IUBio Biosequences .. Software .. Molbio soft .. Network News .. FTP

PFU/Particle ratios

James Crowe jcrowe at nih.gov
Tue Aug 1 07:01:01 EST 1995


Just a few thoughts on pfu from an applied perspective:

PFU involves both virus and host contributions. The factors on the host
side permitting or limiting plaque formation are not well understood, but
are clearly significant. Our laboratory uses these phenotypic phenomema as
the basis for development of experimental vaccines for respiratory viruses.

By random chemical mutagenesis or multiple passages in cell culture, one
can generate plaque morphology or conditional lethal mutants, with
phenotypes such as small-plaque, temperature sensitive, cold-adapted,
cell-line adapted, animal host-range restricted, etc (until recently these
neg. sense single strand viruses were not amenable to cDNA rescue). These
phenotypes can generally be detected in a range of cell types, but not
always.

For instance, some mutants are able to plaque well on Vero but not at all
on HEp-2. Plaque size also varies depending on cell type (the method of
plaque detection becomes important here, for instance, we find plaque size
determination to be infinitely more reproducible with a specific method of
detection like immunperoxidase staining of fixed monolayers with monoclonal
antibodies). Also, the degree of temperature sensitivity of ts mutants
varies depending on the cell type. Temperature sensitivity is defined by
efficiency of plaque formation (pfu at a temp. relative to the pfu at a
permissive temperature). By quantitating virus stocks by pfu in several
cell types at up to ten temperatures simultaneously (as we sometimes do)
one can determine that many of these mutants have an infinitely variable
pfu/particle ratio depending on cell type and temperature. (Other factors
are important too, like confluency and passage level of cells, status and
type of medium esp buffer system, type of overlay, length of incubation,
contaminants, etc. Therefore we find that one can only really make
comparisons of plaque phenotypes within a test, as compared to known
control virus stocks, not between tests).

If plaque formation is dependent on such conditons, what is the relevance
of pfu? I think that you have to think of the biological relevance of in
vitro markers for in vivo replication. It is obvious that the whole
Jennerian approach is base on host-range restricted phenomena. Also,the
site of replication is important, for ex. respiratory viruses in general
replicate in respiratory epithelia (therfore one might expect human
epithelial cell lines to be best in vitro marker system for these viruses).
Different sites of the body have not only different cell types, but also
different conditions. For instance, we have demonstrated that ts mutants
with 36 degree C shutoff temperatures will replicate in the upper airway
(gradient of temp from RT at the nares to about 34 in the posterior
oropharynx) but replicate poorly at the 37.5 core body temp found in the
lower airways.

For live attenuated vaccine development, the ultimate goal is to
identifying viruses that infect the natural host (for ex. human infants
being immunized) but are growth restricted, and therefore attenuated. One
can standardize procedures for quantitating pfu in a relevant cell type at
relevant temperatures and conditions and follow ability to replicate in
vivo. We have identified in vitro markers (based on plaque morphology, ts
phenotype, etc),that correlate in a linear fashion with replication and
reactogenicity in human vaccinees. This makes pfu relevant to us.

Apologies for rambling.

J. Crowe, RVS, LID, NIAID
jcrowe at nih.gov






More information about the Virology mailing list

Send comments to us at biosci-help [At] net.bio.net