The reverse transcriptase activity in cell-free medium of chicken embryo fibroblast cultures is not associated with a replication-competent retrovirus

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Abstract

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.

Study design: The kinetics of RT activity produced by 10-day-old chicken embyro 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.

Conclusion: The RT activity produced by chicken primary cell cultures is not associated with a retrovirus that can replicate in human cells.

Introduction

Of the commonly used viral vaccines licensed in the US, measles, mumps and influenza are produced in chicken cells derived from embryonated eggs (Plotkin and Mortimer, 1994). Recently, highly-sensitive PCR-based assays have been developed (Silver et al., 1993, Pyra et al., 1994, Heneine et al., 1995) that can detect RNA-dependent DNA polymerase or RT (Baltimore, 1970, Temin and Mizutani, 1970) in the equivalent of one to ten virus particles. One such assay designated as the PERT (Pyra et al., 1994) was used to detect RT activity in live, attenuated viral vaccines grown in chicken cells (Böni et al., 1996b). This RT activity has recently been found to be associated with a particle that contained RNAs related to the EAV-0 family (Weissmahr et al., 1997). Sequences of this latter family are prevalent in the genus Gallus and have been described as ancient proviral genomes (Dunwiddie et al., 1986, Resnick et al., 1990, Boyce-Jacino et al., 1992). Early studies using traditional RT assays had reported a particle-associated RT activity in the allantoic fluid of embryonated chicken eggs from avian retrovirus-free chickens and in concentrated medium of primary and secondary chicken embryo fibroblast cultures (Bauer and Hofschneider, 1977, Bauer et al., 1977, Bauer et al., 1978a, Bauer et al., 1978b). No infectious retrovirus was found associated with the RT activity using avian target cells (Bauer et al., 1978a).

The recent discovery of the RT activity in chicken cell-produced vaccines provided the impetus for the current re-evaluation of this RT activity using more sensitive assays in order to assure vaccine safety. Since the RT activity in these vaccines is due to its cell substrate, the major safety concern is the presence of a potential retrovirus that can replicate in human cells. We have therefore searched for replication-competent retroviruses in primary CEF cultures using four different human cell lines as well as PBMC as target cells. The human target cells were chosen to detect retroviruses of types C, D, E and F, which represent the different types of known primate infectious retroviruses (Hooks and Gibbs, 1975, Todaro et al., 1978, Sherwin and Todaro, 1979, Rhim et al., 1982, Dalgleish et al., 1984, Teich, 1984, Sommerfelt and Weiss, 1990).

Section snippets

PBRT assay

The PBRT assay was performed as previously described (Maudru and Peden, 1997). Briefly, RT reactions were conducted using 10 μl of undiluted or 1:10 diluted sample in a total volume of 25 μl containing a final concentration of 10 mM Tris–HCl, pH 8.3, 50 mM KCl, 5 mM MgCl2, 0.35% Triton X-100, 200 μM of each dNTP, 2 mM freshly-diluted DTT, 0.13 μM oligodeoxynucleotide RT primer (designated as M1 by Maudru and Peden (1997)), 8 U of RNasin (Promega, Madison, WI) and 0.28 pmol of MS2 RNA template

Kinetics of RT activity in medium of primary CEF cultures

The kinetics of PBRT activity produced in CEF cultures from 10-day-old embryos is shown in Fig. 1. Similar kinetics were seen using different number of cells, however the level of RT activity reflected the cell concentration, i.e. 10×106<20×106, <30×106. The results of the lowest and highest cell concentrations are shown in the figure. In all cases, RT activity was detected on day 1, increased to peak at day 5, and decreased thereafter. The peak RT activities in the CEF cultures ranged from 10−6

Discussion

A major safety concern associated with live, attenuated viral vaccines is the potential presence of adventitious agents including retroviruses derived from the cell substrate that may infect human cells and persist long-term as an integral part of the host genome. This is accomplished through the retrovirus replication cycle (Varmus and Swanstrom, 1984, Varmus and Swanstrom, 1985), which is characteristic of all retroviruses and depends in part upon the enzymatic activity of RT (Baltimore, 1970

Acknowledgements

We thank Theodore Bryan and Marilyn Lundquist for technical assistance; Drs Muhammad Shahabuddin, Hana Golding, Peter Patriarca and William Egan for discussions and comments on the manuscript; and Drs Dennis Trent, M. Carolyn Hardegree and Kathyrn Zoon for encouragement and support for this work. The PBRT work was supported in part by a grant from the National Vaccine Program Office to K.W.C. Peden.

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