Microbiology 6/e

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MULTIPLICATION
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Remember.
Multiplication

• Viruses are obligate intracellular parasites that
  can reproduce only within a host cell.
• They do not have
• Enzymes for metabolism
• Do not have ribosomes
• Do not have the equipment to make proteins
• Viruses use a lock and key fit to identify hosts

Viruses - when in host cell, they will take
control of synthetic and genetic machinery of
host cell.
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LESSON OUTCOME

• General steps in multiplication/replication of
  virus
• Multiplication in animal virus
• - differences between naked and enveloped virus

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Viral Multiplication general phases in the
life cycle of viruses

• Multiplication/Replication cycles involving 5
  steps
• Adsorption the attachment of viruses to host
  cells
• Penetration entry of virions (or their genome)
  into host cells
• Synthesis new nucleic acids, capsid proteins,
  and other viral components- transcription,
  translation and genome replication
• Maturation assembly of newly synthesized viral
  components into complete virions
• Release departure (pemergian) of new virions
  from host cells

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Animal viruses general multiplication steps

• Adsorption the attachment of viruses to host
  cells
• Penetration entry of virions (or their genome)
  into host cells
• Uncoating to separate nucleic acid from protein
  coat - envelop and capsid are dissolved the
  nucleic acid is released
• Synthesis new nucleic acids, capsid proteins,
  and other viral components- transcription,
  translation and genome replication
• Maturation assembly of newly synthesized viral
  components into complete virions
• Release departure of new virions from host cells

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1. Adsorption

• Invasion begins when the virus encounters a
  susceptible host cell and adsorbs specifically to
  receptor sites on the cell membrane
• The membrane receptors that viruses attach to are
  usually glycoproteins the cell requires for its
  normal function.
• Ex. - rabies virus ? the acetylcholine receptor
  of nerve cells.
• - human immunodeficiency virus (HIV or AIDS
  virus) ? CD4 protein on certain white blood
  cells.

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Adsorption

• The mode of attachment varies between the two
  general types of viruses (naked and enveloped)
• In enveloped virus - influenza virus and HIV,
  glycoprotein spikes bind to the cell membrane
  receptors.

(Spikes that recognize membrane protein receptor)
Enveloped viruses
The configuration of the spike has a
complementary fit for cell receptors. The process
in which the virus lands on the cell and plugs
into receptors is termed docking.
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Adsorption

• Naked nucleocapsids (adenovirus, for example) use
  molecules on their capsids that adhere to cell
  membrane receptors

Protruding molecules - spike
(Specific membrane protein involved with cell
adhesion)
An adenovirus has a naked capsid that adheres to
its host cell by nestling surface molecules on
its capsid into the receptors on the host cells
membrane.
Naked viruses
(Attachment sites on surfaces of capsids)
Viral recognition of an animal host cell
Rhinoviruses have canyons or depressions, in
the capsid that attach to specific membrane
proteins on host cell membrane
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Adsorption

• Virus can invade its host cell - through making
  an exact fit with a specific host molecule.
• Host range, may be as
• i) restricted as hepatitis B, which infects only
  liver cells of humans
• ii) intermediate like the poliovirus, which
  infects intestinal and nerve cells of primates
  (humans, apes, and monkeys)
• iii) broad as the rabies virus, which can infect
  various cells of all mammals.
• Host cells that lack compatible virus receptors
  are resistant to adsorption and invasion by that
  virus why human liver cells are not infected by
  the canine hepatitis virus and dog liver cells
  cannot host the human hepatitis A virus.

