Endogenous Avian Leukosis Virus Research Articles

Short communication: diversity of endogenous avian leukosis virus subgroup E elements in 11 chicken breeds.

Avian leukosis virus subgroup E (ALVE) as a kind of endogenous retroviruses extensively exists in chicken genome. The insertion of ALVE has some effects on chicken production traits and appearance. Most of the work on ALVEs has been done with commercial breeds. We present here an investigation of ALVE elements in seven Chinese domestic breeds and four standard breeds. Firstly, we established an ALVE insertion site dataset by using the obsERVer pipeline to identify ALVEs from whole-genome sequence data of eleven chicken breeds, seven Chinese domestic breeds, including Beijing You (BY), Dongxiang (DX), Luxi Game (LX), Shouguang (SG), Silkie (SK), Tibetan (TB) and Wenchang (WC), four standard breeds, including White Leghorn (WL), White Plymouth Rock (WR), Cornish (CS), and Rhode Island Red (RIR). A total of 37 ALVE insertion sites were identified and 23 of them were novel. Most of these insertion sites were distributed in intergenic regions and introns. We then used locus-specific PCR to validate the insertion sites in an expanded population with 18~60 individuals in each breed. The results showed that all predicted integration sites in 11 breeds were verified by PCR. Some ALVE insertion sites were breeds specific, and 16 out of 23 novel ALVEs were found in only one Chinese domestic chicken breed. We randomly selected three ALVE insertions including ALVE_CAU005, ALVE_ros127, and ALVE_ros276, and obtained their insertion sequences by long-range PCR and Sanger sequencing. The insertion sequences were all 7525 bp, which were full-length ALVE insertion and all of them were highly homologous to ALVE1 with similarity of 99%. Our study identified the distribution of ALVE in 11 chicken breeds, which expands the current research on ALVE in Chinese domestic breeds.

Retroviral Association with Ovarian Adenocarcinoma in Laying Hens

The laying hen has been used as a model for ovarian adenocarcinoma (OAC) in women. Previous work has shown an association between expression of endogenous retroviral proteins and elevated envelope mRNA and occurrence of OAC has been demonstrated in humans, but causality has not been demonstrated. The objective of this study was to determine whether there is a similar association between retrovirus presence and OAC in a commercial laying hen flock at the University of Illinois Poultry Research facility with a history of a high prevalence of OAC in its aged hens. Laying hens of three age strata were randomly selected for a cross-sectional study. Blood samples were collected, and serum was tested for antigens of endogenous or exogenous avian leukosis virus (ALV) by ELISA. Birds were humanely euthanized, and spleens, ovaries, and any tissues with gross lesions were sampled. Ovaries and tissues with gross lesions were examined histologically and spleens were used for RT-PCR to detect endogenous ALV via ALV-E env mRNA expression. Overall, hens with OAC were 5.2 times more likely to be ALV positive than hens without OAC (95% C.I. = 2.06-13.14). Holding age stratum constant, OAC positive hens were 3.6 times more likely to be positive for ALV via antigen-capture ELISA (95% C.I. 1.08- 11.96). Endogenous ALV-E in hens may be analogous to the human endogenous retroviruses, which have also been associated with OAC in women. Further studies to establish causation are warranted to better understand the potential for laying hens to serve as a laboratory model for viral-induced ovarian tumors in humans.

Identification and characterisation of endogenous Avian Leukosis Virus subgroup E (ALVE) insertions in chicken whole genome sequencing data

