MCB 204 :

INTRODUCTORY VIROLOGY

(3 Units)

OUTLINEIntroduction.General characteristics of viruses to include plant, animal and bacterial virusesViral replication, spread and cytopathic effects.Virus classificationPurification and assay of viruses.Regulation of lytic development and maintenance of the Lysogenic state in bacteriophagesLambda, P2 and 14 single stranded DNA and RNA phage viroids as pathogens.Principles of isolation, cultivation and maintenance of plant and animal cells in vivo.Application of cell culture technique in virology.Viruses as agents of diseases in animals.

Introduction.• A virus is a small, infectious agent that is made up of

a core of genetic material surrounded by a shell of protein with size ranging from 10 - 400 nm.

• Viruses are found wherever there is life and have probably existed since living cells first evolved

• The origin of viruses is unclear because they do not form fossils.

• Molecular techniques have been used to compare the DNA or RNA of viruses and are a useful means of investigating how they arose.

• There are three main hypotheses that try to explain the origins of viruses

• Regressive hypothesis • Viruses may have once been small cells that

parasitised larger cells.• Over time, genes not required by their

parasitism were lost. • This is also called the degeneracy hypothesis

or reduction hypothesis. • Cellular origin hypothesis • Some viruses may have evolved from bits of

DNA or RNA that "escaped" from the genes of a larger organism.

• The escaped DNA could have come from plasmids (pieces of naked DNA that can move between cells) or transposons (molecules of DNA that replicate and move around to different positions within the genes of the cell).

• Once called "jumping genes", transposons are examples of mobile genetic elements and could be the origin of some viruses.

• They were discovered in maize by Barbara McClintock in 1950.

• This is sometimes called the escape hypothesis.

Coevolution hypothesis • This is also called the virus-first hypothesis and

proposes that viruses may have evolved from complex molecules of protein and nucleic acid at the same time as cells first appeared on earth and would have been dependent on cellular life for billions of years.

General characteristics of viruses to include plant, animal and bacterial viruses

• The genetic material may be either deoxyribonucleic acid (DNA) or ribonucleic acid (RNA).

(which is responsible for carrying forward hereditary traits from parent cells to offspring)

• Viruses are at the borderline between living and nonliving matter.

• When they infect a host cell, they are able to carry on many life functions, such as metabolism and reproduction.

• But outside a host cell, they are as inactive as a grain of sand. ( can exist in two phases – Extracellular form ‘crystal’, - Intracellular ‘ active nucleic acid’

• Viruses cause disease by infecting a host cell and taking over its biochemical functions.

• In order to produce new copies of itself, a virus must use the host cell's reproductive "machinery.“

• The newly made viruses then leave the host cell, sometimes killing it in the process, and proceed to infect other cells within the organism.

• Viruses can infect plants, bacteria, and animals.

• Animal viruses cause a variety of diseases, including AIDS (acquired immuno deficiency syndrome), hepatitis, chicken pox, smallpox, polio, measles, rabies, the common cold, and some forms of cancer.

• Viruses that affect bacteria are called bacteriophages, or simply phages (pronounced FAY-jez). Phages are of special importance because

• The tobacco mosaic virus, one of the most studied of all viruses, infects tobacco plants.

Characteristic of plant Viruses• All plant viruses are RNA viruses but their

some exception to this rule• Penetration of plant viruses is usually

hindered by the fact that plant are protected by complex outer layers including cell walls.

• Entry of a plant virus into its host requires presence of mechanical damage to the cell wall.

• Plant viruses gain entrance to their host through lesions

• They develop necrotic spot around the primary lesion or bruised area

• Plant viruses are spread by vectors e.g Aphids.

• They enter into the leaf tissue through injures

• The virus multiplies in the alimentary tract of the insect.

Some human diseases caused by viruses• AIDS • chicken pox • colds • Colorado tick fever • encephalitis • fever blisters • genital warts • gastroenteritis • genital herpes • German measles • hepatitis • influenza • leukemia • liver cancer • measles • mumps • oral herpes • polio • rabies • smallpox • virus hemorrhagic fever • yellow fever

Structure of viruses • Although viral structure varies considerably among

different types of viruses, all viruses share some common characteristics.

• All viruses contain either RNA or DNA surrounded by a protective protein shell called a capsid.

• The genetic material in a virus may take one of four forms: a double strand of DNA, a single strand of DNA, a double strand of RNA, or a single strand of RNA.

