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 VIROLOGY-LECTURE TWO  
 BASIC VIROLOGY: 
 REPLICATION OF
 VIRUSES 

Virology Lecture 1
BASIC VIROLOGY: DEFINITIONS, CLASSIFICATION, 
MORPHOLOGY AND CHEMISTRY  

Virology Lecture 3
 DNA VIRUS REPLICATION 
 STRATEGIES  

Virology Lecture 4
 RNA VIRUS REPLICATION
 STRATEGIES  

Virology Lecture 5
 ONCOGENIC VIRUSES 
 Virology Lecture 6
 SEVEN  HUMAN  
 IMMUNODEFICIENCY VIRUS  
 AND AIDS  

Virology Lecture 7
 PICORNAVIRUSES - 
 PART ONE  
 ENTEROVIRUSES  
Virology Lecture 

 HERPES VIRUSES  

Virology Lecture 9

INFLUENZA VIRUS


Virology Lecture 10
Virology Lecture 11




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  VIROLOGY - LECTURE TWO  

  BASIC VIROLOGY: REPLICATION OF VIRUSES  

Dr. Margaret Hunt 

MEDICAL MICROBIOLOGY, MBIM 650/720  LECTURE 51

READING: Murray et al., Microbiology, 3rd. Ed., Chapter 7

TEACHING OBJECTIVES

 An overall view of the replication cycle of viruses

 

 

PRINCIPAL EVENTS INVOLVED IN REPLICATION

1. ADSORPTION

Virus attaches to the cell surface. Attachment is via ionic interactions which are temperature-independent.

Viral attachment protein recognizes specific receptors on the cell surface (These may be protein or carbohydrate or lipid components of the cell surface).

Cells without the appropriate receptors are not susceptible to the virus.

 

2. PENETRATION (Virus enters the cell)

Enveloped viruses

(A) Entry by fusing with the plasma membrane. Some enveloped viruses fuse directly with the plasma membrane. Thus, the internal components of the virion are immediately delivered to the cytoplasm of the cell.

(B) Entry via endosomes at the cell surface

Some enveloped viruses require an acid pH for fusion to occur and are unable to fuse directly with the plasma membrane. These viruses are taken up by invagination of clathrin coated pits into endosomes. As the endosomes become acidified, the latent fusion activity of the virus proteins becomes activated by the fall in pH and the virion membrane fuses with the endosome membrane. This results in delivery of the internal components of the virus to the cytoplasm of the cell

Non-enveloped viruses

Non-enveloped viruses may cross the plasma membrane directly or may be taken up via clathrin-coated pits into endosomes. They then cross (or destroy) the endosomal membrane.

 

3. UNCOATING

Nucleic acid has to be sufficiently uncoated that virus replication can begin at this stage. When the nucleic acid is uncoated, infectious virus particles cannot  be recovered  from the cell - this is the start of the ECLIPSE phase - which lasts until new infectious virions are made.

 

4. SYNTHESIS OF VIRAL NUCLEIC ACID AND PROTEIN

A lot of strategies are used, some will be discussed later.

 

5. ASSEMBLY/MATURATION

New virus particles are assembled. After assembly they may undergo a maturation process.

6. RELEASE

Virions may be released due to cell lysis, or, if enveloped, may bud from cell. Budding viruses do not necessarily kill the cell. Thus, some budding viruses may be able to set up persistence.

hiv_bud.jpg (41452 bytes) Transmission electron micrograph of HIV-1, budding and free   CDC

hivbud-kun.jpg (42511 bytes) HIV budding from human lymph tissue (TEM x133,335) Copyright Dr Dennis Kunkel, University of Hawaii (used with permission)

 

All proteins in a mature virus particle are said to be structural proteins - even if they make no contribution to the morphology or rigidity of the virion - non-structural proteins are those viral proteins found in the cell but not packaged into the virion.

 

Not all released virus particles are infectious. The ratio of infectious to non-infectious particles varies with the virus and with growth conditions.

Some methods (e.g. electron-microscopy) enable every virion to be counted but are not informative about infectivity. Other methods (e.g. hemagglutination) are a less sensitive measure of how much virus is present, but again are not informative about infectivity. Other methods, e.g. plaque assay, measure the number of infectious virus particles.

EFFECT OF VIRUSES ON HOST MACROMOLECULAR SYNTHESIS

Many viruses inhibit host RNA, DNA or protein synthesis (or any combination of these). The mechanisms by which the virus does this vary widely.

Cytopathic effect.  The presence of the virus often gives rise to morphological changes in the host cell. Any detectable changes in the host cell due to infection are known as a cytopathic effect. Cytopathic effects (CPE) may consist of cell rounding, disorientation, swelling or shrinking, death, detachment from the surface, etc.

Many viruses induce apoptosis (programmed cell death) in infected cells. This can be an important part of the host cell defense against a virus - cell death before the completion of the viral replication cycle may limit the number of progeny and the spread of infection. (Some viruses delay or prevent apoptosis - thus giving themselves a chance to replicate more virions.)

Some viruses affect the regulation of expression of the host cell genes which this can have important results both for the virus's ability to grow, and in terms of the effect on the host cell.

Assays for plaque-forming units.

The CPE effect can be used to quantitate infectious virus particles by the plaque-forming unit assay.

Cells are grown until they form a monolayer of cells covering a plastic bottle or dish. They are then infected with the virus. The liquid growth medium is replaced with a semi-solid one so that any virus particles produced as the result of an infection cannot move far from the site of their production. A plaque is produced when a virus particle infects a cell, replicates, and then kills that cell. Surrounding cells are infected by the newly replicated virus and they too are killed. This process may repeat several times. The cells are then stained with a dye which stains only living cells. The dead cells in the plaque do not stain and appear as unstained areas on a colored background. However viruses which do not kill cells may not produce plaques.

plaque.jpg (141065 bytes)  A plaque assay. Serial dilutions of virus have been plated on confluent monolayer cultures of cells. The cells are stained after a period of time in which a single virus infects a cell, produces new virus particles and infects surrounding cells. The white areas show areas of the culture in which the cells have been killed. Each "plaque" is the result of the presence of one original infectious virus particle.

 

USEFUL WEB SITES:

http://www.cdc.gov

http://www.tulane.edu/~dmsander/garryfavweb.html


 

 


 






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