The reverse transcriptase activity in cell-free medium of chicken embryo fibroblast cultures is not associated with a replication-competent retrovirus.
BACKGROUND
Reverse transcriptase (RT) activity has previously been reported in concentrated medium of primary chicken embryo cell cultures using the traditional RT assay. Recently, using the newly-developed and highly-sensitive product-enhanced reverse transcriptase (PERT) assay, RT activity has been detected in live, attenuated vaccines grown in chicken cell substrates. Furthermore, this activity has been associated with particles that contain RNA related to an ancient, endogenous avian retrovirus family designated as EAV-0.


OBJECTIVE
To investigate whether the RT activity present in vaccines produced in specific pathogen-free chicken cell substrates is associated with an infectious retrovirus that can replicate in human cells.


METHODS
The kinetics of RT activity produced by 10-day-old chicken embryo fibroblast (CEF) cultures was determined by analyzing cell-free medium in a PCR-based RT (PBRT) assay. Material containing the peak PBRT activity was used as the inoculum to infect various human cell lines and peripheral blood mononuclear cells. Filtered supernatants from control and test cultures were analyzed for the presence of replication-competent retroviruses by the PBRT assay. The cells were monitored for other adventitious agents by routine observation for cytopathic effect (CPE) and by transmission electron microscopy (TEM) at culture termination.


RESULTS
The PBRT activity did not increase above the background level in the human target cells through at least five cell passages, thus indicating the absence of a replicating retrovirus. No other adventitious agents were detected based upon TEM analysis and the absence of CPE.


CONCLUSIONS
The RT activity produced by chicken primary cell cultures is not associated with a retrovirus that can replicate in human cells.
Laboratory of Retrovirus Research, US Food and Drug Administration, Bethesda, MD 20892, USA. khan@a1.cber.fda.gov
A S
AS
Khan
T
T
Maudru
A
A
Thompson
J
J
Muller
J F
JF
Sears
K W
KW
Peden
D000818:Animals; D002478:Cells, Cultured; D002642:Chick Embryo; D018920:Coculture Techniques; D003470:Culture Media; D003588:Cytopathogenic Effect, Viral; D006801:Humans; D007700:Kinetics; D008854:Microscopy, Electron; D012194:RNA-Directed DNA Polymerase / Q000378:metabolism*; D012190:Retroviridae / Q000201:enzymology / Q000502:physiology*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013047:Specific Pathogen-Free Organisms; D014407:Tumor Cells, Cultured; D014779:Virus Replication
D003470:Culture Media; D012194:RNA-Directed DNA Polymerase