• The size of the genetic material of viruses is often quite small. Compared to the 100,000 genes that exist within human DNA, viral genes number from 10 to about 200 genes

•

• Viruses exist in one of three forms, as shown in Figure below;

• They are named on the basis of their general shape as rodlike, icosahedral (having 20 sides), or spherical.

• Some viruses also have an outer covering known as an envelope that surrounds the capsid.

• The outer surface of some kinds of viral particles contain threadlike "spikes" that are often used in helping a virus invade a host cell (for example, the spherical virus in Figure 1).

• Helical• These viruses are composed of a single type of capsomer

stacked around a central axis to form a helical structure, which may have a central cavity, or hollow tube.

• This arrangement results in rod-shaped or filamentous virions:

• These can be short and highly rigid, or long and very flexible.

• The genetic material, in general, single-stranded RNA, but ssDNA in some cases, is bound into the protein helix by interactions between the negatively charged nucleic acid and positive charges on the protein.

• Overall, the length of a helical capsid is related to the length of the nucleic acid contained within it and the diameter is dependent on the size and arrangement of capsomers.

• The well-studied tobacco mosaic virus is an example of a helical virus.

• Icosahedral• Most animal viruses are icosahedral or near-spherical

with icosahedral symmetry.• A regular icosahedron is the optimum way of forming

a closed shell from identical sub-units• The minimum number of identical capsomers required

is twelve, each composed of five identical sub-units.• Many viruses, such as rotavirus, have more than

twelve capsomers and appear spherical but they retain this symmetry.

• Capsomers at the apices are surrounded by five other capsomers and are called pentons.

• Capsomers on the triangular faces are surrounded by six others and are call hexons

Complex

Structure of viruses

Complex• These viruses possess a capsid that is neither purely

helical nor purely icosahedral, and that may possess extra structures such as protein tails or a complex outer wall.

• Some bacteriophages, such as Enterobacteria phage T4, have a complex structure consisting of an icosahedral head bound to a helical tail, which may have a hexagonal base plate with protruding protein tail fibres.

• This tail structure acts like a molecular syringe, attaching to the bacterial host and then injecting the viral genome into the cell.

• The poxviruses are large, complex viruses that have an unusual morphology.

• The viral genome is associated with proteins within a central disk structure known as a nucleoid.

• The nucleoid is surrounded by a membrane and two lateral bodies of unknown function

• . The virus has an outer envelope with a thick layer of protein studded over its surface.

• The whole virion is slightly pleiomorphic, ranging from ovoid to brick shape.

• Mimivirus is the largest known virus, with a capsid diameter of 400 nm.

• Protein filaments measuring 100 nm project from the surface. The capsid appears hexagonal under an electron microscope, therefore the capsid is probably icosahedral.

• Envelope• Some species of virus envelop themselves in a

modified form of one of the cell membranes, either the outer membrane surrounding an infected host cell or internal membranes such as nuclear membrane or endoplasmic reticulum, thus gaining an outer lipid bilayer known as a viral envelope.

• This membrane is studded with proteins coded for by the viral genome and host genome; the lipid membrane itself and any carbohydrates present originate entirely from the host.

• The influenza virus and HIV use this strategy. Most enveloped viruses are dependent on the envelope for their infectivity.

Viral Genome• Viruses are exceptionally flexible with respect

to the nature of their genomes.• There are 4 possible nucleic acids, which are: Single stranded DNA (ssDNA) Double stranded DNA (dsDNA) Single stranded RNA (ssRNA) Double stranded RNA (dsRNA)

• All four types are found in Animal viruses but most Plant viruses have single ssRNA genome and most bacteria viruses contain dsDNA

• The genome may be linear or circular.• Some switch from linear to circular forms• dsRNA are known to infect animals, plants,

fungi and at least one bacteria species• ssRNA are more common

Replication of Viruses

• Virus replication take place only in living cells and their replication is usually in two ways

• Lytic pathway• Lysogenic pathway• Virus replication is facilitated by the host cell

which provides the required energy and synthetic machinery for the replication.

Lysogenic Pathway• Many DNA phages are called TEMPERATE PHAGES• They have 2 replicative options.• They replicate lytically as a virulent phages or they

can remain within the host without destroying it.• They accomplish this by integrating their genome

into the host chromosome.• The relationship between a temperate phage and

its host is called LYSOGENY.• The form of virus that remains within its host is

called PROPHAGE.

