ACELLULAR MICROORGANISMS-VIRUSES

Viruses (Latin virus-poison or venom) are unique acellular entities consisting of either DNA or RNA enclosed in a protein coat, and obligate intracellular parasites that only replicate within living cells. Definition of Viruses Viruses are very small, infectious, obligate intracellular molecular parasites, which do not respire, move or grow. The virus genome is composed either of DNA or RNA and directs the viral replication by the synthesis of virion components within an appropriate host cell. Progeny viruses are formed by de novo assembly from newly synthesized components within the host cell. Lwoff (1957) Viruses are infection potentially pathogenic nucleoprotein with only one type of nucleic acid which reproduce from their genetic material and unable to grow and divide and devoid of enzyme. Luria, Darnell (1968) Viruses are entitles, whole genome of which are elements of nucleic acid that replicate inside the living cell using the cellular synthesis machinery and causing the synthesis of special element that can transfer the viral genome to other cell.

Unique properties of viruses--1. Viruses are acellular, neither prokaryotic nor eukaryotic. 2. They are ultramicroscopic structure and they are not seen under ordinary microscope.

3. They are visible under electron microscope. 4. Their size ranging from 20-300nm. 5. Viruses are existing in extracellular as well as intracellular. 6. Do not completely fulfill the characteristic of life. 7. Viruses are very simple in structure; it has nucleocapsid that is protein capsid surrounding genetic material. 8. Genetic material either DNA or RNA may be either single stranded or double stranded. 9. Viruses are metabolically inert because they lack enzyme. 10. Obligate intracellular parasite of bacteria, protozoa, fungi, algae, plant and animals. 11. It utilizes the host protein synthesis machinery and other enzyme for replication. 12. Viruses are not inhibited by antibiotics. 13. They are only inhibited by Interferons. 14. They complete their life cycle either by lytic or lysogenic manner. 15. They have no cytoplasm, no internal organelles. HISTORICAL BACKGROUND In fact prevention of viral diseases came years before the discovery of viruses. Europeans were first protected from viral diseases when Edward Jenner 1798 develops small pox vaccine. Discovery of viruses was made possible with the invention of porcelain bacterial filter in 1884 by Charles Chamberland and tobacco mosaic virus was first to be studied.

The Beginning of Virology The generally recognized beginning of Virology is a paper presented to the St. Petersburg Academy of Science on the 12th February 1892 by Dmitri Iwanowski (18641920), a Russian botanist. He showed that extracts from diseased tobacco plants could transmit disease to other plants after passage through ceramic filters fine enough to retain the smallest known bacteria. Martinus W. Beijerinck father of enrichment culture technique discovered Tobacco mosaic virus 1898.

Six years later in Holland, Martinus Beijernick (1851-1931) confirmed Iwanowski's results on tobacco mosaic virus. He developed with the term "contagium vivum fluidum" ('soluble living germ') as first the idea of the virus. Agents that pass through filters that retain bacteria came to be called ultrafilterable viruses, appropriating the term virus from the latin for "poison".

The same year (1898), the German scientists Friedrich Loeffler (1852-1915) and Paul Frosch, both former students and assistants of Robert Koch (1843-1910), observed that a similar agent was responsible for foot-and-mouth disease. In spite of these findings, there was resistance to the idea that these mysterious agents might have anything to do with human diseases.

Bacterial viruses were first described by Frederick Twort (in 1915;) and

Felix d'Hrelle (in 1917; ). D'Hrelle named them bacteriophages because of their ability to lyse bacteria on the surface of agar plates. Rapidely many scientists utilized these viruses as model systems to investigate many aspects of virology, including virus structure, genetics, and replication. The chemical nature of viruses was established by Wendell M Stanley when he first crystallized the tobacco mosaic virus in 1935. In 1938 Bawden and Pirie separate the tobacco mosaic virus particle into protein and Nucleic acid.

