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PFU does NOT = infectious particles

DANDERSON at PRL.PULMONARY.UBC.CA DANDERSON at PRL.PULMONARY.UBC.CA
Thu Jul 27 10:50:12 EST 1995


In article <demers-2407952306580001 at demers.cts.com>, demers at cts.com (Bill Demers) writes:
>> Suppose a purified virus prep has a particle number:pfu ratio of 50:1.
>> 
>> Does this mean that 49 out of 50 particles are non-infectious and most of
>> the prep is "junk"
>> or
>> that pfu is a concentration of virus required to detect a single
>> infectious event, ie. 50 infectious particles exposed to cells has the
>> chance of forming a single plaque.

ryan at mbcf.stjude.org wrote in response:

>short answer: 
>yes, it means that 98% of the particles are  non-infectious "junk".

>somewhat longer answer: 
>The strategy of the plaque assay is to isolate (by limiting dilution)
>individual infectious virus particles. Because of this limiting dilution, 
>a scorable event (a plaque) is interpreted as the result of a single
>_independently infectious_ particle. Snip.....

I disagree!  

The plaque assay is a measure of infectivity and as such is reported 
as a UNIT of infectivity.  This unit does not reflect the number of 
infectious particles in a solution but the number of particles which 
will give an investigator a relative quantity of virus/volume in 
THEIR assay.  

Indeed there are non-infectious particles in virus solution and thus, 
the definition of infectious units to particle ratio.  It is 
important to remember that this ratio can vary significantly 
depending upon your virus, cell line of propagation and culture 
techniques (i.e. the pH of the innoculum is critical for determining 
accurate virus concentrations).  However, the ratio of particles to 
PFU reflects the number of particles necessary to develop a plaque.  
Furthermore, plaque development is dependent upon the steps necessary 
for virus replication.  I will use Picornaviruses as an example 
(remembering that the steps of infection I will use as an example, 
are often debated) .
 
First, the infectious virus has to bind to a receptor, be 
endocytosed, uncoat and extrude it's nucleic acid into the cytoplasm, 
translate the viral message, transcribe it's RNA (both negative and 
positive strand genome), undergo further genomic amplification and 
translation, and be assembled in to progeny virus particles.  The 
first few steps (up to transcription of positive strand genome) of 
such an infection are completely dependent upon the first infectious 
particle.  Until there is synthesis of nascent positive strand RNA 
the only viral genome to perform this is the infecting genome. 

Considering the presence of cellular RNases, DAI kinase, 2'5' 
Oligo-Adenylate synthase, other innate anti-viral mechanisms, and the 
efficiency of replication it is virtually impossible for the 
infectious process to be 100% efficient at all of the above steps.

My main point is that not all infectious particles in an inoculum are 
going to bind, be endocytosed, uncoat, translate and transcribe in a 
"permissive cell."   Thus, a single plaque forming unit (PFU) does 
not reflect a single infectious particle, but is instead the number 
of infectious particles which results in a PRODUCTIVE infection.  

Real Life:  

We have two HeLa cell lines, one recently purchased from ATCC (two 
years ago) and one which has been maintained in the laboratory for 
years.  Both have been frozen down in working and stock preparations, 
enough to last for 15 to 20 years (barring thawing), thus preventing 
genetic drift of the cell over these years.  If I plaque my stock  
coxsackievirus B3 on both cell lines, making sure that the same 
number of cells are plated, and they are at the same level of 
confluence at the time of inoculation, I get two different answers 
for my virus concentration.  In fact the PFU/ml can vary by up to 2 
logs.  The same virus aliquot and different cells!!!!!  The 
efficiency of infection and/or replication is NOT the same in these 
two cell lines!!

Where does that leave us!  Questioning the gold standard of 
determining virus concentration, and a neat model to study!  To 
question though, is science!

Dan Anderson
The University of Nebraska Medical Center
The University of British Columbia



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