Evidence of avian leukosis virus subgroup E and endogenous avian virus in measles and mumps vaccines derived from chicken cells: investigation of transmission to vaccine recipients

Abstract

Reverse transcriptase (RT) activity has been detected recently in all chicken cell-derived measles and mumps vaccines. A study of a vaccine manufactured in Europe indicated that the RT is associated with particles containing endogenous avian retrovirus (EAV-0) RNA and originates from the chicken embryonic fibroblasts (CEF) used as a substrate for propagation of the vaccine. We investigated the origin of RT in measles and mumps vaccines from a U.S. manufacturer and confirm the presence of RT and EAV RNA. Additionally, we provide new evidence for the presence of avian leukosis virus (ALV) in both CEF supernatants and vaccines. ALV pol sequences were first identified in particle-associated RNA by amplification with degenerate retroviral pol primers. ALV RNA sequences from both the gag and env regions were also detected. Analysis of hypervariable region 2 of env revealed a subgroup E sequence, an endogenous-type ALV. Both CEF- and vaccine-derived RT activity could be blocked by antibodies to ALV RT. Release of ALV-like virus particles from uninoculated CEF was also documented by electron microscopy. Nonetheless, infectivity studies on susceptible 15B1 chicken cells gave no evidence of infectious ALV, which is consistent with the phenotypes of the ev loci identified in the CEF. PCR analysis of ALV and EAV proviral sequences in peripheral blood mononuclear cells from 33 children after measles and mumps vaccination yielded negative results. Our data indicate that the sources of RT activity in all RT-positive measles and mumps vaccines may not be similar and depend on the particular endogenous retroviral loci present in the chicken cell substrate used. The present data do not support transmission of either ALV or EAV to recipients of the U.S.-made vaccine and provide reassurance for current immunization policies.

FIG. 1

(A) Detection of RT activity in uninoculated CEF supernatants and measurement of RT titer in MMR by the Amp-RT assay. Lane 1, HIV-1 as positive control; lane 2, culture medium as negative control; lane 3, water as assay reagent control; lane 4, culture supernatant of uninoculated CEF; lanes 5 to 8, end-point 10-fold dilutions of 1 μl of reconstituted MMR. (B) RT-PCR analysis of chicken β-actin mRNA sequences. Lane 1, particle-associated CEF RNA isolated from uninoculated culture supernatant; lane 2, cellular RNA from CEF cells; lane 3, assay reagent control. (C) RT-PCR amplification by generic retroviral pol primers by an RNA template-specific method. Lane 1, particle-associated RNA from CEF supernatants; lane 2, HIV-1 RNA as positive control; lane 3, RNA from supernatant of uninfected cell line A3.01 as negative control; lane 4, water as assay reagent control; lanes 5 to 8, duplicate RT-PCR controls of lanes 1 to 4, respectively, done with no RT in the reaction mixture; M, molecular weight marker. (D) Southern-blot hybridization of RT-PCR products shown in panel C with a specific HIV-1 pol probe.

FIG. 2

Analysis of ALV and EAV RNA sequences from uninoculated CEF supernatants. (A) RT-PCR products of ALV gag and env RNA sequences. CEF, RNA from CEF supernatant; RSV-B, Rous sarcoma virus B as positive control. (B) RT-PCR products of EAV pol and EAV element RNA. M, molecular weight marker; H₂O, reagent control.

FIG. 3

EM analysis of pellets of supernatants of CEF cultures (A and B) and of CEF (C). Arrows indicate ALV-like particles. Bar, •••.

FIG. 4

Typing of ev loci in CEF DNA by locus-specific PCR analysis. Test results for each locus show diagnostic PCR fragments of specific sizes that indicate the presence or absence of the locus. Lane 1, 50- to 2,500-bp DNA molecular size marker; lane 2, ev-1 test result showing fragments for the presence (295 bp) and absence (505 bp) of the locus, indicating heterozygosity; lane 3, ev-3 test result showing a fragment for the presence of the locus (190 bp) and a weaker signal for the negative fragment (270 bp), also suggesting heterozygosity; lane 4, ev-6 test result showing a fragment (300 bp) for the presence of the locus; lane 5, ev-7 test result showing a fragment (330 bp) for the absence of the locus.

FIG. 5

Neutralization of RT activity in culture supernatants of CEF, measles vaccine, AMV RT, and HIV-1 RT by antiserum against AMV RT (antiserum). pre, preimmune serum; OD, optical density.

FIG. 6

Detection of ALV and EAV RNA sequences in single-dose MMR vaccines (Vac). (A) Ethidium bromide-stained RT-PCR products. (B) Hybridization of RT-PCR products to internal oligonucleotide probes.

FIG. 7

PCR analysis of PBMC of MMR recipients for the presence of ALV (A) or EAV (B) proviral DNA sequences. The detection thresholds of known copy numbers of target sequences are shown in the righthand panels. Cont, positive controls containing 1 or 1,000 copies; H₂O, reagent control; pre and post, samples from vaccine recipients obtained before and after MMR vaccination, respectively.