Classification In 1927 Johanson was the first to attempt for the classification of plant viruses. Traditionally viruses have been name according to disease caused by them by adding a suffix virus Polio----poliovirus. In addition the bacteriophage were name after the lab code eg. QB, Qx174,M13, T2,T7 P22

Holmes classification Holmes (1948) used Carolus Linnaeus s system of binomial nomenclature to classify viruses into 3 groups under one order, Virales. They are placed as follows: Order l Suborder ------------------------------------------------------------------------------Phaginae Phytophaginae Zoophaginae Virales

Phaginae Phytophaginae

Attacking on Bacteria Attacking on Plants

Zoophaginae

Attacking on Animals

But this classification is ignored due to some undefined term. In 1962 Andre Lwoff, Robert Horne and Paul Tournier proposed a system of classification of Viruses which is commonly adopted by the Provisional Committee on nomenclature of viruses PNCV formed by the International Association of Microbiologist. LHT based on the

1. Nature of nucleic acid----------------------------DNA/RNA 2. Symmetry------------------------------------------Hedlical, Icosahedral 3. Presence /absence of envelop 4. Diameter of capsid
5

5. Number of Capsomer.

Subphylum
Class OrderFamily Class Orderenvelop)

Deoxyvira

Deoxyhelica (helical) Chitovirales ( envelop) Pox viridae Deoxycubica(cubical) Haplovirales ( no -12 -32 -72 -20 -812 Subphylum Ribovira

Class: Ribohelica (helical symmetry) Order: Rhabdovirales (rod-like viruses) Suborder : Rigidovirales Plant viruses Family: Dolichoviridae 12-13.nm Family :Protoviridae 15 nm Family : Pachyviridae 20 nm

Family: Microvidae capsomeres Family: Parvoviridae Family: Paplloviridae Family: Adenoviridae Family: lridoviridae

Suborder: Flexiviridales Plant viruses Family: Leptoviridae 10-11 nm Family: Mesoviridae 12-13 nm Class Deoxybinala (viruses with head and Family: Adroviridae 15 nm tail) Order Urovirales Family: Phagoviridae (bacteriophages) Order : Sagovirales Family: Myxoviridae Family: Paramyxoviridae Family: Stomatoviridae Class OrderFamily Ribocubica Gymovirales ( envelop) Napoviridae

STRUCTURE OF VIRUS Occurrence:

7

It occur on a very wide host such as plant and animal. They cause very serious diseases in crop plant, ornamental and forest tree resulting in decreased in growth and yield. Morphology: Shape : Viruses are different shapes such as spherical/ cuboid, elongated, flexous or coiled, bullet shaped filamentous. Size: Viruses are of variable sizes range from 10 nm to 300 nm. Viral structure: o The complete assembly of the infectious particle is known as virion. o A virion consist of nucleic acid core surrounded by a protein coat or capsid. The complete set of virion is known as nucleocapsid. In turn nucleocapsid may be naked or enveloped. The envelop is composed of a large number of subunit known as capsomers. Chemically the envelop is made up of protein and glycoprotein. Due to the presenc e of lipid the envelop seen flexible and loosed. Envelop is composed of both host and viral components protein( virus specific and carbohydrate( host specific) There are certain projection of glycoprotein on the envelop known as spikes, where are arranged into distinct units.

 The morphological types of virus observed through electron microscopy and crystallography have been categorized into the following 3 groups.

HELICAL VIRUSES The helical viruses are elongated rod shape rigid or flexible. Their capsid is a hallow cylinder with a helical structure. Capsid consists of monomer arranged helically in rotational axis. Capsid may be naked or enveloped.

1.NAKED VIRUSES One of the examples of naked viruses is the TMV. o For the first timed Stanley 1935 isolated TMV in the crystalline form from the leaf sap of infected tobacco plant. This virus is rod shaped measuring 280 nm X 150 180nm. It consist of a protein tube with a lumen of 20 A which encloses a sshexix of coiled RNA. Protein coat of the virus contain a number of identical subunit which are arranged in helical manner. Capsid consist of several capsomer each compose of a few monomer. 49 monomer( each with mol. Wt. 12000) take three turn of the helix and give total 2130 subunit of a rod. Each subunit is made up of 158 amino acid residue forming single polypeptide chain. Genetic material is ssRNA.

