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BACTERIOPHAGES AND VIRUSES

Study of viruses is called VIRIOLOGY. Study of viruses as a causative agent of infections began in late 19th 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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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.

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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.

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 BACTERIOPHAGESThe 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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MORPHOLOGICAL GROUPS OF BACTERIOPHAGES :On the basis of EM studies, Bradley (1967) has described the following six morphological types of bacteriophages.

TYPE A B C D E F GMORPHOLOGY hexagonal

head, a rigid tail with contractile sheath and tail fibers

a hexagonal head but lacks contractile sheath. Its tail is flexible and mayor may have tail fibe

hexagonal head and a tail shorter than head. Tail lacks contractie sheath and mayor may not have tail fiber,

head which is made up of capsomers but lacks tail,

head made up of small capsomers but contains no tail

Filamentous phage

Pleomorphic, no capsid

NUCLEIC ACID AND NO. OF STRANDS

ds DNA dsDNA dsDNA ssDNA ssRNA ssDNA dsDNA

EXAMPLE T2, T4, 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.

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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. 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).

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

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

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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.

CLASSIFICATION OF BACTERIOPHAGESBacteriophages 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 PhagesFollowing are the examples of dsDNA phages:(i) T-odd phage of E.coli e.g. T1, T3, T5, T7(ii) T-even phage of E.coli e.g.T2, T4, T6(iii) The other E.coli phages e.g., P1, P2, Mu, φ80.(iv) The phages of Bacillus subtilis e.g., PBSI, PBSX, PBSI, SPOI, SPO2.(v) The phage of Shigella a e.g., P2(vi) The phage of Salmonella e.g., PI, P22.

(vii) The phage of Haemophilus e.g., HPl.(viii) The phage of Pseudomonas e.g., PM2.3. The ssRNA phagesExamples of the ssRNA bacteriophages are as below:(i) Group I : E. coli. phages such as f2, MS2, M12, R17, fr, etc.(ii) Group II : The QP phages.

4. The dsRNA phagesExample: The φ6 bacteriophage.

FAMILIES OF BACTERIOPHAGES (Representative):

NON ENVELOPED ENVELOPEDdsDNA dsDNAMyoviridae:T2 Plasmoviridae:MV-L2

Styloviridae:P2

NON ENVELOPED ENVELOPED

ssDNA ssRNA dsRNAMicroviridae:ØX174 Leviviridae:MS2 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.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 - TMVIn 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.eduThis 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

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 MicrobiologyThe 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.

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(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 AcidsViral 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) andDouble 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 EnzymesSeveral 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'.Such enzymatic activities have not been detected in plant or bacterial viruses, except for the dsRNA viruses of plants.

PLANT VIRUSESPlant 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.

Virion Morphology

Enzyme Product OR Function VirusEnzymes affecting interaction of host cell surface with virionsNeuraminidase.Endoglycosidase.Fusion factor.

NANA split off from surface polysaccharides. Degradation of surface. Modification of lipid bilayer.

Ortho - and paramyxovirus. E. coli phages. Paramyxovirus.

DNA- mRNA transcription enzymesDNA-dependent RNA polymerase. dsRNA transcriptase.ssRNA transcriptase.

Transcribes ss mRNA.Transcribes ss mRNA.Transcribes (+) strand ss mRNA.

Poxvirus, Phages N4, SPO2. dsRNA viruses.ssRNA (+) viruses.

Enzymes adding specific terminal groups to viral mRNA Nucleotide Phosphohydrolase.Guanylyl transferase. RNA methylases.Poly (A) polymerase.

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.

Viruses synthesizing mRNA in virions (e. g. poxviruses and reoviruses).

RNA- DNA transcription enzymes Reverse transcriptase. RNase H (with above).Polynucleotide ligase.

DNA-RNA hybrids; dsDNA. Degrades RNA in RNA-DNA hybrids. Closes ss breaks in dsRNA.

Retroviruses.-do--do-

Nucleic acid replication or processing enzymes DNA-dependent DNA polymerase.DNases (exo- and endo-). Endoribonucleases.

Synthesizes dsDNA.Break DNA strands and crosslinks.mRNA processing.

Hepatitis B.Pox-, retro- adenoviruses.Ss mRNA viruses (e. g: poxviruses).

Other enzymes Protein kinasestRNA aminoaovlases.

Phosphorylate proteins.,Aminoacylate tDNA. Retro-, orthomyxo- paramyxo,herpes- and adenovirusesRetroviruses

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.

N=NAKED, E=ENVELOPED

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.

GENERAL PROPERTIES OF MAJOR GROUPS OF ANIMAL VIRUS

DNA VIRUSES

GROUPNO.OF STRANDS

SYMMETRY N/E SHAPE STRUCTURE SIZE IN NM NUCLEIC ACID

Parvovirus 1 Icosahedron N Spherical 20 Linear ssDNA

Papovirus 2 Icosahedron N Spherical 40-55 Circular dsDNA

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 Circular dsDNA

RNA VIRUSESReovrus 2 Icosahedron N Spherical 80 RNA

Segmented 10-13 molecules

Orbivirus 2 Icosahedron N Spherical 60 RNAPicarnovirus 1 Icosahedron N Spherical 20-30 Plus Strand

RNATogavirus 1 Icosahedron E Spherical 40-70 Plus Strand

RNARetrovirus 1 Icosahedron E Roughly spherical 100 Plus strand

RNA

Orthomyxovirus

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

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 (25-50 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.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 VirusesMembers 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.

Viroids and PrionsViroids: 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 endsDiagram 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 carried on hands or tools during propagation or cultural practices, and by vegetative propagation. No specific insect or other vectors of viroids are known.VirusoidsSimilar 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