THE TOPIC IS PRESENTED BY FATIMA KHAN:

ROLL NO:-11021514-007

TOPIC:-

CULTIVATION & STRUCTURE OF VIRUSES:

VIRUSES:

DEFINITION:-

“Viruses are simple acellular entities of one or more molecules of either DNA or RNA.They are reproduced only within living cells & are obligatory intracellular parasites”

Viruses lead a kind of “borrowed life”

Viruses exist in two phases:

Extra cellular phase:- in this phase ,they posses few enzymes .Virions are regarded as extracellular phase of virus ,they take viral genome from cell to cell & protect the genome in inhospitable environment to which virus cannot replicate.

Intra cellular phase:-in this phase virus exist as replicating nucleic acid that induce host metabolism to synthesize its components &eventually complete virus particles are released.

GENERAL PROPERTIES:-

Viruses differ from other living cells by at least two ways:

1. Their simple , acellular organization.2. Their inability to reproduce independently of host cells & carry

out cell division as prokaryotes & eukaryotes do.

CULTIVATI ON:-Detection of viruses---------disease identification:-

Diseases like flu & cold are viral diseases & are easily diagnosable[straight forward diagnosis].

Koch`s postulate are applicable to bacteria & other pathogens but for viral detection these postulates are non-applicable because like bacteria ,viruses are non-cultureable.So to resolve this problem THOMAS M. RIVER`S expanded Koch`s postulates to include virus.

“filtrates of infectious material shown not to contain bacterial or other cultivable organisms must produce specific antibodies in appropriate animals”

Virus cultivation using an EMBRYONATED EGGS:-

for many years ,researchers have cultivated animal viruses by inoculating suitable host animals or embryonated eggs.To prepare the eggs for virus cultivation ,the shell is first disinfected with iodine & penetrated with small sterile drill. viruses may be reproduce in certain parts of embryo eg: myxoma virus grows well on chorioallantoic membrane ,whereas mump virus prefer alantoic cavity.After inoculation ,hole is sealed by gelatin.The infection cause a local lesion known as pock,whose appearance is characristics of virus.

Virus cultivation of MONOLAYERS OF ANIMAL CELLS:-

Animal viruses have been grown in tissue culture on monolayers of animal cells.This is made possible by development of growth media for animal cells & by advent of antibiotics that can prevent bacterial & fungal contaimination.A layer of animal cells & viruses inoculum is covered on petri dish & viruses are allowed time to settle & attach to cells.Then cells are covered by thin layer of agar to limit virion spread so that only adjacent cells are infected by newly produced virions. As a result localized areas of cellular destruction & lysis called plaques are formed. Some cytological techniques may be used in clinical laboratory for identification of viruses from sample of patients.

CYTOPATHIC EFFECT:- “when viruses replicate in host cells often a noticeable detorition or structural changes occurs , this is called cytopathic effect”

PLAQUES:-

CYTOPATHIC EFFECTS :-

AGAR CULTURE OF BACTERIOPHAGES:- Bacterial cells are cultivated in both broth & agar culture containing young & active growing cells. Due to lysis so many cells are destroyed that turbidity of bacterial cells become clear.Agar cultures are prepared by mixing bacteriophages sample with cool,liquid agar & suitable bacterial culture.this mixture is poured in petri dish & after hardening ,bacteria in top of agar grow & reproduce forming a lawn.When virion come on top layer ,it infect adjacent host cells & lysis result in clearing of lawn(plaque).

NORMAL MAMMALIAN CELLS:-

APPEARANCE OF TISSUE CULTURE CELLS:-

PHAGE PLAQUE (ON LAWN OF E.COLI):

PLANT VIRUSES CULTIVATION:-

Plant viruses are cultivated in variety of ways.Plant tissue culture, culture of separated cells , or culture of protoplast may be used.Virus can be grown in whole plant.leaves are mechanically induced by viruses through rubbing. A localized NECROTIC LESION ,may developed due to rapid death of cells in infected area. Besides lesions pigments may

appear. Necrotic lesions on plant:-

T1 phage T2 phage

T3 phage T4 phage

STRUCTURE OF VIRUSES:-

SIZE:- In 1950`s TMW & other viruses were finally identified.Viruses ranges in sizw from 17-300 nm in diameter.Smallest virus is 17nm in

size (the size of ribosomes) & largest is 1000nm(the size of smallest bacteria).Viruses are barely visible to light microscope & mostly are visible via EM.

GENERAL STRUCTURAL PROPERTIES:-

1. NUCLEOCAPSID CORE:- The nucleocapsid is composed of nucleic acid either DNA or RNA,held within a protein coat called capsid,which protect viral genetic material & aid in transfer b/w host cells.The capsid surrounds the virus & is composed of finite no. of protein subunits called “capsomers” which usually associate with close to nucleic acids.

TYPES OF CAPSIDS:- There are following types of capsids:-

A- ISOCAHEDRAL CAPSID:-

The capsid of most isocahedral viruses is in the shape of a regular polyhedron with 20 triangular faces and 12 corners. The capsomers of each face form an equilateral triangle. An example of a isocahedral virus is the adenovirus. Another is the poliovirus.

B-HELICAL CAPSID:-

Other capsids are helical and shaped like hollow protein cylinders, which may be either rigid or flexible.

Eg: Tobacco mosaic virus.