Spread of VirusesViral pathogenesis refers to the processes and

event that lead to disease production following an interaction between a virus and the host

Infection by a virus does not always lead to disease

Virus enters the host through breaks in the skin, mucus membrane, the oral cavity , the respiratory tract and the urethra

Whether or not the host would be susceptible to the infection depends on certain host and viral factors

Host Factors Immune status of the host: Good immunity

prevent the virus from reaching the target organs.

Viral infections are more severe in immunosuppressed than in immuno competent individual.

Age of the host: In general the very young and the very old are more susceptible to virus infection

Partly because of the immaturity of the immune system in a very young and the weakening of the immune system in the very old.

Presence or Absence of cellular receptors: Virus infection of a cell can only take place only if the cells have receptors for the virus

The general health of the host: Including nutritional status

Host that are in poor health or malnourished are more

susceptible to viral infection.

Viral Factors Virulence: This refers to the disease producing ability of

the virus A virulent strain of a virus is more likely to produce

disease in the host. A non virulent strain may not produce disease or may

produce mild disease

The size of the inoculums: Exposure to a high infective dose of a virus is more likely to result to disease than low dose

Route of entry: Entry of viruses into a host by certain routes may not result into infection e.g

Most of the enveloped viruses that enter the host via the mouth are destroyed by digestive enzymes and can not produce infection.

CYTOPATHIC EFFECTS

• A cytopathic effect is an observable morphological change that occurs in cells because of viral replication. E.g

• The swelling of cells, binding together of cells following a lytic replication of a virus etc.

• The result is usually death of the cell which is referred to as CYTOCIDAL INFECTION/ CELL DEATH

Possible mechanism of host cell damage

• Many viruses can inhibit host DNA and protein synthesis

• Cell endosome may be damaged resulting in the release of hydrolytic enzymes and cell destruction

• Viruses can alter plasma membrane• High concentration of proteins from several

viruses can have a direct toxic effect on cells and organisms

• Intracellular structures called inclusion bodies are formed during virus infections , These inclusion bodies can disrupt cell structure

• Chromosomal disruptions result from viral infection

• The infected cell which eventually causes cancer

CLASSIFICATION OF VIRUSES

• Viruses are not considered to be strictly living, and their classification is complex issue

• As with true organisms we have species, genera, families and orders of viruses

• But none of the higher grouping ( Class, Phylum and Kingdom) familiar from convectional biological taxonomy, are not used for viruses

• A proposal of non- Latinised viral binomials has been proposed

• Factors taken into account in the classification of viruses include:–Type of host– Pathogenecity (type of disease caused by

infection)–Ecological characteristic–Recently sequencing of viral genomes has

meant that insight are being gained in this area (Baltimore)–Physiochemical characteristic

TYPE OF HOST • Plant ,Animals or Bacteria

• PATHOGENECITY• Viruses affecting same tissues and producing

similar symptoms are grouped together• Examples : Influenza, Rinoviruses,

Adenoviruses are grouped together as viruses affecting the respiratory tract.

• Enterovirus, herpesimplex virus, mumps are referred to as viruses affecting central nervous system (CNS) .

• DISADVANTAGE• 1. Some viruses that affect more than one organ

in a single infection will belong to several group • Example: Measles virus affect the respiratory

tract, the eye, the skin and CNS• 2. Viruses whose pathogenecity is unknown can

not be classified

Ecological Characteristics• Ecological features of viruses such as the

involvement of vectors of vertebrates reservoirs in their transmission cycle or maintenance in nature can be used

• Viruses are classified into;• Arboviruses and Non-arboviruses• Roboviruses and Non-roboviruses

• Arboviruses: Viruses that are transmitted biologically between blood sucking arthropods

• Example; Yellow fever virus transmitted by Aedes mosquito

• Roboviruses: Viruses that are transmitted with rodent reservoir like the rat from their droppings (faeces) or fur

• Example : Lassa fever virus• Infected rodents are asymptomatic and shed

the virus in their urine

Physicochemical characteristic• Physicochemical properties are the most reliable

and satisfactory parameters for classifinying viruses

• Viruses are classified into families with parameters such as :

• Nucleic acid type• Nucleic acid size• Morphology• Mode of replication• Genome size• Strandedness• Capsid symmetry