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2. Penetration/Uncoating

• Animal viruses do not have a mechanism for
  injecting their
• nucleic acid into host cells nucleic acid and
  capsid usually
• penetrate animal cells.
• Penetration -i) endocytosis
• ii) direct fusion of viral envelop
  with host cell membrane
• Endocytosis the entire virus (including the
  envelope) is engulfted
• by the cell enclosed in a vacuole or vesicle
• - Most naked viruses enter cell by endocytosis
  in which virions are captured by pitlike regions
  on cell surface enter the cytoplasm within a
  membranous vesicle
• - Enveloped viruses the envelope fuse with
  hosts plasma membrane or by endocytosis. In
  endocytosis the envelope will fuse to vesicle
  membrane
• Uncoating is a process that releases the viral
  nucleic acid into cytoplasm.
• - when enzymes dissolve envelope and capsid, the
  virus is said to be uncoated.
• - Naked viruses by proteolytic enzymes, host or
  virus
• - Enveloped viruses (poxviruses) by a specific
  enzyme encoded by viral DNA
• Viral entry into the host cell - direct fusion of
  the viral envelope with the host cell membrane
  (as in influenza and mumps viruses) - the
  envelope merges directly with the cell membrane,
  ? release the nucleocapsid into the cells
  interior.

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Penetration/Uncoating
VIRAL ENTRY INTO HOST CELL
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3. Synthesis

• The synthetic and replicative phases of animal
  viruses are highly regulated and extremely
  complex at the molecular level. Free viral
  nucleic acid - control over the hosts synthetic
  and metabolic machinery depending on the virus
  genome (DNA or RNA)
• The DNA viruses (except poxviruses) enter the
  host cells nucleus and are replicated in the
  nucleus, transcription in nucleus
• RNA viruses (except retroviruses), are
  replicated in the cytoplasm, transcription in
  cytoplasm.
• RNA VIRUS REPLICATION AND PROTEIN SYNTHESIS
• Almost immediately upon entry, the viral nucleic
  acid alters the genetic expression of the host
  and instructs it to synthesize the building
  blocks for new viruses.
• 1. The RNA of the virus becomes a message for
  synthesizing viral proteins (translation). -
  Viruses with positive-sense RNA molecules already
  contain the correct message for translation into
  proteins.
• - Viruses with negative-sense RNA molecules must
  first be converted into a positive-sense message.
  
• 2. Some viruses come equipped with the necessary
  enzymes for synthesis of viral components others
  utilize those of the host.
• 3. In the next phase, new RNA is synthesized
  using host nucleotides. Proteins for the capsid,
  spikes, and viral enzymes are synthesized on the
  hosts ribosomes using its amino acids.

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4. Maturation

• Maturation Once all viral nucleic acid,
  enzymes, and other proteins have been completely
  synthesized, assembly of components into complete
  virions begins.
• DNA virus assembly take place in nucleus
• RNA virus assembly take place in cytoplasm

Assembly of Viruses Host Cell as Factory
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5. Release

• Release The release of new virions through a
  membrane may or may not destroy the host cell.
  Adenoviruses bud from host cell in a controlled
  manner (ex. shedding) which does not lyse host
  cells vs release through lysis destroy the host
  cells
• To complete the cycle, assembled/matured viruses
  leave their host in one of two ways.
• i) Non-enveloped and complex viruses that reach
  maturation in the cell nucleus or cytoplasm are
  released when the cell lyses or ruptures. - (cell
  lysis)
• ii) Enveloped viruses are released by budding or
  exocytosis from the membranes of the cytoplasm,
  nucleus, or endoplasmic reticulum?
• - The nucleocapsid binds to the membrane, which
  curves completely around it and forms a small
  pouch.
• Pinching off the pouch
• releases the virus with its
• envelope. Budding of
• enveloped viruses
• causes them to be shed
• gradually, without the
• sudden destruction of the cell.

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Release

• Regardless of how the virus leaves, most active
  viral infections are ultimately lethal/deadly to
  the cell because of accumulated damage.
• Lethal damages include a permanent shutdown of
  metabolism and genetic expression, destruction of
  cell membrane and organelles, toxicity of virus
  components, and release of lysosomes.
• A fully formed, extracellular virus particle that
  is virulent (able to establish infection in a
  host) is called a virion
• The number of virions released by infected cells
  is variable, controlled by factors such as the
  size of the virus and the health of the host
  cell. About 3,000 to 4,000 virions are released
  from a single cell infected with poxviruses,
  whereas a poliovirus-infected cell can release
  over 100,000 virions - even a small number of new
  virions happens to meet another susceptible cell
  and infect it, the potential for rapid viral
  proliferation is immense.