BackgroundEndogenous retroviruses (ERVs) are the remnants of retroviral infections which can elicit prolonged genomic and immunological stress on their host organism. In chickens, endogenous Avian Leukosis Virus subgroup E (ALVE) expression has been associated with reductions in muscle growth rate and egg production, as well as providing the potential for novel recombinant viruses. However, ALVEs can remain in commercial stock due to their incomplete identification and association with desirable traits, such as ALVE21 and slow feathering. The availability of whole genome sequencing (WGS) data facilitates high-throughput identification and characterisation of these retroviral remnants.ResultsWe have developed obsERVer, a new bioinformatic ERV identification pipeline which can identify ALVEs in WGS data without further sequencing. With this pipeline, 20 ALVEs were identified across eight elite layer lines from Hy-Line International, including four novel integrations and characterisation of a fast feathered phenotypic revertant that still contained ALVE21. These bioinformatically detected sites were subsequently validated using new high-throughput KASP assays, which showed that obsERVer was highly precise and exhibited a 0% false discovery rate. A further fifty-seven diverse chicken WGS datasets were analysed for their ALVE content, identifying a total of 322 integration sites, over 80% of which were novel. Like exogenous ALV, ALVEs show site preference for proximity to protein-coding genes, but also exhibit signs of selection against deleterious integrations within genes.ConclusionsobsERVer is a highly precise and broadly applicable pipeline for identifying retroviral integrations in WGS data. ALVE identification in commercial layers has aided development of high-throughput diagnostic assays which will aid ALVE management, with the aim to eventually eradicate ALVEs from high performance lines. Analysis of non-commercial chicken datasets with obsERVer has revealed broad ALVE diversity and facilitates the study of the biological effects of these ERVs in wild and domesticated populations.

Diversity of endogenous avian leukosis virus subgroup E (ALVE) insertions in indigenous chickens

BackgroundAvian leukosis virus subgroup E (ALVE) insertions are endogenous retroviruses (ERV) that are restricted to the domestic chicken and its wild progenitor. In commercial chickens, ALVE are known to have a detrimental effect on productivity and provide a source for recombination with exogenous retroviruses. The wider diversity of ALVE in non-commercial chickens and the role of these elements in ERV-derived immunity (EDI) are yet to be investigated.ResultsIn total, 974 different ALVE were identified from 407 chickens sampled from village populations in Ethiopia, Iraq, and Nigeria, using the recently developed obsERVer bioinformatics identification pipeline. Eighty-eight percent of all identified ALVE were novel, bringing the known number of ALVE integrations to more than 1300 across all analysed chickens. ALVE content was highly lineage-specific and populations generally exhibited a large diversity of ALVE at low frequencies, which is typical for ERV involved in EDI. A significantly larger number of ALVE was found within or near coding regions than expected by chance, although a relative depletion of ALVE was observed within coding regions, which likely reflects selection against deleterious integrations. These effects were less pronounced than in previous analyses of chickens from commercial lines.ConclusionsIdentification of more than 850 novel ALVE has trebled the known diversity of these retroviral elements. This work provides the basis for future studies to fully quantify the role of ALVE in immunity against exogenous ALV, and development of programmes to improve the productivity and welfare of chickens in developing economies.

Endogenous avian leukosis virus subgroup E elements of the chicken reference genome

The chicken reference genome contains 2 endogenous avian leukosis virus subgroup E (ALVE) insertions, but gaps and unresolved repetitive sequences in previous assemblies have hindered their precise characterization. Detailed analysis of the most recent reference genome (GRCg6a) now shows both ALVEs within contiguous chromosome assemblies for the first time. ALVE6 (ALVE-JFevA) and ALVE-JFevB are both located on chromosome 1, with ALVE6 close to the p-arm telomere. ALVE-JFevB is a structurally intact element containing the ALVE gag, pol, and env genes and is capable of forming replication competent viruses. In contrast, ALVE6 contains a 3,352 bp 5′ truncation and lacks the entire 5′ long terminal repeat and gag gene. Despite this, ALVE6 remains able to produce intact envelope protein, likely due to a mutation in the recognition site for a known inhibitory miRNA (miR-155). Whole genome resequencing data sets from layers, broilers, and 3 independent sources of wild-caught red junglefowl were surveyed for the presence of each of these reference genome ALVEs. ALVE-JFevB was found in no other chicken or red junglefowl genomes, whereas ALVE6 was identified in some layers, broilers, and native breeds but not within any other red junglefowl genome. Improved assembly contiguity has facilitated better characterization of the 2 ALVEs of the chicken reference genome. However, both the limited ALVE content and unique presence of ALVE-JFevB suggests that the reference individual is unrepresentative of ancestral Gallus gallus ALVE diversity.