VIRUS PURIFICATION• Viral particles can be extracted or isolated from

various cellular component of the host cell• The following methods are usually employed• 1. Differential and Density gradient

Centrifugation• 2. Precipitation of viruses• 3. Denaturation of contaminant• 4. Enzymatic digestion of host cell

constituents

Differential and Density gradient Centrifugation

• This is simply a process whereby the host cells containing the viral particles are disrupted through repeated centrifugation

• It is an initial purification step to separate viral particles from the host cell

Centrifugation

FIG 2.7

FIG 2.8

Precipitation of Viruses• Although after centrifugation some cell

components still remain in the virus preparation• Viruses are separated from cellular components by

adding ammonium sulphate or polyethylene glycol

Denaturation of Contaminants• The remaining cellular impurities may be treated

with pH or Temperature that will denature them• Organic solvent like butanol and chloroform can be

added to remove lipids by emulsifying them i.e breaking them into tiny pieces

Enzymatic Digestion of host Cell

• This is the last step used in the purification of viruses

• It is done to remove any remaining cellular protein and host nucleic acids

• Enzymes like ribonuclease and trypsin often degrade cellular RNA’S and proteins respectively while leaving viral particles unaltered

Virus Assay• The quantity of viruses in a sample can be

determined in two (2) ways:• Direct count• Indirect count• DIRECT COUNT• Viral particles can be counted using an electron

microscope with the aid of latex beads

INDIRECT COUNT• There are a number of indirect count method in

determining the number of viruses in a sample• These include: • I. Determination of Plague Forming Unit (PFU)• Several dilution of viruses are plated out with

appropriate cells. • Each plague in a layer of host cells is assumed to have

risen from the replication of a single viron particle e.g• If there are 50 plagues from 0.1ml of 10-6 dilutions .• The PFU per ml will be 50PFU/0.1ml X 10- 6 = 5.0 X

108 PFU/ml.

• 2 . Hemagglutination assay:• This is based on the fact that many viruses can

bind to surface of red blood cells.• The quantity of virus is determined by the

highest dilution of virus that form hemagglutination

• Lethal Infections Dose [LD50] :• The quantity of virus or viral particle in a

suspension that will lead to the death of 50% of the host cell

MAINTAINANCE OF LYSOGENY STATE

• Whether or not lysogeny occurs is determined by the action of a complex repression system

• Temperate viruses can have a dual existence• Under one set of condition ,it is an

independent entity that is able to control its own replication

• But when its DNA is incorporated into the host genetic material , replication is then under the control of the host cell

• One of the best studied temperate phages is Lambda (ʎ) phages which infects E coli .

• Morphologically, lambda particles looks like those of many other bacterophage

• The virus particles has an icosahedral head 64nm in diameter , and a tail 150nm which has helical symmetry

• Attached to the tail is a single 23nm long fiber• In addition to the major proteins of the coat,

there are a number of minor coat proteins

• The genome is a double stranded DNA molecule with a single stranded tail of 12 nucleotides in length which are complementary

• The ends of the DNA are said to be cohesive• Thus, when the two ends of the DNA are free

in the host cell, they associate and form a double stranded circle.

• The lambda genome has two sets of gene• One controlling lytic growth.• The other lysogenic growth• Upon infection, genes promoting both lytic

growth and lysogenic integration are expressed.

• Which pathway succeeds is determined by the competing action of these early gene products and by the influence of host factors

• The genetic map of lambda phage genome consist of several operons (a cluster of genes whose expression is controlled by a single operator)

• Each of which controls a set of related functions

• Upon injection , transcription of the phage genes which code for the lambda repressor occurs

• If repressor builds up before lytic functions are expressed , lytic reproduction is blocked

• The repressor protein blocks the transcription of all

later lambda genes

• Thus preventing expression of the genes involved in

the lytic cycle

• In lysogenic cell, the cI gene is responsible for

products that are repressive in function

• Two of the protein synthesized early in infection are

involved in positive regulation of the cI gene

• The positive regulatory proteins that the lambda

produces are themselves regulated by the host

and by another lambda protein called the Cro

• It is located adjacent to the cI genes

• The proximity of the regulatory genes for

repressor (cI) and Croprotein (cro gene) is the

key to the genetic switch between lytic and

lysogeny reproduction

LYTIC GROWTH OF LAMBDA AFTER INDUCTION

• Multiplication of lambda occurs only after the repressor is activated

• Agents which induce lysogenic cell to produce phage are agents which damage DNA

• Eg: Ultraviolet radiation, X- rays or DNA damaging chemicals such as Nitrogen mustard

• Upon DNA damage, a host defense mechanism called SOS response is brought into play

• An array of 10-20 bacterial genes is turned on, some of which help the bacterium survive radiation.

