1. MULTIPLICATION

2. 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
19_04SimpViralReproCycle_A.swf
Viruses - when in host cell, they will take
control of synthetic and genetic machinery of
host cell.

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

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

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

6.  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.

7.  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.

8.  Naked nucleocapsids (adenovirus, for example) use molecules
on their capsids that adhere to cell membrane receptors
Protruding
(Specific molecules -
membrane protein spike
involved with cell
adhesion)
An adenovirus has a naked capsid that
(Attachment sites on Naked viruses
adheres to its host cell by nestling
surfaces of capsids)
surface molecules on its capsid into the
Viral recognition of an animal host cell: receptors on the host cell’s membrane.
Rhinoviruses have “canyons” or
depressions, in the capsid that attach to
specific membrane proteins on host cell
membrane

9.  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.

10.  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 host’s 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 cell’s interior.

11. VIRAL ENTRY INTO HOST CELL

12.  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
host’s synthetic and metabolic machinery; depending on the virus genome (DNA or
RNA)
 The DNA viruses (except poxviruses) enter the host cell’s 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 host’s ribosomes using its
amino acids.

13.  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

14.  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.

15.  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.

17. 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 host’s 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
Replication of an enveloped dsDNA animal virus are not derived from the plasma
(e.g. herpesvirus) membrane. Herpesvirus has an
envelop that is derived from the
nuclear membrane.

18. 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
i. Early events – take place before the synthesis of viral DNA and
results in production of enzymes and proteins for viral DNA
replication
ii. Late events – after the synthesis of viral DNA, results in
production of structural proteins needed for building new
capsids.

19. 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.

20. 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:
1. (+) 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.
2. dsRNA (+) sense are transcribed into
ssDNA with help of reverse transcriptase
(e.g. retrovirus – HIV)
2. (-) sense RNA make (+) sense RNA which
are mRNA (e.g. measles and influenza)
 Nucleic acid replication and assembly of
nucleocapsid - cytoplasm

21. 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

22. MULTIPLICATION (II)

23.  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

24.  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

25. Point of entry
REVISION for virus:
Point of exit
for virus:

26. REMEMBER….
The assembly of newly formed viral particles
i.cytoplasm – eg. Poxvirus, poliovirus
ii.Cell nucleus – eg. Human adenovirus nucleocapsids
iii.Plasma membrane of host – eg. HIV at the inner surface of host
cell’s cell membrane
The source to form new viral particles
i.Proteins and glycoproteins – coded by viral genome
ii.Envelope lipids and glycoproteins – synthesized by host cell
enzymes and are present in the host cell plasma

27.  Bacteriophages means “eaters of bacteria”
The bacteriophages – discovered by Frederick Twort and Felix d’Herelle 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

28.  Bacteriophages have specialized structures for attaching to
bacterial cell walls – adsorption involve attachment of specific
tail fibers to bacteria’s cell wall
 They adsorb to host bacteria using specific receptors on the
bacterial surface

29. Penetration
 Bacteriophages have a mechanism for injecting their nucleic acid into host cells
(nucleic acid and capsid usually penetrate animal cells)
Only nucleic acid penetrate
This eliminates the need for uncoating. into host cell.
Penetration of a bacterial cell by a T-even bacteriophage.
(a)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.
(b)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.

30.  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.

31.  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

32.  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 Temperate
phage phage

33.  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 cell’s 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

34. Temperate phage – Virulent phage –
lambda phage T4 phage
Induction:
The stimulation of a prophage
to initiate a lytic cycle
Induction: Due to lack of
nutrients for bacterial growth
Prophage: or the presence of chemical
viral DNA toxic to lysogen
within the host
genome
Lysogen: the
bacterium that has
combination of
temperate phage DNA
and host.

35. 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
4. Maturation – T4 that bind to specific
head is assembled in receptor sites on the
host cell cytoplasm host cells.
from new capsid
protein, phage tails
from new formed base
plates, sheaths and 2. Penetration –
collars. After heads lysozyme, weakens
and tails are attached the bacterial cell
walls – for T4
3. Synthesis – transcription of phages DNA are
phage DNA to mRNA, introduce into the
translated on host ribosomes periplasmic space
to synthesize capsid proteins
and viral enzymes

36. Replication of a temperate bacteriophage: Following
adsorption and penetration, the virus undergoes prophage
formation
Temperate phages have
alternative replication cycle
i)A productive lytic cycle
ii)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 - λ phage of E. coli.

37.  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.

38. Comparison of animal virus and
bacteriophage multiplication

39.  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 .

40. 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

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