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Release
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Modes of infection and replication of animal
viruses enveloped virus, DNA genome

• The enveloped viruses enter the host cell through
• i) endocytosis into host cell cytoplasmic
• ii) the fusion of virus envelop with the hosts
  cell/plasma membrane
• Penetration involves nucleocapsid only
• Replication and transcription takes place in
  nucleus
• Translation in the cytoplasm ?capsid and protein
  are synthesize in cytoplasm
• Maturation assembly of nucleocapsid of new
  virus particle in nucleus
• Some viruses have envelopes that are not
  derived from the plasma membrane. Herpesvirus has
  an envelop that is derived from the nuclear
  membrane.

Replication of an enveloped dsDNA animal virus
(e.g. herpesvirus)
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Synthesis in DNA animal viruses

• Synthesis of new genetic material and proteins
  depends on the viruses
• Generally, DNA animal viruses replicate their DNA
  in host cell nucleus with aid of viral enzymes
  and synthesize their capsid and other proteins in
  the cytoplasm with aid of host cell enzymes
  typical of adenoviruses, hepadnaviruses,
  herpesviruses and papovaviruses.
• Assembly of nucleocapsid in nucleus
• dsDNA viruses replication proceeds in a complex
  series of steps designated as early and late
  transcription and translation
• Early events take place before the synthesis of
  viral DNA and results in production of enzymes
  and proteins for viral DNA replication
• Late events after the synthesis of viral DNA,
  results in production of structural proteins
  needed for building new capsids.

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Modes of infection and replication of animal
viruses - enveloped virus, RNA genome
Nucleic acid synthesis cytoplasm Assembly of
nucleocapsid - cytoplasm
General features in the multiplication cycle of
an enveloped animal virus. Using an RNA virus
(rubella virus), the major events are outlined,
although other viruses will vary in exact details
of the cycle.
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Synthesis in RNA animal viruses
Modes of infection and replication of animal
viruses enveloped virus, RNA genome

• Synthesis in RNA animal viruses takes place in a
  greater variety of ways than found in DNA
  viruses
• () sense RNA acts as mRNA (e.g. picornaviruses)
  and viral proteins are synthesize immediately
  after penetration and uncoating. The nucleus of
  host cell is not involved.
• dsRNA () sense are transcribed into ssDNA with
  help of reverse transcriptase (e.g. retrovirus
  HIV)
• (-) sense RNA make () sense RNA which are mRNA
  (e.g. measles and influenza)
• Nucleic acid replication and assembly of
  nucleocapsid - cytoplasm

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Modes of infection and replication of animal
viruses RNA genome

• The broadest variety of RNA genomes is found
  among viruses are those that infect animals.
• The genome of class IV can directly serve as mRNA
  and can be translated into viral protein
  immediately after infection.
• A (-) sense RNA is synthesized as template for
  replication of more () sense RNA

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MULTIPLICATION (II)
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ANNOUNCEMENT

• PRACTICAL 5
• - Each group is required to bring their own
  sample as shown in pg 13 in the lab manual.
• - Each group bring 3 samples soil, sewage water
  and chicken faeces

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LESSON OUTCOME

• Bacteriophage multiplication steps
• Lytic cycle and Lysogenic cycle
• Virulent, temperate virus, prophage definition
• Different bacteriophage and animal
  multiplication how the viruses enter the host
  cells, release

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Point of entry for virus
REVISION
Point of exit for virus
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REMEMBER.

