Variety of Endogenous Proviruses in the Genomes of Chickens of Different Breeds

Retroviruses and retrovirus-derived vectors integrate nonrandomly into the genomes of host cells with specific preferences for transcribed genes, gene-rich regions, and CpG islands. However, the genomic features that influence the transcriptional activities of integrated retroviruses or retroviral vectors are poorly understood. We report here the cloning and characterization of avian sarcoma virus integration sites from chicken tumors. Growing progressively, dependent on high and stable expression of the transduced v-src oncogene, these tumors represent clonal expansions of cells bearing transcriptionally active replication-defective proviruses. Therefore, integration sites in our study distinguished genomic loci favorable for the expression of integrated retroviruses and gene transfer vectors. Analysis of integration sites from avian sarcoma virusinduced tumors showed strikingly nonrandom distribution, with proviruses found prevalently within or close to transcription units, particularly in genes broadly expressed in multiple tissues but not in tissue-specifically expressed genes. We infer that proviruses integrated in these genomic areas efficiently avoid transcriptional silencing and remain active for a long time during the growth of tumors. Defining the differences between unselected retroviral integration sites and sites selected for long-terminal-repeat-driven gene expression is relevant for retrovirus-mediated gene transfer and has ramifications for gene therapy.

Section: Methodsmentioning

confidence: 99%

Section: Production Of Transforming Virus and Tumor Inductionmentioning

confidence: 99%

Proviruses Selected for High and Stable Expression of Transduced Genes Accumulate in Broadly Transcribed Genome Areas

Plachý

Kotáb

Divina

et al. 2010

“…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). Since any given chicken may contain several different loci, the phenotypes of endogenous particles expressed by vaccine substrates may vary with the embryo pool used.…”

mentioning

confidence: 99%

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

Hussain

Johnson

Freire

et al. 2003

Yellow fever (YF) vaccines are highly efficacious in preventing mortality and morbidity in humans living in or traveling to regions of tropical Africa and South America where YF is endemic (3,20). To date, more than 400 million doses of YF vaccines have been administered (20). Currently licensed YF vaccines include the attenuated 17D strain, which is propagated in chicken embryos. Vaccine preparation typically includes inoculation of 7-to 9-day-old embryonated eggs with the vaccine strain. Infected embryos are harvested, pooled, and then homogenized. Homogenates are clarified by low-speed centrifugation, and the supernatant fluid containing the vaccine harvest is titrated and lyophilized under standardized conditions (33).Because of the risk of contamination with substrate-derived avian pathogens, regulations for vaccine manufacture require all avian embryos used for propagation of vaccine virus to be derived from a closed, specific pathogen-free (SPF) flock devoid of known avian pathogens including exogenous retroviruses of the avian leukosis virus (ALV) and the reticuloendotheliosis virus groups (32). Despite the use of SPF chicken flocks, low-level reverse transcriptase (RT) activity, an indication of retroviruses, was recently detected in YF vaccines and other chick cell-derived measles and mumps vaccines produced by several manufacturers in Europe and the United States (5,19,28). 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. These manufacturing regulations do not address the presence of endogenous retroviruses because such particles have not previously been known to be associated with chick cell-derived vaccines.Both EAV and ALV-E are members of endogenous retroviral families present in the chicken germ line. Little is known about the EAV family, which is distinct from but related to the ALV family. EAV elements are present in at least 50 copies per chicken genome (23). The majority of EAV loci remain uncharacterized, and the nucleotide sequences of full-length EAV genomes are not available. Partial EAV sequences show numerous frameshift mutations or sizable deletions in the env region (6).

“…In contrast, several complete loci (e.g., ev-2, ev -11, ev-12, ev-18, and ev-21) yield particles which, in vitro and in vivo, are infectious to a variety of subgroup E-receptive fowl (4,10,13,26,42). In White Leghorns, ev-1 is ubiquitous and the other loci are less prevalent and vary in number depending on the chicken line (21,40). Any given chicken may contain several different loci.…”

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confidence: 99%

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

Johnson

Heneine

2001

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...