10

2 ENVELOP VIRUSES: When the helical viruses are enclosed with in an envelop they are known as enveloped helical viruses --- influenza virus. The envelop is composed of a viral protein and host cell component lipid and carbohydrate.. The envelop consist of numerous spikes. The helical capsid exist infolded form inside the envelop.

11

POLYHEDRAL ( ISOHEDRAL) There are several animal, plant and bacterial virus which have either naked or enveloped icosahedral shape. o Polyhedral structure has the 3 possible symmetry such as tetrahedral, octahedral and icosahedral. o Viruses are more or less spherical, therefore icosahedral symmetry is the best one for packaging and bonding of subunit. o Several intra molecular bond of low energy is formed. o An icosahedraron is a regular polyhydron with 20 triangle faces and 12 corner. o The capsomer of each faced forms an equatorial triangle and 12 intersecting point.

12

 There are two types of capsomer one is pentamer and other is hexamer 12 capsomer in OX 174

32 capsomer in polio virus 162 capsomer in herpes virus 252 capsomer in adeno virus NAKED VIRUS: Naked are turnip yellow mosaic viruses, polio virus. Adenovirus are large 80 nm icosahedral contain ds DNA. The capsid has the rugid like capsomer each containing pentamer and hexamer. Total 32 hexamer and 12 pentamer are found in capsid. Hexamer are polygonal disc of 8 nm diameter with hole of 2.5 nm diameter in centre. Surface of capsid these spike like structure form 12 point.

Envelop:
13

Icosahedral viruses for example herpes virus capsid is enclosed inside the envelop of 30 nm thickness that is made up of glycoprotein lipid complex. The envelop consist of spikes on its surface capsid is spherical and of about 100nm diameter enclosed dense core of dsRNA

COMPLEX SYMMETRY: Complex viruses are divided into two groups those without identifiable capsid and those with capsid to which other structure attached. 1. Vaccinia virus is an example of a v irus without a define capsid. The nucleic acid is surrounded b y several coats. 2. Bacteriophage of T even series( T2,T4,T6) are example of complex viruses with capsid attached

14

3. Structure of T4 bacteriophage. The head capsid has the form of a prolate icosahedrons. The tail bacteriophage show a combination and helical symmetry. The head capsid has the form of prolate icosahedrons. The head contain DNA. Head tail connectorThe head tail connector has a collar Tail _ The tail consist of contractile sheath surrounding an inner core. It is connected to the collar at upper end and the base plate at the lower end. The base plate- The base plate is hexagonal and has a tail pin. Tail fibre--- from corner of the base plate is along tail fibre. The tail fibre recognize specific receptor site on the host cell wall during attachment. CULTIVATION OF VIRUSES Viruses are obligate intracellular parasites. They cannot grown on any laboratory culture medium. It requires the living cell for its growth and multiplication. Three type viruses Bacteriophage

15

 Animal viruses Plant viruses

CULTIVATION OF BACTERIOPHAGE The cultivation of bacteriophage is the simplest and can be done in the laboratory by first cultivating the bacteria. On the ordinary laboratory media. The nutrient media is seeded with the required bacteria and a confluent growth is obtained in a peteiplate after incubating it for 24 hour. Simultaneously the viruses that infect the particular bacteria are also added in it. Observation: After incubation the confluent growth of bacteria with intermit ant clear zone seen. These clear zone are produced due to the bacteriolysis with the infected virus and are known as plaque. The bacteriophage can also be cultivated in the nutrient broth. First of all the bacteria ( E.coli) is inoculated into nutrient broth which is then incubated at 37 C for 24 hours to get maximum growth or turbidity. Now the virus infecting the bacteria (lambda phage) is inoculated into the nutrient broth containing E.coli and further incubated for 24 hours. The growth of viruses is indicating by the clearing of the turbidity by bacteriolysis with the infected virus. The cells that are killed get settle at the bottom.

CULTIVATION OF ANIMAL VIRUSES The animal viruses can be cultivated by either of three method.

16

CULTIVATION IN EMBRYONATED EGG The embryonated hen egg was first used for the cultivation of viruses by Good Pasture in 1931 and the method was further developed by Burnet. The embryonated egg offer several sites for the cultivation of viruses. The growing chick embryos are utilized for this purpose and the incubating at 35 C. The route of inoculation is important and inoculum may be introduced by the following routes.