• The assembly of newly formed viral particles
• cytoplasm eg. Poxvirus, poliovirus
• Cell nucleus eg. Human adenovirus nucleocapsids
• Plasma membrane of host eg. HIV at the inner
  surface of host cells cell membrane

• The source to form new viral particles
• Proteins and glycoproteins coded by viral
  genome
• Envelope lipids and glycoproteins synthesized
  by host cell enzymes and are present in the host
  cell plasma

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Bacteriophages

• Bacteriophages means eaters of bacteria
• The bacteriophages discovered by Frederick
  Twort and Felix dHerelle in 1915 it first
  appeared that the bacterial host cells were being
  eaten by some unseen parasite, hence the name
  bacteriophage was used.
• Most bacteriophages (or phage) contain
  double-stranded DNA, although single-stranded DNA
  and RNA types exist as well.
• It is known that every bacterial species is
  parasitized by various specific bacteriophages.
• Bacteriophages are of great interest to medical
  microbiologists because they often make the
  bacteria they infect more pathogenic for humans.
• - The most widely studied bacteriophages are
  those of the intestinal bacterium
• Escherichia coli especially the T-even phages
  such as T2 and T4
• The multiplication of T-even bacteriophages
  -similar stages as the animal viruses described
  earlier

Have been used as a model systems for animal and
plant viruses
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Adsorption

• Bacteriophages have specialized structures for
  attaching to bacterial cell walls adsorption
  involve attachment of specific tail fibers to
  bacterias cell wall
• They adsorb to host bacteria using specific
  receptors on the bacterial surface

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Penetration

• Bacteriophages have a mechanism for injecting
  their nucleic acid into host cells (nucleic
  acid and capsid usually penetrate animal cells)
• This eliminates the need for uncoating.

Only nucleic acid penetrate into host cell.

• Penetration of a bacterial cell by a T-even
  bacteriophage.
• After adsorption, the phage plate becomes
  embedded in the cell wall, and the sheath
  contracts, pushing the tube through the cell wall
  and membrane and releasing the nucleic acid into
  the interior of the cell.
• Section through E. coli with attached phages.
  Note that these phages have injected their
  nucleic acid through the cell wall and now have
  empty heads.

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Synthesis

• Entry of the nucleic acid causes the cessation of
  host cell DNA replication and protein synthesis.
  Soon the host cell machinery is used for viral
  replication and synthesis of viral proteins.

Maturation

• As the host cell produces new phage parts, the
  parts spontaneously assemble into bacteriophages.
• An average-sized Escherichia coli cell can
  contain up to 200 new phage units at the end of
  this period.

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Release

• Eventually, the host cell becomes so packed with
  viruses that it lyses (splits
• open) - releasing the mature virions.
• The process is hastened by viral enzymes produced
  late in the infection cycle
• that digest the cell envelope, thereby
  weakening it. Upon release, the virulent
• phages can spread to other susceptible
  bacterial cells and begin a new infection.

Involve lysozyme to break the host cell wall
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Bacteriophages

• Bacteriophages can be classified as virulent or
  temperate
• Virulent phage (or lytic phage) lyse and destroy
  bacteria they infect
• Temperate phage able to undergo lytic cycle and
  lysogenic cycle.
• - temperate phage exhibit lysogeny the state
  whereby the DNA of temperate bacteriophages
  integrate into the host DNA
• - no replication of new viruses and cell lysis
• The host cells are called lysogenic cells
• The viral DNA within the bacteria chromosome is
  called prophage

Virulent phage
Temperate phage
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Temperate phages

• Undergo adsorption and penetration into the
  bacterial host but are not replicated or released
  immediately.
• - viral DNA enters an inactive prophage state,
  during which it is inserted into the bacterial
  chromosome. This viral DNA will be retained by
  the bacterial cell and copied during its normal
  cell division so that the cells progeny will
  also have the temperate phage DNA.
• - This condition, in which the host chromosome
  carries bacteriophage DNA, is termed lysogeny.
• Because viral particles are not produced, the
  bacterial cells carrying temperate phages do not
  lyse, and host cells appear entirely normal. On
  occasion, in a process called induction, the
  prophage in a lysogenic cell will be activated
  and progress directly into viral replication and
  the lytic cycle.
• - Lysogeny is a less deadly form of parasitism
  than the full lytic cycle and is thought to be an
  advancement that allows the virus to spread
  without killing the host.
• Because of the intimate association between the
  genetic material of the virus and host, phages
  occasionally serve as transporters of bacterial
  genes from one bacterium to another and
  consequently can play a profound role in
  bacterial genetics. This phenomenon, called
  transduction, is one way that genes for toxin
  production and drug resistance are transferred
  between bacteria

