Assessing the roles of endogenous retrovirus EAV-HP in avian leukosis virus subgroup J emergence and tolerance.
ABSTRACT: Avian leukosis virus (ALV) subgroup J is thought to have emerged through a recombination event between an unknown exogenous ALV and the endogenous retrovirus elements designated EAV-HP. All EAV-HP elements identified to date in the chicken genome show large deletions, including that of the entire pol gene. Here we report the identification of four segregating chicken EAV-HP proviruses with complete pol genes, one of which shows exceptionally high sequence identity and a close phylogenetic relationship with ALV-J with respect to the env gene. Embryonic expression of EAV-HP env has been suggested as a factor associated with immunological tolerance induction in a proportion of ALV-J-infected meat-type chickens. In support of this, env gene transcripts expressed from two of the four newly identified EAV-HP proviruses were demonstrated in chicken embryos. However, when ALV-J-infected outbred meat-type chickens were assessed, the presence of intact EAV-HP proviruses failed to directly correlate with ALV-J tolerance. This association was further examined using F(2) progeny of two inbred lines of layer chicken that differed in EAV-HP status and immunological responses to ALV-J. Immunological tolerance developed in a small proportion of F(2) progeny birds, reflecting the expected phenotypic ratio for inheritance of a double-recessive genotype; however, the status of tolerance did not show any direct correlation with the presence of the intact EAV-HP sequence. Nevertheless, identification of an intact chicken EAV-HP locus showing a uniquely close relationship to the ALV-J prototype clone HPRS-103 in the env region provides the strongest evidence of its contribution to the emergence of ALV-J by recombination.
Project description:Avian leukosis virus subgroup J (ALV-J), an exogenous avian retrovirus, is thought to have evolved by recombination with the highly identical env gene of the endogenous avian retrovirus EAV-HP. Embryonic expression of EAV-HP env has been suggested to be associated with the induction of immunological tolerance, a feature observed in a significant proportion of meat-type chickens infected with ALV-J. In support of this hypothesis, we demonstrate that EAV-HP loci, some of which could be associated with tolerance, are still segregating within the chicken population.
Project description:The existence of novel endogenous retrovirus elements in the chicken genome, designated EAV-HP, with close sequence identity to the env gene of avian leukosis virus (ALV) subgroup J has been reported (L. M. Smith, A. A. Toye, K. Howes, N. Bumstead, L. N. Payne, and K. Venugopal, J. Gen. Virol. 80:261-268, 1999). To resolve the genome structure of these retroviral elements, we have determined the complete sequence of two proviral clones of EAV-HP from a line N chicken genomic DNA yeast artificial chromosome library and from a meat-type chicken line 21 lambda library. The EAV-HP sequences from the two lines were 98% identical and had a typical provirus structure. The two EAV-HP clones showed identical large deletions spanning part of the gag, the entire pol, and part of the env genes. The env region of the EAV-HP clones was 97% identical to the env sequence of HPRS-103, the prototype subgroup J ALV. The 5' region of EAV-HP comprising the R and U5 regions of the long terminal repeat (LTR), the untranslated leader, and the 5' end of the putative gag region were 97% identical to the avian retrotransposon sequence, ART-CH. The remaining gag sequence shared less than 60% identity with other ALV sequences. The U3 region of the LTR was distinct from those of other retroviruses but contained some of the conserved motifs required for functioning as a promoter. To examine the ability of this endogenous retroviral LTR to function as a transcriptional promoter, the EAV-HP and HPRS-103 LTR U3 regions were compared in a luciferase reporter gene assay. The low luciferase activity detected with the EAV-HP LTR U3 constructs, at levels close to those observed for a control vector lacking the promoter or enhancer elements, suggested that these elements function as a weak promoter, possibly accounting for their low expression levels in chicken embryos.
Project description:Compared to other avian leukosis viruses (ALV), ALV-J primarily induces myeloid leukemia and hemangioma and causes significant economic loss for the poultry industry. The ALV-J Env protein is hypothesized to be related to its unique pathogenesis. However, the molecular determinants of Env for ALV-J pathogenesis are unclear. In this study, we compared and analyzed GP37 of ALV-J Env and the EAV-HP sequence, which has high homology to that of ALV-J Env. Phylogenetic analysis revealed five groups of ALV-J GP37 and two novel ALV-J Envs with endemic GP85 and EAV-HP-like GP37. Furthermore, at least 15 virus-adapted mutations were detected in GP37 compared to the EAV-HP sequence. Further analysis demonstrated that three tyrosine-based motifs (YxxM, ITIM (immune tyrosine-based inhibitory motif) and ITAM-like (immune tyrosine-based active motif like)) associated with immune disease and oncogenesis were found in the cytoplasmic tail of GP37. Based on the potential function and distribution of these motifs in GP37, ALV-J Env was grouped into three species, inhibitory Env, bifunctional Env and active Env. Accordingly, 36.91%, 61.74% and 1.34% of ALV-J Env sequences from GenBank are classified as inhibitory, bifunctional and active Env, respectively. Additionally, the Env of the ALV-J prototype strain, HPRS-103, and 17 of 18 EAV-HP sequences belong to the inhibitory Env. And models for signal transduction of the three ALV-J Env species were predicted. Our findings and models provide novel insights for identifying the roles and molecular mechanism of ALV-J Env in the unique pathogenesis of ALV-J.