17

CULTIVATION IN CHORIOALLANTOIC MEMBRANE Chorioallantoic membrane is the respiratory organ richly supplied with blood. Being easily approachable through the shell it is commonly used.

For cultivation of viruses--The egg is incubated for 12 days. After this circular part is marked the egg where Chorioallantoic is well developed. A hole is made at the centre of air sac at the top. The piece of the shell is removed without disturbing the shell membrane and a drop of saline is put on it to exposed membrane and then with the pipette or syringe 0.05ml of inoculum is introduced on the Chorioallantoic membrane and the egg is incubated at the optimum temperature for as long as necessary for virus to be cultivated. The shell is cut with scissor and the Chorioallantoic membrane is removed in buffer saline in petri plate and checked for viruses.

CULTIVATION IN AMNIOTIC CAVITY For this the egg is incubated for 30 days and the point is marked where the embryo is well developed. A hole is made in the air sac. The half of the rectangular are is drilled out without disturbing the shell membrane. the Chorioallantoic membrane is pulled and 0.05 to 0.25 ml of inoculum is injected by syringe into the amniotic cavity. The CAM is then allowed to fall down to its original place. After inoculation egg is incubated for 3 to 5 days. The egg is removed when the embryo is killed. The amniotic membrane is cut and fluid is drained out and is examined for the virus. CULTIVATION IN ALLONTIC CAVITY
18

For this the egg is incubated for 10-11 days. A point is marked at the distinct part where Chorioallantoic is well developed. Similar hole is made in air sac and 0.1 to 0.2 ml of inoculum is injected by syringe to a depth of 2 mm and then the opening is sealed with transparent tape and egg is removed shell is cut with scissor and allantoic fluid is drained with a capillary pipette and examined for the virus.

CULTIVATION IN YOLK SAC For this egg is incubated for 5-9 days. After incubation a hole is made in the centre of air sac and the inoculation is made by the using syringe directly into the yolk sac. The egg is then incubated, the yolk sac is removed when embryo dies and is examined for the virus. Yolk sac inoculation is used for the cultivation of some viruses Diagram

CULTIVATION OF VIRUSES IN ANIMAL

19

The earliest method for the cultivation of virus causing human diseases was inoculation into human ,volunteer. Due to the risk involved human is available and when the virus is relatively harmless. Monkey were used for the isolation of poilo virus by Landstainer and Popper 1909 but due to their cost and risk to handler monkey fluid only limited application in virology. This use of white mice pioneered by Theiler 1903 extended the scope of animal inoculation greatly mice still the . Most widely employed animals in virology. Mice may be inoculated by several routes: 1. Intra cerebral 2. Sub cutaneous 3. Intra peritoneal 4. Intra nasal.

Tissue culture: Cultivation of bits of tissues and organs in vitro had been used by physiologists and surgeons for the study of morphogenesis and wound healing. The first application of tissue culture in virology was by Steinhardt and colleagues (1913), who maintained the vaccinia virus in fragments in rabbit cornea. Maitland (1928) used chopped tissue in nutrient media for cultivation of vaccinia viruses. The turning point which made tissue culture the most important method for cultivation of virus was the demonstration by Enders, Weller and Robins (1949), that poliovirus can be grown in tissue culture in non-neural origin. There are three types of tissue cultures: 1) Organ culture: Small bits of organs can be maintained in vitro for days and weeks, preserving their original architecture and function. Formalin is used for the preservation. Organ culture is useful for the isolation of some

20

viruses which appear to be highly specialized parasites of certain organs. Example: Tracheal ring organ culture is employed for the isolation of corno virus, a respiratory pathogen. 2) Explant culture: Fragments of minced tissues can be grown as explants embedded in plasma clots. They may also be cultivated in suspension. This was originally called as tissue culture. This method is now seldom employed in virology. Example: Adenoid tissue explant culture was used for the isolation of adenovirus. 3) Cell culture: The cell culture is the method routinely employed nowadays for identification and cultivation of viruses. Cells of various types of tissues of animals may be cultivated. But more commonly, fibroblast and muscle epithelial cells are used for the propagation of virus.