Detection and characterization of endogenous avian leukosis virus obtained from contaminated avian vaccines

Detection and characterization of endogenous avian leukosis virus obtained from contaminated avian vaccines

Endogenous Avian Leukosis Virus in Combination with Serotype 2 Marek's Disease Virus Significantly Boosted the Incidence of Lymphoid Leukosis-Like Bursal Lymphomas in Susceptible Chickens.

In 2010, sporadic cases of avian leukosis virus (ALV)-like bursal lymphoma, also known as spontaneous lymphoid leukosis (LL)-like tumors, were identified in two commercial broiler breeder flocks in the absence of exogenous ALV infection. Two individual ALV subgroup E (ALV-E) field strains, designated AF227 and AF229, were isolated from two different breeder farms. The role of these ALV-E field isolates in development of and the potential joint impact in conjunction with a Marek's disease virus (MDV) vaccine (SB-1) were further characterized in chickens of an experimental line and commercial broiler breeders. The experimental line 0.TVB*S1, commonly known as the rapid feathering-susceptible (RFS) line, of chickens lacks all endogenous ALV and is fully susceptible to all subgroups of ALV, including ALV-E. Spontaneous LL-like tumors occurred following infection with AF227, AF229, and a reference ALV-E strain, RAV60, in RFS chickens. Vaccination with serotype 2 MDV, SB-1, in addition to AF227 or AF229 inoculation, significantly enhanced the spontaneous LL-like tumor incidence in the RFS chickens. The spontaneous LL-like tumor incidence jumped from 14% by AF227 alone to 42 to 43% by AF227 in combination with SB-1 in the RFS chickens under controlled conditions. RNA-sequencing analysis of the LL-like lymphomas and nonmalignant bursa tissues of the RFS line of birds identified hundreds of differentially expressed genes that are reportedly involved in key biological processes and pathways, including signaling and signal transduction pathways. The data from this study suggested that both ALV-E and MDV-2 play an important role in enhancement of the spontaneous LL-like tumors in susceptible chickens. The underlying mechanism may be complex and involved in many chicken genes and pathways, including signal transduction pathways and immune system processes, in addition to reported viral genes.IMPORTANCE Lymphoid leukosis (LL)-like lymphoma is a low-incidence yet costly and poorly understood disease of domestic chickens. The observed unique characteristics of LL-like lymphomas are that the incidence of the disease is chicken line dependent; pathologically, it appeared to mimic avian leukosis but is free of exogenous ALV infection; inoculation of the nonpathogenic ALV-E or MDV-2 (SB-1) boosts the incidence of the disease; and inoculation of both the nonpathogenic ALV-E and SB-1 escalates it to much higher levels. This study was designed to test the impact of two new ALV-E isolates, recently derived from commercial broiler breeder flocks, in combination with the nonpathogenic SB-1 on LL-like lymphoma incidences in both an experimental egg layer line of chickens and a commercial broiler breeder line of chickens under a controlled condition. Data from this study provided an additional piece of experimental evidence on the potency of nonpathogenic ALV-E, MDV-2, and ALV-E plus MDV-2 in boosting the incidence of LL-like lymphomas in susceptible chickens. This study also generated the first piece of genomic evidence that suggests host transcriptomic variation plays an important role in modulating LL-like lymphoma formation.

Reviving rare chicken breeds using genetically engineered sterility in surrogate host birds

In macrolecithal species, cryopreservation of the oocyte and zygote is not possible due to the large size and quantity of lipid deposited within the egg. For birds, this signifies that cryopreserving and regenerating a species from frozen cellular material are currently technically unfeasible. Diploid primordial germ cells (PGCs) are a potential means to freeze down the entire genome and reconstitute an avian species from frozen material. Here, we examine the use of genetically engineered (GE) sterile female layer chicken as surrogate hosts for the transplantation of cryopreserved avian PGCs from rare heritage breeds of chicken. We first amplified PGC numbers in culture before cryopreservation and subsequent transplantation into host GE embryos. We found that all hatched offspring from the chimera GE hens were derived from the donor rare heritage breed broiler PGCs, and using cryopreserved semen, we were able to produce pure offspring. Measurement of the mutation rate of PGCs in culture revealed that 2.7 × 10-10 de novo single-nucleotide variants (SNVs) were generated per cell division, which is comparable with other stem cell lineages. We also found that endogenous avian leukosis virus (ALV) retroviral insertions were not mobilized during in vitro propagation. Taken together, these results show that mutation rates are no higher than normal stem cells, essential if we are to conserve avian breeds. Thus, GE sterile avian surrogate hosts provide a viable platform to conserve and regenerate avian species using cryopreserved PGCs.

