Analysis of a coded panel of licensed vaccines by polymerase chain reaction-based reverse transcriptase assays: A collaborative study

Characterization of Endogenous Avian Leukosis Viruses in Chicken Embryonic Fibroblast Substrates Used in Production of Measles and Mumps Vaccines

“…Following transfer, the DNA was UV cross-linked to the membrane and the blot was prehybridized at 65°C for 1 h with 100 ml of 2ϫ SET (0.05 M NaCl, 0.003 M Tris-Cl, and 0.2 mM EDTA) containing 200 mg of heparin. The digested DNA was hybridized overnight at 65°C with the 2.2-kb Multiprime (Boehringer-Mannheim) 32 P-labeled evpol-3ЈLTR probe generated from PCR amplification of CEF DNA using the evpolF8 and evLTRR2 primers (Table 1). Following hybridization, the membranes were given three 15-min washes with 2ϫ SSC (1ϫ SSC is 0.15 M NaCl plus 0.015 M sodium citrate)-0.5% sodium dodecyl sulfate and then one wash with 0.1ϫ SSC-0.2% sodium dedecyl sulfate at 65°C.…”

“…A 200-ng portion of each oligonucleotide probe was end labeled at 37°C for 30 min with 2 U of T4 polynucleotide kinase (New England Biolabs) and 50 Ci of [␥- 32 P]ATP in a total volume of 50 l. Probes were purified on Sephadex G-50 spin columns (Pharmacia Biotech) to remove unbound [␥-32 P]ATP. Hybridizations were performed using 67 ng of the labeled oligonucleotide probe.…”

“…Both ev-1-and ev-3-like RNA sequences were identified, as well as two other RNA sequences with intact pol regions, presumably of ev-18 and ev-19 origin. Inoculation of susceptible quail fibroblasts with CEF culture supernatants from both 5-azacytidine-induced and noninduced CEF led to ALV infection, confirming the presence of infectious ALV-E. Our data demonstrate that both defective and nondefective ev loci can be present in CEF vaccine substrates and suggest that both ev classes may contribute to the ALV present in vaccines.Reverse transcriptase (RT) activity, an indication of the presence of retroviruses, was recently detected in chick cellderived live, attenuated vaccines including those produced by European and U.S. manufacturers for measles, mumps, and yellow fever (8,32,41). Chicken embryos and chicken embryonic fibroblasts (CEFs) from controlled breeding flocks are used in vaccine manufacture to propagate high-titer attenuated vaccine inocula.…”

“…Reverse transcriptase (RT) activity, an indication of the presence of retroviruses, was recently detected in chick cellderived live, attenuated vaccines including those produced by European and U.S. manufacturers for measles, mumps, and yellow fever (8,32,41). Chicken embryos and chicken embryonic fibroblasts (CEFs) from controlled breeding flocks are used in vaccine manufacture to propagate high-titer attenuated vaccine inocula.…”

Detection of Avian Retroviruses in Vaccines by Amplification on DF-1 Cells with Immunostaining and Fluorescent Product-Enhanced Reverse Transcriptase Endpoint Methods

“…The detection of RT activity in yellow fever, measles, and mumps vaccines (6,7) clearly demonstrated a need for effective methods to test for the presence of replication-competent retroviral contaminants. It was discovered upon further investigation that nonreplicative endogenous avian retroviral elements not associated with infectious retroviruses were responsible for the positive RT signals observed in these vaccines (8,9,10,11,12).…”