KSHV/HHV8 DNA and plasmids

Pat Moore psm9 at columbia.edu
Mon Dec 22 09:34:06 EST 1997


For KSHV/HHV8 PCR controls and probes, there are a number of sources of
reagents. In addition to Brian Foley's note regarding the NIH AIDS
Reagent Program (http://www.niaid.nih.gov/reagent) where Don Ganem has
deposited an EBV-/KSHV+ cell line, Ethel Cesarman has deposited several
KSHV+/EBV+ (BC-1 and BC-2) cell lines and our lab has deposited a
KSHV+/EBV- cell line  (BCP-1) at ATCC.  Either BCP-1 or BCBL-1 can be
used for the IFA serologic assay (Keddes pg 918-24, or Gao pg925-28,
Nature Medicine, 1996;2. ABI, Rockville MD also markets a lytic antigen
IFA which I haven't worked with and cannot vouch for. This might avoid
biohazard problems outlined below.)

We deposited the overlapping genomic clones for the BC-1 strain of KSHV
(a mixture of cosmid and lambda clones) for the entire BC-1 genome in
the AIDS Reagent Program, so that pretty much everyone should have
reasonable access to KSHV DNA without having to go back to primary
tissues for either gene cloning or probes.  These reagents are freely
available for noncommercial research purposes only.  

A note of concern regarding the cell lines:  KSHV is an unclassified
biohazard.  While preliminary evidence suggests that some of these cell
lines are nonproductive and that the virus is not easily transmitted,
caution is appropriate in working with them.  We advise working under
BL-3 conditions or at least BL-2 with BL-3 lab practices (e.g.
sterilization of all items taken out of the biosafety hood), a lab
monitoring program and training of employees working with the virus.  If
you are not working on virologic experiments, you may just want to work
with the DNA clones to avoid these concerns.    

Regarding PCR-based experiments in searching for the virus in cell
lines, tissues, etc., there has been alot of PCR-based work published in
reputable journals which is not reproducible and probably resulted from
inapparent PCR contamination.  Although PCR is useful for fishing
expeditions, more stringent confirmation for the presence of the virus
is required before work is published.  Preferably, Southern or northern
hybridization data.




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