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

Abstract

Previous findings of low levels of reverse transcriptase (RT) activity in chick cell-derived measles and mumps vaccines showed this activity to be associated with virus particles containing RNA of both subgroup E endogenous avian leukosis viruses (ALV-E) and endogenous avian viruses (EAV). These particles originate from chicken embryonic fibroblast (CEF) substrates used for propagating vaccine strains. To better characterize vaccine-associated ALV-E, we examined the endogenous ALV proviruses (ev loci) present in a White Leghorn CEF substrate pool by restriction fragment length polymorphism. Five ev loci were detected, ev-1, ev-3, ev-6, ev-18, andev-19. Both ev-18 and ev-19 can express infectious ALV-E, while ev-1, ev-3, and ev-6 are defective. We analyzed the full-length sequence of ev-1 and identified an adenosine insertion within the pol RT-␤ region at position 5026, which results in a truncated RT-␤ and integrase. We defined the 1,692-bp deletion in the gag-pol region of ev-3, and we found that in ev-6, sequences from the 5 long terminal repeat to the 5 pol region were absent. Based on the sequences of the ev loci, RT-PCR assays were developed to examine expression of ALV-E particles (EV) in CEF supernatants. 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. The use of chicken embryos and CEF in vaccine manufacturing requires that they be derived from closed, specific-pathogen-free source chickens that are free of known exogenous retroviral chicken pathogens, including the reticuloendotheliosis virus and the avian leukosis virus (ALV) groups (45).Investigations of the origin of RT activity in the measles vaccine found evidence of particles containing endogenous avian virus (EAV) RNA in a vaccine manufactured in Europe (44), while evidence of both EAV and endogenous avian leukosis virus (ALV-E) was found in a vaccine made in the United States (41). While vaccine manufacturing regulations require elimination of exogenous retroviral infections from source chickens, these regulations do not address the presence of endogenous retroviruses because such particles were not previously known to be associated with chick c...

Viral Nucleic Acids in Live-Attenuated Vaccines: Detection of Minority Variants and an Adventitious Virus

“…The detection of ALV and EAV particles in live vaccines derived from chicken embryo fibroblast has been reported previously and investigated extensively (14,15,17). Seroconversion to ALV and EAV was not detected among a TOT refers to total nucleic acids directly extracted from the vaccine suspensions.…”

“…Avian leukosis virus (ALV) and endogenous avian virus (AEV) have been reported in attenuated vaccines grown in chicken embryo fibroblasts (CEF), but extensive testing has also ruled out human infections (14,15). Vaccine-associated ALV and AEV are thought to originate from endogenous retroviruses in the chicken germ line (14,15,17).…”

Naturally Occurring Multiple Perineuriomas in a Chicken (Gallus domesticus)

“…The ev-1 locus ubiquitously identified in CEF of White Leghorns expresses few or no particles, because the locus has a +1 frameshift mutation in the pol RT-b region, which resulted in a truncated RT-b subunit and integrase. 3,14,24 Recently, however, we isolated a strain of ALV (TymS_90) from layer chickens affected with fowl glioma in Japan. 10 This strain is different from FGV, and viral genome analysis suggests a recombinant ALV derived from ev-1 and other strains of avian leukemia and sarcoma viruses.…”

Identification and Characterization of Avian Retroviruses in Chicken Embryo-Derived Yellow Fever Vaccines: Investigation of Transmission to Vaccine Recipients

“…The origin of RT activity has thus far been examined in measles vaccines produced in chicken embryonic fibroblasts (CEF). These studies have shown that this RT activity was associated with particles containing RNA from endogenous avian virus (EAV) and endogenous ALV (ALV-E) (17,28,30). These findings were consistent with current vaccine manufacturing regulations that require the elimination of exogenous retroviral infections from source chickens.…”

“…The defective nature of ev-1 is a result of a ϩ1 frameshift mutation at nucleotide position 5026 within the pol region. This signature mutation truncates the ␣ subunit of RT (RT-␣) and inactivates integrase (17). ev-1 is ubiquitous in White Leghorns, and the other loci are less prevalent and vary in number depending on the chicken line (14).…”