An endogenous retroviral element exerts an antiviral innate immune function via the derived lncRNA lnc-ALVE1-AS1

Endogenous retroviruses (ERVs) constitute an important component of animal and human genomes and are usually silenced by epigenetic mechanisms in adult cells. Although ERVs were recently reported to be linked to early development, tumorigenesis and autoimmune disease, their impacts on antiviral innate immunity and the underlying mechanisms have not been elucidated. Here, we provide the first direct evidence of an endogenous retroviral element affecting antiviral innate immunity via its derived antisense long non-coding RNA (lncRNA). We found that an antisense lncRNA, which is called lnc-ALVE1-AS1 and is transcribed from the endogenous avian leukosis virus in chromosome 1 (ALVE1), distinctly inhibited the entry and replication of exogenous retroviruses in chicken embryonic fibroblasts (CEFs). This behaviour is at least in part attributed to the induction of an antiviral innate immune pathway by ALVE1 activation, suggesting that an activated endogenous retroviral element may induce antiviral defence responses via its derived antisense lncRNA. We also found that lnc-ALVE1-AS1 mediated these effects by activating the TLR3 signalling in the cytoplasm. Our results provide novel insights into the antiviral innate immune function of ERVs, suggesting that ERVs may play an important role in antiviral defences and provide new strategies for the development of new vaccines.

MORPHOLOGICAL AND IMMUNOHISTOCHEMICAL CHARACTERIZATION OF A RHABDOMYOSARCOMA WITH SYSTEMIC METASTASIS IN AN AVIAN LEUKOSIS VIRUS (ALV) INFECTED CHICKEN

Immunohistochemistry features and cross-reactivity of commercially available antibodies are poorly understood in avian species. Here, we present a confirmative diagnosis of a rare systemically metastatic rhabdomyosarcoma in a seven-month-old male domestic chicken (Gallus domesticus). The animal had multiple grey to dark-red masses in the pectoral muscle, coelomic cavity, and multiple visceral organs. Microscopically, these masses were composed of spindloid neoplastic cells arranged into interwoven and patternless arrays with the presence of binucleated to multinucleated and karyomegalic neoplastic cells. A systemically metastatic rhabdomyosarcoma associated with avian leukosis viruses (ALV) infection was diagnosed by immunohistochemically demonstrating neoplastic cells dispersedly positive for desmin and p27 major capsid protein of ALVs, but diffusely negative for cytokeratin, vimentin, [Formula: see text]-smooth muscle actin, MyoD1, S-100, and neuron-specific enolase. These tumors and internal organs were further characterized positive for subgroups A, B, C, D, or E ALVs, but negative for subgroup J ALV, by polymerase chain reactions. The report suggests the potential of using IHC for a confirmative diagnosis of rhabdomyosarcoma and ALV infection in chickens. The role of endogenous and exogenous ALVs in the rhabdomyosarcoma is also discussed in the present report.

Further observations on serotype 2 Marek's disease virus-induced enhancement of spontaneous avian leukosis virus-like bursal lymphomas in ALVA6 transgenic chickens

Breeders of the 2009 generation of Avian Disease and Oncology Laboratory transgenic chicken line ALVA6, known to be resistant to infection with subgroups A and E avian leukosis virus (ALV), were vaccinated at hatch with a trivalent Marek's disease (MD) vaccine containing serotypes 1, 2, and 3 Marek's disease virus (MDV) and were maintained under pathogen-free conditions from the day of hatch until 75 weeks of age. Spontaneous ALV-like bursal lymphomas, also termed lymphoid leukosis (LL)-like lymphomas, were detected in 7% of the ALVA6 breeders. There was no evidence of infection with exogenous and endogenous ALV as determined by virus isolation tests of plasma and tumour tissue homogenates. For the next three generations, serotype 2 MDV was eliminated from the trivalent MD vaccine used. Results show, for the first time, that removal of serotype 2 MDV from MD vaccines eliminated spontaneous LL-like lymphomas within 50 to 72 weeks of age for at least three consecutive generations. Two experiments were also conducted to determine the influence of in ovo vaccination with serotype 2 MD vaccines on enhancement of spontaneous LL-like lymphomas in ALVA6 chickens. Chickens from the 2012 generation were each inoculated in ovo or at hatch with 5000 plaque-forming units of serotype 2 MDV. Results indicate that by 50 weeks of age the incidence of spontaneous LL-like lymphomas in chickens inoculated in ovo with serotype 2 MDV was comparable with that in chickens inoculated with virus at hatch, suggesting that the augmentation effect of serotype 2 MDV is independent of age of vaccination.