Types of cell cultures: On the basis of origin, chromosomal characters, and the number of generations through which they can be maintained, cell cultures are classified in three types. 1) Primary cell culture: 2) Diploid cell culture: 3) Continuous cell culture:

DETECTION OF VIRAL GROWTH Detection of viral growth in cell culture : Virus growth in cell culture can be detected by the following methods. 1. Cytopathic effect:

21

Many viruses causes morphological changes in cultured cell in which they grow. These changes can ve examination of the cultures. readily observed by microscopic

These changes are known as cytopathic effect and viruses causing CPE produce d by different groups of viruses are characteristics and help in the presumptive identification of virus isolates Eg. Adenovirus produce resembling bunches of grapes. 2. Metabolic inhibition: In normal cell the medium turns acid due to cellular metabolism. When virus grow in cell culture cell metabolism is inhibited and there is no acid production . this can be made out by the color of the indicator( phenol red) incorporated in the medium . large granular clump

3. Heam adsorption: When heamagglutinating viruses such as influenza and para influenza virus grow in the cell culture their presence can be indicated by the addition of Guinea pig RBC to the culture. The viruses are multiplying in the culture the RBC will adsorbed on to the surface cell this is known as heamadsorption. 4. Interference: The growth of a non-cytopathogenic virus in cell culture can be tested by the subsequent challenges with a known cytopathogenic virus. The growth of first virus will inhibit infection by the second virus by interference. 5. Transformation:
22

Tumor forming (oncogenic) viruses induce cell transformation and loss of contact inhibition so that growth appear in a piled up fashion producing microtumors. 6. Immunofluorescence: Cell from virus infected cultures can be stained by fluorescent conjugated antiserum and examined under the UVmicroscope for the presence of viruses antigen. This give positive result. 7. Heam agglutination: It is test used to detect viral growth and also aid in positive diagnosis of particular viruses. Eg influenza, mump etc. These viruses agglutinate human RBC and chick RBC.

BACTERIOPHAGES In 1915 for the first time Frederick W. Twort in England observed that bacterial colonies sometime were lysed. The lytic effect was transmitted from on e colony to the other. The transmission of lytic effect continued even after the dilution and passing them through bacterial filter. However after heating the solution destroyed. the lytic effect was

23

 He thought that the lytic effect would have been done due to the virus. In 1917 DHerelle at pasture institute rediscovered this phenomenon i.e. Twort-DHerelle phenomenon and coined the term bacteriophage. T4 BACTERIOPHAGE There are 3 even number T-phages ( T2 T4 and T6) and 4 odd numbered T phage that infect E.coli. Out of the 7 coliphage of T-series the virulent T4 phage is most studied. Morphology: The T4 phage is tadpole shaped and consist of the five important sub structure such as the head, head tail connector, tail plate, and fibers. The viral particles is naked icosahedral and tailed.

Head: The head is elongated bipyramidal, hexagonal like prism consisting of two 10 faces equatorial bands. It consists of pyramidal vertex at either end. Size of head is 95 x 65 nm which consist of about 2000 identical protein subunit capsomer.

Tail:

24

The phage consist of a long helical tail which is connected to the head with a connector having a color with attached whisker. Around tail , fibre remain folded and held at mid point by the whiskers. Size of the tail is 80 x 18 nm. It consist of an inner tubular core which is surrounded by a contractile sheath. Protein subunit( 144 arranged in 24 ring each consisting 6 subunit constitute the sheath. The sheath connect the head at one end and base plate at the other end. Base plate: From the head at distal end there is hexagonal base plate attached to an end of tail. The base plate contains six spikes or tail fibre at its six corners.

Spikes: . The spikes are 130 nm x 2 nm in size. The spikes have two part, the proximal half fibre and the distal half fibre. The proximal and the later help in recognition of specific receptor site present on the cell surface of the bacterial cell wall. Nucleic ac id: A ds DNA molecule of about 50 micro meter is tightly packed inside the head. The DNA is about 1000 time larger than the phage itself. It is circular and terminally redundant.