• However, one result of DNA damage is that a bacterial protein called RecA is turned into a special type of protease which participate in the destruction of the lambda repressor

• With lambda repressor damage ,the inhibition of expression of lambda lytic gene is abolished

• The protease activity of RecA , brought about by DNA damage, normally play an important role in the cells response to DNA damaging agents, by participating in the break down of a host protein

• LexA, which represses a set of host genes involved in DNA repairs

• Once the lambda repressor has been inactivated, the positive and negative control exerted by this repressor are abolished and the new transcriptional events can be inactivated

• These inevitably leads to lysis because even if the lambda repressor is made, it is inactivated.

VIROIDS• It is a infectious RNA particle smaller than a

virus that causes disease in plants• A viroid is similar to a virus in that it can

reproduce only inside a host cell as particles of RNA

• It differs from a virus in that each RNA particles contains a single specific RNA

• It does not have a capsid or an envelope • NB: some viruses do not have an envelope.

Detection and Cultivation of Viruses• Viruses are extremely small in size and hence

can not be seen with the light microscope• They may be detected by electron

microscope, in addition many indirect technique of detecting viruses

• Many of indirect methods depends on the ability to cultivate viruses invitro on a living cell system

• E.g bacteriophage may be cultivated on susceptible bacterial culture

• Viruses that effect vertebrate are generally cultivated on tissue or cell culture or on embryonated egg

• The culture of plant viruses is normally carry out in interplant cell

• Usually the viral effect on the host cell are often observable thereby providing it means of detecting the presence of viruses

Cultivation of Viruses • Bacteriophages may be cultured on lawn plate

of susceptible bacterial culture • In this technique a suspension of virus

sensitive bacteria is inoculated with virus and then spread on a solid nutrient medium after being serially diluted

• This is then incubated at appropriate temperature that will allow the normal multiplication of the bacteria cell

• Each virus then infect and lyses a bacterium cell releases progeny

• The process continued until the time the bacterial lawn begins to appear

• A small zone of clearance will have developed around the original viable infected cells

• Each of such cleared zone is called Plague, It represent one bacteriophages

• The number of isolated phages can be use to estimate the concentration of bacteriophages in the original suspension by multiply the count by dilution factor

Isolation of Viruses

• To isolate viruses from natural habitat, a sample of material

from the natural habitat is shaken with water( if solid and

not liquid)

• The supernatant is treated with chloroform which

eliminate most of the bacteria cells

• A suspension is then centrifuge and the supernatant

obtained can be sterilized by membrane filtration

• Aliquots (small volume) of the filtrate are then mixed with suspension of susceptible bacteria

• The suspension is then plated out on agar by lawn plate method

• Any plague that appear represent phage particle that were present in the natural material

• Phage from single plague can be isolated by stabbing phage with a sterile inoculating needle and suspending the adhering material in small volume

• Purification of the phage is then achieved by repeated serial isolation from single phage

• To prepare large batches of phage for chemical and physical analysis

• A culture of bacteria growing exponentially is liquid medium is inoculated with phage particles

• The series of cycle of phage growth result in lysis of most or all the cells in the culture

• The culture is then freed of remaining cell and cellular debris by low speed centrifugation and sterilization by membrane or treatment with chloroform

Purification and Precipitation of Viruses

• The phage suspension can then be treated with ammonium sulphate (68-88%)to precipitate the viral particle

• Ammonium ion can be remove by dialysis against appropriate buffer

• Viruses can be stored at a temperature of 40OC to activate the viruses

Inactivation of viruses• This is a process by which a virus is deprive of

its ability to infect or replicate• This is very useful for vaccine production• The process of inactivation can be carry out by

any of this means:• HEAT: Rapid inactivation occur at temperature

above 80OC • ANTISERA: Phage activity can be neutralized

by a serum containing antibody homologous to the surface antigen of the phage

• IONIZATION: Ionizing particles such as alpha, beta and gamma ray can be use to inactivate viruses

• CHEMICAL AGENTS: Some heavy metals , formaldehyde are directly active against the phage.