Novel Models of Avian Leucosis Virus-induced CarcinogenesisNovel Models of Avian Leucosis Virus-induced Carcinogenesis

The LSCC-SF-MC29 cell culture model system was further characterized by studies on the provirus content of the cells, the host range and the subgroup specificity of the produced virus. The transforming potential of the Mc29 virus was evaluated by the focus-forming and colony-forming assays on primary cell cultures and continuous cell lines of avian and mammalian origin. The in ovo effects of the myelocytomatosis virus Mc29 on 15I line White Leghorn chicken embryos were studied by routine histopathological methods. Six avian leucosis virus-specific proviral sequences were detected by PCR analysis in the genome of LSCC-SF-MC29 cells. The presence of a Mc29 provirus-specific sequence located in the gag-myc region was confirmed. Using primers designed to differentiate ALV subgroups, amplification product was obtained with subgroup B/D-specific PCR primers. As it was expected, the subgroup E-specific PCR primers amplified the endogenous ALV sequences. In vitro studies on the host range of Mc29 virus showed that the primary cultures of chicken and hamster cells and a continuous hamster cell line were susceptible, while the cultures of primary quail cells and of a permanent line of duck embryo cells were resistant to the transforming effect of the virus. In ovo, the inoculated Mc29 virus induced hyperplasic and preneoplastic lesions in the embryonal liver and pancreas and myxomas of the neck.

Early Detection of Avian Oncogenic Viruses from Blood of Apparently Healthy Chickens

Viral neoplasms in commercial poultry are mainly caused by members of two families with Marek's disease virus (MDV) belonging to Herpesviridae and reticuloendotheliosis virus (REV), avian leukosis virus subgroups A to E and avian leukosis virus subgroup J (ALV-J) belonging to Retroviridae. This study was con- ducted to know the status of neoplasms caused by avian oncogenic viruses in commercial chickens. 25 blood sam- ples were collected from a broiler breeder flock that appeared healthy but chickens from the flock on necropsy showed visceral tumours. PCR was employed on blood DNAs of these chickens for detection of avian oncogenic viruses which eventually detected the presence of multiple oncogenic virus infection in most birds. MDV specific primers that could differentiate between pathogenic and non-pathogenic serotype-1 virus detected MDV in four blood DNAs. REV could be detected from all the 25 blood DNAs and endogenous ALV was detected in 21 blood DNAs signifying the slow transforming nature of the ret- roviruses that may take months to perpetuate visible tumours. It was concluded that concurrent presence of multiple oncogenic viruses in the same bird is more common than the presence of single virus. Thus, for early disease control programs, this moderately simple PCR diagnostic technique can be utilized to identify the birds undergoing latent infection with avian oncogenic viruses.

Detection and molecular characterization of recombinant avian leukosis viruses in commercial egg-type chickens in China