25

26

LIFE CYCLE OF BACTERIOPHAGE Bacteriophage exhibits two different types of life cycle1. Lytic or Virulent cycle 2. Temperate or Avirulent or Lysogenic cycle: Lytic or Virulent cycle: In virulent cycle there is intracellular multiplication of phage followed by the lysis and release of progeny virion. This is called lytic cycle

Temperate or Avirulent or Lysogenic cycle: In Lysogenic cycle the phage DNA becomes integrated with the bacterial genome, replicating without any cell lysis. LIFE CYCLE: Multiplication of Bacteriophages The replication of virulent phage was initially using T even numbered (T2,T4,T6) phage of E.coli. The multiplication cycle of phage occur in five steps1. Attachment or Adsorption 2. Penetration 3. Biosynthesis of phage component 4. Maturation 5. Release of progeny phage particle

Attachment or Adsorption: The first step in infection of host bacterial cell by phage is adsorption.
27

 Phage particle come into contact with bacterial cell by random collision. A phage attaches to the surface of the bacterium by the tail. Adsorption depends on the presence of chemical group called as receptor on the surface of bacterial cell. The receptor of bacterial cell is a lipopolysaccharide. Host specificity of phage its affinity at the adsorption. Infection of bacterium by naked phage genetic material is known as transfection.

Penetration: Attachment is followed by injection of genetic material (nucleic acid /DNA) in to the bacterial cell . The phage DNA is injected into the bacterial cell through the hollow core. Penetration may be enhanced by the presence of phage tail lysozyme with break small portion of the cell wall for the entry of phage DNA. After penetration of DNA the empty head and tail of phage remain outside the bacterial cell is called shell. If many phages are attached to the bacterial cell multiple holes are produced on the bacterial cell with the consequent leakages of cell component. Bacterial lysis occurs without viral multiplication. Phage such as T1 and T5 that do not have contractile sheath also inject their nucleic acid through the cell envelop by adhesion site between the inner and outer membrane.
28

Biosynthesis of phage component: After the infection and penetration of DNA transcription of part of viral genome produce early mRNA molecules which is translated into a set of early protein. These cause the switch off host cell macromolecule synthesis, degrade the host DNA/ chromosome and start the synthesis of viral components. Viral DNA replicate and also produce the late mRNA molecule transcribe from gene which specify the protein of phage coat. The late messages are translated into subunit of capsid. Rest of the structure gets condensed to form phage head, tail and tail fibre. Maturation: The phage DNA, head and tail protein are synthesized separately in the bacterial cell. DNA condense into compact polyhedron and packaged into head and finally the tail structure are added. The process of assembly of the phage from its component is called maturation. Release of progeny phage particle By the sudden explosion or breakaging the bacterial cell wall. o Lysozyme synthesized within the cell caused the bacterial cell wall to breakdown and newly produced Bacteriophage are release from the host cell

29

Lysogenic cycle: Lysogeny is a process in which the viral nucleic acid does not usurp the functions of the host bacterium's synthetic processes but becomes an integral part of the bacterial chromosome. As the bacterium reproduces, viral nucleic acid is transmitted to the daughter cells at each cell division. In the lysogenic state the virus is simple one of the bacterial genes. Under certain natural conditions or artificial stimuli (such as exposure to ultraviolet light), the synthesis of virus may, take over, and lyses occurs

30

Step involved in lysogenic cycle 1 2 3 4 5 Attachment injection of phage DNA circularization of Phage DNA Host chromosome Binnary fission-prophage Excision of prophase

31

ATTACHMENT

REPLICATION
NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNN

PENETRATION

Lysogenic Cycle

nnn nnn nnn nnn nnn nnd ddd

INTEGRATION

32

Cells and Viruses Characteristic Structure Cell Cell membrane, cytoplasm; eukaryotes also contain nucleus and organelles Independent cell division either asexually or sexually DNA and Yes; in multicellular organisms, cells increase in number and differentiate Use yes Virus

Reproduction

Genetic Code Growth Development

Obtain Energy

and

Response Environment Change Over Time

to yes

yes

33

34