C-VIRUSES WITH ENVELOP:-

Many viruses have an envelope” an outer membranous layer surrounding the nucleocapsid.”Enveloped viruses have a roughly spherical but somewhat variable shape even though their nucleocapsid can be either icosahedral or helical.

Eg: Influenza virion, herpes & HIV virus.

D-VIRUSES WITH COMPLEX CAPSIDS:-

Have capsid symmetry that is neither purely icosahedral nor helical.They may possess tails and other structures (e.g., many bacteriophages) or have complex, multilayered wall surrounding

the nucleic acid (e.g., poxviruses such as vaccinia).

PROTOMERS:-

“Subunits of proteins which aggregate to form capsomers & which in turn aggregate to form capsid.”

OUTER COVERING (COAT):-

HELICAL CAPSIDS:- These capsids have shape like hollow tube with protein walls.eg:- TMV. A single type of protomer associate together in helical or spiral arrangement to produce a long, rigid tube, 15-18 nm in diameter & 300 nm long. The RNA genetic material is wound spirally & toward inside of capsid. Not all helical capsids are rigid & some are flexible too. Eg : capsids of influenza, is enclosed in thin flexible envelop.

ISOCAHEDRAL CAPSIDS:-

It is nature`s most favourite shape because it is most efficient way to enclose a space.Hexagons & proteins Pentagons are used for construction purposes.As we know capsomers are made up of protomers(five/six subunits).

CAPSOMERS

Pentamer hexamer

(pentone with 5 subunit) (hexone with 6 subunit)

The icosahedron of adeno virus is constructed of 42 capsomers; larger icosahedra are made ,if more hexamers are used to form the edges and faces .

NUCLEIC ACIDS:-

Viruses are exceptionally flexible with respect to the nature of their genetic material. They employ all four possible nucleic acid types:

a) single-stranded DNA,

b) double-stranded DNA,

c) single-stranded RNA,

d) double-stranded RNA.

All four types are found in animal viruses.Plant viruses most often have single-stranded RNA genomes.The size of viral genetic material also varies greatly. The smallest genomes (those of the MS2 and QB viruses) are around 110 daltons, just large enough to code for three to four proteins. At the other extreme, T-even bacteriophages, herpesvirus, and vaccinia virus have genomes of 1.0 to 1.6 × 10 ^8 daltons and may be able to direct the synthesis of over 100 proteins.

DNA VIRUSES:-

ss linear & circular DNA:-

Tiny DNA viruses like φX174 and M13 bacteriophages or the parvoviruses possess single-stranded DNA (ssDNA) genomes. Some of these viruses have linear pieces of DNA, whereas others use a single, closed circle of DNA for their genome.

ds linear & closed DNA:-

Most DNA viruses use double-stranded DNA (dsDNA) as their genetic material. Linear dsDNA, variously modified, is found in many viruses; others have circular dsDNA.

The lambda phage has linear dsDNA with cohesive ends—single-stranded complementary segments 12 nucleotides long—that enable it to cyclize when they base pair with each other.

ss RNA virus:-

Most RNA viruses employ single-stranded RNA (ssRNA) as their genetic material.

The RNA base sequence may be identical with that of viral mRNA, in which case the RNA strand is called the plus strand or positive strand (viral mRNA is defined as plus or positive).

Positive-sense (5' to 3') viral RNA signifies that a particular viral RNA sequence may be directly translated into the desired viral proteins.

Therefore, in positive-sense RNA viruses, the viral RNA genome can be considered viral mRNA, and can be immediately translated by the host cell.

However, the viral RNA genome may instead be complementary to viral mRNA. and then it is called a minus or negative Negative-sense (3' to 5') viral RNA is complementary to the viral mRNA and thus must be converted to positive-sense RNA by an RNA polymerase prior to translation. Negative-sense RNA (like DNA) has a nucleotide sequence complementary to the mRNA that it encodes.

Polio, tobacco mosaic, brome mosaic, and Rous sarcoma viruses are all positive strand RNA viruse.

rabies, mumps, measles, and influenza viruses are examples of negative strand RNA viruses.

Many of these RNA genomes are segmented genomes,that is, they are divided into separate parts.

It is believed that each fragment or segment codes for one protein.

Usually all segments are probably enclosed in the same capsid .

Many animal viruses, some plant viruses, and at least one bacterial virus are bounded by an outer membranous layer called an envelope.

animal virus envelop.

projection of proteins in the form of spikes.

Pleomorphic variable shapes of virus due to flexible & membranous envelop.

bullet-shape rabbies virus have constant characteristics shape due to rigid envelop.

INFLUENZA VIRUS:-

VIRAL ENZYMES:-

Are located within the capsid, or even on capsid & envelop.

Mainly involved in nucleic acid replication.

e.g: the influenza virus uses RNA as its genetic material and carries an RNAdependent RNA polymerase that acts both as a replicase and as an RNA transcriptase that synthesizes mRNA under the direction of its RNA genome.

VIRUSE WITH COMPLEX SYMMETRY:- pox viruses & bacteriophages are examples of complex symmetry viruses.pox viruses have intracellular envelop covering the nucleocapsids & bacteriophages have tail structure present.They posses two type of symmetry i.e; icosahedral (on head) & helical (on tail).

pox viruses:-

bacteriophages:-