Two natural recombinant avian leukosis viruses (ALVs) were isolated from Chinese commercial egg-type chickens in 2009, which suffered from haemangiomas and myelocytomas. Sequence analysis of the complete proviral genomes revealed several unique genetic characteristics of the present two isolates, demonstrating that the two viruses were derived from recombination between earlier Chinese ALV-J and endogenous virus sequences. The two recombinant viruses presented typical genetic organization of replication-competent genus Alpharetrovirus, and the gag and pol genes were well conserved with those of ALVs. The env genes of the two viruses were composed of the internal identical sequences (about 240 bp) of endogenous viruses, and the rest of the sequence belonged to subgroup J ALVs. The long terminal repeats of the two viruses were more closely related to HPRS-103 and earlier Chinese ALV-J than other subgroup ALVs, and multiple transcription regulatory elements of ALV-J were highly conserved. In addition, the two viruses shared an almost identical 3′-untranslated region (UTR) sequence with earlier Chinese ALV-J strains and the US strain 4817, containing a ~127 bp deletion in the E element region. However, further comparison with endogenous ALV indicated that the 3′-UTR sequences with ~127 bp deletion of ALV-J were most probably derived from endogenous viruses by recombination. These results suggested that the two isolates can be characterized as recombinant ALV-J with the internal env gene and 3′-UTR sequence of endogenous ALV.

Survey of Endogenous Virus andTVB*Receptor Status of Commercial Chicken Stocks Supplying Specific-Pathogen-Free Eggs

Endogenous avian leukosis virus (ALVE) and the ALVE receptor (TVB*S1) status of six commercial chicken lines supplying specific-pathogen-free eggs were analyzed. All commercial chicken lines are certified free of the avian leukosis virus (ALV) by screening for expression of the p27 protein using the standard enzyme-linked immunosorbent assay. The commercial chicken lines A, E, and F contained replication competent ALVE inserts. Line A was fixed for ALVE21, and lines E and F were segregating for ALVE10. In addition, ALVE1 was detected in all the chicken lines. Chicken lines B, D, and F were essentially fixed for the TVB*S1 allele that confers susceptibility to ALVE, whereas lines A, C, B, and E were resistant, containing either the TVB*S3 or TVB*R alleles. The results show that lines selected to be ALV p27 negative give rise to two different genotypes. One genotype lacks the TVB*S1 receptor for ALVE. Chicken lines with the TVB*S1 negative genotype can retain replication competent endogenous virus inserts such as ALVE2, 10, or 21 and still display the p27 negative phenotype. These replication competent ALVE viruses are phenotypically p27 negative in the absence of the TVB*S1 receptor because their chromosomal integration sites restrict transcription and subsequent production of the p27 protein and virus particles to levels below the detection limit. If the TVB*S1 receptor is present, the limited production of ALVE virus particles reinfects and integrates into more productive chromosomal locations in the cell. Increased production of infective virus particles and detectable levels of p27 follow this reinfection and integration into more active regions of the cells genome. The other genotype observed in the commercial lines retains the ALVE receptor (TVB*S1) but either lacks replication competent inserts or expresses the envelope encoded protein from defective inserts such as ALVE3 or ALVE6. In this phenotype, the env-coded glycoprotein encoded by the defective inserts binds to the TVB*S1 receptor and blocks the reinfection of the replication competent ALVE virus. This receptor interference stops reinfection and subsequent production of detectable virus particles and the p27 protein. Mixtures of different p27 negative phenotypes can result in the p27 positive phenotype and ALVE virus production. For example, mixtures of ALVE receptor positive (TVB*S1) but ALVE negative (p27 negative and envelope negative) chick embryo fibroblasts (CEFs) with fibroblasts that are receptor negative but ALVE positive could generate cells expressing high levels of p27 and ALVE virus. In this situation, the undetectable levels of ALVE virus from the receptor negative CEFs would infect and integrate into the receptor positive CEFs and produce detectable levels of ALVE virus. The implications of these findings for vaccine manufacturers and regulatory agencies are discussed.

Development of an Endogenous Virus–Free Line of Chickens Susceptible to All Subgroups of Avian Leukosis Virus

Primary chicken embryo fibroblasts (CEF) from special specific pathogen-free chicken lines are used for detection of contamination of adult or embryonic tissues, meconium, or tissue culture fluids with avian leukosis viruses (ALV). The suitability and efficiency of such tests depend on the susceptibility of CEF to the various subgroups of exogenous as well as endogenous ALV. The ideal CEF for such tests should be not only susceptible to all retroviruses, but also free of endogenous viruses so that such tests are immune to any interference that may occur between the endogenous and the tested (exogenous) viruses. CEF and/or chickens free of endogenous viruses are also desirable for gene transfer studies using retroviral vectors, such as RNA interference (RNAi) experiments and transgenic work. The absence of ev genes in CEF or chickens can empower clean detection of successful RNAi construct delivery or gene transfer. CEF free of ev genes are also essential reagents routinely used in growing and detecting unknown retroviruses in varied viral assays. This report documents the development of a new line of chickens, 0.TVB*S1, that is free of endogenous viruses and susceptible to all subgroups of ALV identified in chickens.