Project description: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.
Project description:Avian leukosis virus subgroup J (ALV-J) is an immunosuppressive virus that causes considerable economic losses to the chicken industry in China. However, there is currently no effective vaccine to prevent ALV-J infection. In order to reduce the losses caused by ALV-J, we constructed two effective ALV-J vaccines by inserting the ALV-J (strain JL093-1) env or gag+env genes into the US2 gene of the Marek's disease herpesviruses (MDV) by transfection of overlapping fosmid DNAs, creating two recombinant MDVs, rMDV/ALV-gag+env and rMDV/ALV-env. Analysis of cultured chicken embryo fibroblasts infected with the rMDVs revealed that Env and Gag were successfully expressed and that there was no difference in growth kinetics in cells infected with rMDVs compared with that of cells infected with the parent MDV. Chickens vaccinated with either rMDV revealed that positive serum antibodies were induced. Both rMDVs also effectively reduced the rate of positive viremia in chicken flocks challenged with ALV-J. The protective effect provided by rMDV/ALV-env inoculation was slightly stronger than that provided by rMDV/ALV-gag+env. This represents the first study where a potential rMDV vaccine, expressing ALV-J antigenic genes, has been shown to be effective in the prevention of ALV-J. Our study also opens new avenues for the control of MDV and ALV-J co-infection.
Project description: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.
Project description:The EAV-HP group of chicken endogenous retrovirus elements was previously shown to be defective, with large deletions of the pol gene. In this report, we demonstrate that genomes of other Gallus species also maintain EAV-HP elements with similar deletions. The chicken EAV-HP1 locus was detected in both red (Gallus gallus gallus) and Sonnerat's (Gallus sonneratii) jungle fowl with identical integration sites, indicating that these elements had integrated before separation of the Gallus species. Furthermore, we demonstrate for the first time that the G. sonneratii genome carries EAV-HP elements with intact pol regions.
Project description: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-beta region at position 5026, which results in a truncated RT-beta 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.
Project description:The genetic determination of eggshell coloration has not been determined in birds. Here we report that the blue eggshell is caused by an EAV-HP insertion that promotes the expression of SLCO1B3 gene in the uterus (shell gland) of the oviduct in chicken. In this study, the genetic map location of the blue eggshell gene was refined by linkage analysis in an F(2) chicken population, and four candidate genes within the refined interval were subsequently tested for their expression levels in the shell gland of the uterus from blue-shelled and non-blue-shelled hens. SLCO1B3 gene was found to be the only one expressed in the uterus of blue-shelled hens but not in that of non-blue-shelled hens. Results from a pyrosequencing analysis showed that only the allele of SLCO1B3 from blue-shelled chickens was expressed in the uterus of heterozygous hens (O*LC/O*N). SLCO1B3 gene belongs to the organic anion transporting polypeptide (OATP) family; and the OATPs, functioning as membrane transporters, have been reported for the transportation of amphipathic organic compounds, including bile salt in mammals. We subsequently resequenced the whole genomic region of SLCO1B3 and discovered an EAV-HP insertion in the 5' flanking region of SLCO1B3. The EAV-HP insertion was found closely associated with blue eggshell phenotype following complete Mendelian segregation. In situ hybridization also demonstrated that the blue eggshell is associated with ectopic expression of SLCO1B3 in shell glands of uterus. Our finding strongly suggests that the EAV-HP insertion is the causative mutation for the blue eggshell phenotype. The insertion was also found in another Chinese blue-shelled breed and an American blue-shelled breed. In addition, we found that the insertion site in the blue-shelled chickens from Araucana is different from that in Chinese breeds, which implied independent integration events in the blue-shelled chickens from the two continents, providing a parallel evolutionary example at the molecular level.
Project description:The avian leukosis and sarcoma virus (ALSV) group comprises eight subgroups based on envelope properties. HPRS-103, an exogenous retrovirus recently isolated from meat-type chicken lines, is similar to the viruses of these subgroups in group antigen but differs from them in envelope properties and has been assigned to a new subgroup, J. HPRS-103 has a wide host range in birds, and unlike other nontransforming ALSVs which cause late-onset B-cell lymphomas, HPRS-103 causes late-onset myelocytomas. Analysis of the sequence of an infectious clone of the complete proviral genome indicates that HPRS-103 is a multiple recombinant of at least five ALSV sequences and one EAV (endogenous avian retroviral) sequence. The HPRS-103 env is most closely related to the env gene of the defective EAV-E51 but divergent from those of other ALSV subgroups. Probing of restriction digests of line 0 chicken genomic DNA has identified a novel group of endogenous sequences (EAV-HP) homologous to that of the HPRS-103 env gene but different from sequences homologous to EAV and E51. Unlike other replication-competent nontransforming ALSVs, HPRS-103 has an E element in its 3' noncoding region, as found in many transforming ALSVs. A deletion found in the HPRS-103 U3 EFII enhancer factor-binding site is also found in all replication-defective transforming ALSVs (including MC29, which causes rapid-onset myelocytomas).