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Reproduction / multiplication of temperate phage
Temperate phage lambda phage
Virulent phage T4 phage
Induction The stimulation of a prophage to
initiate a lytic cycle
Induction Due to lack of nutrients for bacterial
growth or the presence of chemical toxic to
lysogen
Prophage viral DNA within the host genome
Lysogen the bacterium that has combination of
temperate phage DNA and host.
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Replication of a virulent bacteriophage A
virulent phage undergoes a lytic cycle to produce
new phage particles within a bacterial cell.
Cell lysis releases new phage particles that can
infect more bacteria
T4 virulent (lytic) phages
5. Release Lysozyme breaks down the cell wall,
allowing viruses to escape in the process the
host cell is lysed ? destroy the host
1. Adsorption chemical attraction, specific
protein recognition factors found in tail fibers
that bind to specific receptor sites on the host
cells.
4. Maturation T4 head is assembled in host cell
cytoplasm from new capsid protein, phage tails
from new formed base plates, sheaths and collars.
After heads and tails are attached
2. Penetration lysozyme, weakens the bacterial
cell walls for T4 phages DNA are introduce into
the periplasmic space
3. Synthesis transcription of phage DNA to
mRNA, translated on host ribosomes to synthesize
capsid proteins and viral enzymes
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Replication of a temperate bacteriophage
Following adsorption and penetration, the virus
undergoes prophage formation

• Temperate phages have alternative replication
  cycle
• A productive lytic cycle
• A reductive infection, in which the phage remain
  latent in the host establishing lysogeny
• - Lysogeny occurs when environmental conditions
  are poor. Allowing survival as a prophage in the
  host. - lysogen

Induction Due to lack of nutrients for bacterial
growth or the presence of chemical toxic to
lysogen
Lysogenic phages - ? phage of E. coli.
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Relationship between Temperate phages and
pathogenicity of host cells

• Occasionally phage genes in the bacterial
  chromosome cause the production of toxins or
  enzymes that cause pathology in the human.
• When a bacterium acquires a new trait from its
  temperate phage, it is called lysogenic
  conversion.
• - The phenomenon was first discovered in the
  1950s in the bacterium that causes diphtheria,
  Corynebacterium diphtheriae. The diphtheria toxin
  responsible for the deadly nature of the disease
  is a bacteriophage product.
• ? C. diphtheriae without the phage are
  harmless.
• Other bacteria that are made virulent by their
  prophages are Vibrio cholerae, the agent of
  cholera, and Clostridium botulinum, the cause of
  botulism.

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Comparison of animal virus and bacteriophage
multiplication
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Animal viruses Latent viral infections

• Latent viral infections
• - herpesviruses - herpes simplex virus. These
  dsDNA viruses that can exhibit a lytic cycle and
  also able to remain latent within the cells of
  host. Once activated by a cold, fever, stress or
  immunosuppression, they replicate resulting in
  cell lysis.
• - HIV virus provirus will become latent until
  induction whereby HIV virus show AIDS symptom.
• Latent infection - infection of a cell where the
  replication cycle is not completed, but the virus
  genome is maintained in the host cell without
  replicating or causing harm.

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Phage Growth
Growth curve for a bacteriophage The eclipse
phage represents the time after penetration
through the biosynthesis of mature phages. The
latent period represents the time after
penetration through release of mature phages.
The number of viruses per infected cell is the
viral yield, or burst size
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Estimation of Phage Numbers

• Plaque assay
• Serial dilutions of suspension of phages
• Each dilution is inoculated onto a plate
  containing bacterial lawn
• As a result of infection, new phages will lyse
  the bacteria
• After several round of lysis, the bacterial lawn
  shows clear areas called plaques.
• Plaque-forming units (pfu) counting the no. of
  plaques X dilution factor the no. of phages in
  ml of suspension.

plaques