Development of a Polymerase Chain Reaction to Differentiate Avian Leukosis Virus (ALV) Subgroups: Detection of an ALV Contaminant in Commercial Marek's Disease Vaccines

Avian leukosis viruses (ALVs) are common in many poultry flocks and can be detected using an enzyme-linked immunosorbent assay or any other test designed to identify p27, the group-specific antigen located in gag. However, endogenous retroviruses expressing p27 are often present and can be confused with exogenous ALVs. A more specific and informative assay involves targeting the variable envelope glycoprotein gene (gp85) that is the basis for dividing ALVs into their different subgroups. We designed polymerase chain reaction (PCR) primers that would specifically detect and amplify viruses from each of the six ALV subgroups: A, B, C, D, E, and J. Subgroup B and D envelopes are related, and our B-specific primers also amplified subgroup D viruses. We also designed a set of common primers to amplify any ALV subgroup virus. To demonstrate the usefulness of these primers, we obtained from the Center for Veterinary Biologics in Iowa culture supernatant from chicken embryo fibroblasts infected with an ALV that was found to be a contaminant in two commercial Marek's disease vaccines. Using our PCR primers, we demonstrate that the contaminant was a subgroup A ALV. We cloned and sequenced a portion of the envelope gene and confirmed that the ALV was a subgroup A virus. Unlike typical subgroup A viruses, the contaminant ALV grew very slowly in cell culture. We also cloned and sequenced a portion of the long terminal repeat (LTR) from the contaminant virus. The LTR was found to be similar to those LTRs found in endogenous ALVs (subgroup E) and very dissimilar to LTRs normally found in subgroup A viruses. The E-like LTR probably explains why the contaminant grew so poorly in cell culture.

Isolation and Characterization of an Adventitious Avian Leukosis Virus Isolated from Commercial Marek's Disease Vaccines

Commercial Marek's disease (MD) vaccines produced by two manufacturers were tested for possible contamination with avian leukosis virus (ALV). Samples of MD vaccines manufactured by two companies (A and B) were received from a breeder company; samples were also received directly from vaccine company B. Using virus isolation tests, samples initially tested positive for subgroup E (endogenous) ALV. However, upon repassage, the vaccines also tested positive for exogenous ALV. The isolated exogenous ALV proved to be a subgroup A virus, as determined by flow cytometry using polyclonal chicken antibodies specific for various subgroups of ALV, and by DNA sequencing of the envelope glygoprotein (gp85). The exogenous ALV isolated from MD vaccines was inoculated in chickens from ADOL lines 15I(5) x 7(1) and 0 to determine its pathogenicity and compare it with that of Rous-associated-virus-1 (RAV-1), the prototype strain of ALV-A. Each chicken from each line was inoculated with approximately 10,000 infectious units of RAV-1 or the ALV-A isolated from vaccines termed B-39 virus at 7th day of embryonation. At hatch, and at 4, 8, and 16 wk of age, chickens were tested for viremia and cloacal shedding; chickens were also observed for ALV-induced tumors within 16 wk of age. Viremia and cloacal shedding results suggest that chickens from both lines were susceptible to infection with either virus. Within 16 wk of age, the proportion of ALV tumors induced by strain B-39 in line 0 and line 15I5 x 7(1) chickens was 0% and 12%, respectively, compared with 62% and 67% in chickens inoculated with RAV-1. The data indicate that commercial MD vaccines produced by two manufacturers were contaminated with endogenous subgroup E and an exogenous subgroup A ALV. Further, data from biological characterization suggest that the ALV-A isolated from commercial MD vaccines is of low oncogenicity, compared with that of RAV-1. GenBank accession numbers: The gp85 gene sequences of ALV isolated from commercial Marek's disease vaccines have been deposited in GenBank and assigned the following accession numbers: A46 subgroup A, DQ412726 ; B53 subgroup A, DQ412727; A46 subgroup E, DQ412728; B53 subgroup E, DQ412729.

