Replication of viruses

First of all the DNA viruses : these viruses replicate inside the nucleus of the cell
using the host cell enzymes like (topoisomerase / gyrase / helicase ) , exception to
that is the Poxvirus which is not produced in the nucleus , its replication occurs in
the Cytoplasm .

Some of them are fully dependent , and some are partially dependent on the host
enzymes like herpes viruses in which the virus produce some of the enzymes
needed like DNA polymerase .

Are these enzymes available at all times ?

The answer in no , normal cells use these enzymes in the S phase during
replication , and they are inactive during other periods , so these viruses must
trigger the cell to activate the enzymes , and this mechanism may trigger
oncogenesis (uncontrolled cell replication ).

RNA viruses :

These viruses replicate in the cytoplasm , because they need RNA polymerase
which is located in the cytoplasm , exception to that are the Retroviruses ,
influenza viruses , these are RNA viruses that are replicating in the nucleus .

If the virus is RNA (-) , RNA dependent RNA polymerase also called (Transcriptase )
converts it into RNA + .

It is not necessary that the same enzyme used for transcription for host proteins
is the same as the one used by the virus .

Assembly of the virus

this depends on whether the virus is helical or icosahedral .

Helical : the nucleocapsid is formed with the nucleic acid in a stepwise matter (
together ) .
Icosahedral : the nucleocapsid is made before the nucleic acid , then the nucleic
acid is inserted into the nucleocapsid .

After assembly modifications may occur like cleavage or glycosylation . cleavage

occur by proteases whether from the cell enzymes or from the viral enzymes .

Realease of the virus

How these viruses are released ?

Lysis (burst ) : by death of the cells , like bacteriophages , this mechanism is

used for both naked and enveloped viruses .
Budding : this mechanism is used by enveloped viruses , doeas this
mechanism cause death of the cell ?

If the budding was too fast causing instability of the membrane , then death
occurs , if the budding was in a slow manner , then no death occur .

How budding occurs ?

First of all there must be insertion of some viral proteins on the membrane , these
proteins are spikes and matrix proteins . Then the nucleocapsid forms .
Does budding cause release of the virus ?

If budding is from the plasma membrane then the virus is released , if the budding
happened from other membrane like golgi of endoplasmic reticulum the no
release happened here .

Outcome of viral replication

Lytic viruses : replication of the virus will kill the host cell , this type is
productive ( new copies of the virus are released )
Budding viruses : slow budding the cell survives , this type is called
persistent infection , like hepatitis B infection which cause chronic
infection , in contrast to hepatitis A which is an acute infection ( lytic virus ).
This mechanism is productive .
Some of the viruses use an enzyme called integrase enzyme , the simply
insert their genetic material into the host cell DNA , they are called
proviruses ( not true viruses because they lack an envelope ) , the cause
latent infection . If this virus infects a bacterial cell it is called lysogeny .
this mechanism is not productive . The classical example on lysogeny is
botolunism toxin , which is produced by chlostridium after being infected
by a bacteriophage . In human cells , these virus may not insert their
genetic material into the cell , but the enter a long term relationship until
the virus is reactivated again , and then the virus will start replication, the
classical example of reactivation is varicella zoster infection , in children it
causes chicken pox , but the virus stays inside the neuronal cells in an
inactive form ( latent phase ) then after decades , the virus reactivates itself
and cause a new type of disease called ( ZOSTER ) . another example is
herpes simplex virus which cause mouth ulcers , when reactivated it cause
the same disease ( mouth ulcers ) in contrast to varicella which has two
different disease from the primary infection ( chicken pox ) and the
secondary one ( zoster ) .
If the cell cannot support the invading virus (non permissive ) , some
viruses can cause cell death , this case is called Abortive infection .
 Some viruses cause oncogenesis leading to cancer , this happens by
inactivation of some proteins that are inhibitory of the cell cycle , leading to
uncontrolled replication of the cell and cancer .

Viral growth curve

Why the curve declined from 10 0 ?

This part of the curve resembles the uncoating phase or disintegration .

Then the curve rises resembling the biosynthesis phase .

Blue line : intracellular count .

Red line extra cellular count .

There are two zones to describe :

Eclipse : this zone starts from the count zero ( after uncoating ) and ends
when the virus starts its assembly .
 Latent period : from the beginning ( virus inside ) ending when the virus is
being released extracellularly , it includes the eclipse period in it .
IF the virus undergo budding , these two lines overlap because assembly
and releasing of the virus occur together .

Cultivation of viruses

Why do I need to culture any virus ?

Diagnosis of diseases .
Production of vaccines .
Studying the characteristics of the virus .

Growing of the viruses can occur in :

Animal cells : used in the past .

Fertilized eggs ( chick embryo )
Tissue culture ( the newest method ) : they are human cells taken outside
the body.

Tissue culture can be divided into :

Primary : cells are taken directly the way they are , the problem here is that
these cells cant grow forever , they have limited number of replication .
Cell line : cancer cells or modified cells that can replicate indefinitely .

How we study these cells ?

Qualitative ( changes in quality ): studying the morphological changes that

happens in these cells , for example lytic viruses will cause lysis of the cell .
Quantitative( number) : example of that is plaque assay .

Plaque assay : only for lytic viruses

Bring the virus diluted ( titer )

Put the virus with the cells of the study .
Put them in agar , why ? to limit the mobility of the virus outside the plate .
 Waiting a period of time
After that check the cells , the virus will infect the cells and cause them to
be destroyed , a clear zone will be found , it is called (plaque zone ) , then
multiplying the number of plaques with a factor .

Other methods for studying viruses :

Some studies use antigens to detect viruses like ( ELISA , fluorescence )

Some use genetic material ( PCR , discussed later)
Heamaglutination for influenza viruses (discussed later ) .