Friday, 25 June 2021

One Step Growth Curve Experiment of Virus

 One step growth experiment is An experiment by which molecular events that are occurring during reproduction of  virus can be observed.
• It reveals the fundamental nature of virus replication process.

• This process was first performed by Ellis & Delbruck in 1939 by using T2 bacteriophages. 

• They also determined the plague counting method for the enumeration of bacteriophages.

• In this experiment, only a single or one cycle of virus growth is observed.

• Therefore, it is called as one step growth experiment. 

• Excess number of host cells are allowed to infect with phage particles.

• This makes the infection synchronous. That means the simultaneous infection of large number of particles to the host cell is taking place.

• Observation made on such host cell culture is similar to observation made on single host cell infected by a phage. 

• In the experiment, excess host cells are infected with phage particles at a ratio of 1:10. 

• This is done to prevent the adsorption of more than one virus per cell. 

• Mixture is incubated for a short period of time (5 min).

• This incubation allows the adsorption of phage particle on host cell. 

• If the bacteria are in excess, all the phage particles will be adsorbed.

• Such mixture is then diluted to such an extent (1:1000) that the virus particles released after first round of replication cannot adsorb to uninfected cell. 

• Thus, only one step of virus growth can occur. 

• Samples of diluted mixture are then removed at regular time interval & used for plaque count. 

• This gives a measure of infectious centers i.e. the infected bacteria & number of virus particles (i.e. No. of plaques per ml). 

• When a log no, of plague forming units/ml is potted against time, a curve is obtained & it is termed as one step growth curve.

 
• This one step growth curve shows the various events that are occurring during the virus replication cycle.

• This curve gives three distinct phases -
   1]. Latent Period
   2]. Burst or Rise Period
   3]. Plateau Period

One step growth curve of virus

I]. Latent Period:

It is the period from infection to cell lysis. 

• During this, there is no release of new virus particles from infected cells.

• Therefore, the plaque count remains constant. 

• T phage has latent period of 22 to 23 min at 37°C.

• This period can be divided into two phases as 'Eclipse' period & 'Intracellular Accumulation' period. 

• Time from infection until intracellular accumulation of phages is called as 'Eclipse' period. 

• T2 bacteriophage has eclipse period of about 11.5 min at 37°C.

• In this, gene expression, protein synthesis & genome synthesis occurs. 

• The time from initiation to the end of intracellular accumulation of phages is called as  'Intracellular Accumulation' period. 

• During this, phage proteins & genomes assemble into new phage particles. 

• T2 bacteriophage requires the period of about 11 to 12 min. at 37° C for this period.

II]. Burst Period or Rise Period

• The time from initiation of infected host cell lysis to the end is called rise or burst period

• At the end of latent period, each infected cell lyses & liberates a crop of new virus particles. 

• During this phase, there is release of new viral particles from infected cells & therefore, plaque count increases rapidly. 

• T2 bacteriophage has the rise period of about 10 min. at 37°C.

• Due to the asynchrony of infection the rise period is slightly extended.

III] Plateau Period :

• This period represents the end of all infected host cell lysis.

• The newly liberated phage particles fail to meet uninfected host cells due to high dilution. 

• Therefore, during this phase, the plaque count remains constant. 

• T2 phage enters in plateau in about 30 min. at 37°C.

Burst Size - Burst size is defined as the number of virus particles produced from the infection of a single cell. The burst size is caleulated using following formula -

• T2 phage has a burst size of less than 100 phages/cell.

• Burst size varies from 20 to 3000 virions/cell for different viruses.

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