Development and validation of a PCR-RFLP assay to evaluate TVB haplotypes coding receptors for subgroup B and subgroup E avian leukosis viruses in White Leghorns

The cellular receptor of subgroup B avian leukosis virus (ALVB) is encoded by a gene at the tumour virus B (TVB) locus. TVB alleles encode specific receptors permitting infection by exogenous ALVB or avian leukosis virus subgroup D (ALVD) as well as endogenous avian leukosis virus subgroup E (ALVE), and thus susceptibility is dominant to resistance. Two single nucleotide polymorphisms at the TVB locus have been reported distinguishing three TVB alleles (TVB*S1, TVB*S3 and TVB*R). We have developed a polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) assay using the two single nucleotide polymorphisms to define three observed allelic haplotypes and to identify the six possible TVB genotypes consisting of the three haplotypes in defined laboratory strains of chickens. One additional potential allelic haplotype and four genotypes were also briefly discussed. Chickens from parents heterozygous for different TVB alleles were challenged with Rous sarcoma viruses of subgroup ALVB and ALVE to induce wing-web tumours. Tumour incidences were evaluated between chickens of the genotypes determined with this newly developed PCR-RFLP assay. Importantly, chickens typed with this assay as TVB*S3/*S3 were resistant to infection by ALVE only, and those TVB*R/*R were resistant to both ALVE and ALVB. Furthermore, a vast majority of chickens with the susceptible TVB*S1/- genotypes developed a tumour. This PCR-RFLP assay enables a relatively rapid assessment of all six anticipated TVB genotypes in experimental strains of chickens undergoing segregation for TVB*S1, TVB*S3, and TVB*R alleles. This non-infectious assay should be further evaluated for the capacity to select and breed commercial chickens for genetic resistance to infections by ALVB, ALVD and ALVE.

Identification and characterization of avian retroviruses in chicken embryo-derived yellow fever vaccines: investigation of transmission to vaccine recipients.

All currently licensed yellow fever (YF) vaccines are propagated in chicken embryos. Recent studies of chick cell-derived measles and mumps vaccines show evidence of two types of retrovirus particles, the endogenous avian retrovirus (EAV) and the endogenous avian leukosis virus (ALV-E), which originate from the chicken embryonic fibroblast substrates. In this study, we investigated substrate-derived avian retrovirus contamination in YF vaccines currently produced by three manufacturers (YF-vax [Connaught Laboratories], Stamaril [Aventis], and YF-FIOCRUZ [FIOCRUZ-Bio-Manguinhos]). Testing for reverse transcriptase (RT) activity was not possible because of assay inhibition. However, Western blot analysis of virus pellets with anti-ALV RT antiserum detected three distinct RT proteins in all vaccines, indicating that more than one source is responsible for the RTs present in the vaccines. PCR analysis of both chicken substrate DNA and particle-associated RNA from the YF vaccines showed no evidence of the long terminal repeat sequences of exogenous ALV subgroups A to D in any of the vaccines. In contrast, both ALV-E and EAV particle-associated RNA were detected at equivalent titers in each vaccine by RT-PCR. Quantitative real-time RT-PCR revealed 61,600, 348,000, and 1,665,000 ALV-E RNA copies per dose of Stamaril, YF-FIOCRUZ, and YF-vax vaccines, respectively. ev locus-specific PCR testing of the vaccine-associated chicken substrate DNA was positive both for the nondefective ev-12 locus in two vaccines and for the defective ev-1 locus in all three vaccines. Both intact and ev-1 pol sequences were also identified in the particle-associated RNA. To investigate the risks of transmission, serum samples from 43 YF vaccine recipients were studied. None of the samples were seropositive by an ALV-E-based Western blot assay or had detectable EAV or ALV-E RNA sequences by RT-PCR. YF vaccines produced by the three manufacturers all have particles containing EAV genomes and various levels of defective or nondefective ALV-E sequences. The absence of evidence of infection with ALV-E or EAV in 43 YF vaccine recipients suggests low risks for transmission of these viruses, further supporting the safety of these vaccines.