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
