
Prion is 100 times smaller than a virus, contains only protein is heterogenous in size and density, and can exist in
many molecular forms. Prions possess molecular weight between 27,000 and 30,000 daltons. Electron microscopic
studies have shown that a large number of prion molecules (-1000) aggregate together to form a composite
structure called 'prion-rods'. The latter are typically 100 to 200 nm in length and 10-20 nm in diameter.
Chemical Nature of Prions
The chemical nature of the prions, as stated earlier, is considered to be proteinaceous and they have no nucleic
acids of their own.
Replication of Prions
If prions lack their own nucleic acids and are merely proteins, a very important question requires an answer. One
hypothesis states that the existence of small piece of DNA gene (also called prp gene) is necessary to encode the
amino acid sequence of prion protein at the time of its replication. This DNA gene is a component of the host
genetic material (host DNA). The prion protein presumably serves as a promoter of DNA gene expression.
Recent studies indicate that prions represent a changed conformation of proteins normally found in cells. Once
prions are produced, they somehow persuade the normal versions of the corresponding protein to assume the
altered conformation and, thereby, become prions.
Pass word: virus
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