In article <1F2FA1A7F98 at prl.pulmonary.ubc.ca>, DANDERSON at PRL.PULMONARY.UBC.CA writes:
> 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.
Hi Dan; I'm not sure I follow what you're saying here. No one suggests
that an infectious titre determined in one assay system will always be
the infectious titre in _any_ assay system. If you're looking for a sort
of global definition of infectivity, applicable in any host cell or
animal, I'm not sure what this would mean or how it would be defined or
measured. A PFU titre (or any measure of infectivity) has meaning only for
the virus/host cell combination in which it was determined. When I've
determined a PFU titre on permissive cultured cells, I don't assume that
one PFU (enough virus to get an infectious hit in a dish) will be
sufficient to infect a mouse: I would usually use many PFU to ensure a
productive mouse infection. That is to say, the animal ID50 may be several
PFU, since the infectious dose must overcome the immune system and other
barriers. But a PFU is still a PFU (an infectious unit in permissive
cultured cells, defined at limiting dilution). The PFU titre _is_ the
number of infectious particles per unit volume, usually defined in the
most permissive system available.
> Indeed there are non-infectious particles in virus solution and thus,
> the definition of infectious units to particle ratio.
I think we all agree on this.
> However, the ratio of particles to
> PFU reflects the number of particles necessary to develop a plaque.
This is where you lose me, Dan. It seems there are two ways to interpret
this comment. (1) you titrate particles by an EM count, and then, knowing
that this virus generally gives a particle/PFU ratio of 50/1 (in our
initial example) you know that you have to dose 500 particles per dish to
have 10 viable particles and to see 10 plagues. I have no argument with
this. (2) this could mean that a single cell must get hit 50 times by
viable virus to accumulate enough virus information to get a productive
infection started. If this were the case I think the conclusion would be
"this virus doesn't form plaques".
> The
> first few steps (up to transcription of positive strand genome) of
> such an infection are completely dependent upon the first infectious
> particle.
This was my point in the earlier post.
> 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.
I can't tell if a particular virus particle that didn't make a plaque
in my experiment might have made one under other circumstances. What a PFU
titre tells me is that at a certain inoculum dilution I can expect to see
a certain number of plaques. It doesn't tell me anything about the virus
particles that don't form plaques: they might have no DNA/RNA in them,
they might be only partially defective due to mutation, they might be
perfectly viable & healthy but failed to find a cell receptor due to rare
bad luck. I can only score as infectious the ones that do productively
infect; a PFU assay says nothing about the others...
The point of my earlier post was that
viruses that can make a plaque do so on their own: since the assay is
done at limiting dilution it rules out multiple-hit phenomena and
reveals only the particles that can infect independently of other virus
particles. Discussion of infections that require multiple hits leads us
away from a strictly quantitative assay such as PFU titration, into
quantal assays such as measuring cell culture ID50 or animal ID50, where
the whole culture dish (or animal) is scored as infected or uninfected.
The presence of a large proportion of defective, potentially complementing
particles might show up in such an infectious assay; they cannot show up
in a plaque assay.
One type of exception to the one-hit comments is the plaque titration of
AAV, or of defective Ad-SV40 hybrids. I think that plaque titrations of
these viruses display two-hit kinetics, since plaques arise only from
cells infected with both defective and helper virus. Aside from cases like
these, plaque titrations of most viruses display one-hit kinetics,
confirmable by testing if plaque numbers are porportional to the first
power (as opposed to the second power for two-hit) of particle
concentration.
Regards, Kevin.
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Kevin W. Ryan
Department of Virology & Molecular Biology
St. Jude Children's Research Hospital
Memphis, Tennessee 38101-0318, U.S.A.
phone: (901) 495-3411
fax: (901) 523-2622
Internet: ryan at mbcf.stjude.org
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