Endogenous Retrovirus ev21 Dose Not Recombine with ALV-J and Induces the Expression of ISGs in the Host.
ABSTRACT: Avian leukosis virus subgroup J (ALV-J) infection can cause tumors and immunosuppression. Endogenous viruses integrate into host genomes and can recombine with exogenous avian leukosis virus (ALV). In this study, we analyzed the interaction of endogenous retrovirus 21 (ev21) with the ALV-J in late-feathering Chinese yellow chicken. Two ALV-J strains M180 and K243 were isolated from late-feathering and fast-feathering Chinese yellow chicken flocks, respectively. The env gene of the two strains showed 94.2-94.8% nucleotide identity with reference ALV-J strains. Compared with the env gene and the LTR of ev21 and M180, the nucleotide identity of LTR was 69.7% and env gene was 58.4%, respectively, especially the amino acid identity of env gene as low as 14.2%. Phylogenetic analysis of the nucleotide sequence of the env gene and the 3'LTR showed that M180 was closely related to ALV-J, and was located in a distinct group with ev21 in the phylogenetic tree. Using co-immunoprecipitation (co-IP), we next demonstrate that the envelope protein of ev21 does not interact with the M180 envelope protein. We further show that the envelope protein of ev21 cannot activate ALV-J LTR promoter activity using luciferase-reporter assays. qPCR and western blot analysis revealed that envelope protein of endogenous ev21 can facilitate the expression of PKR at 6h post ALV-J infection (hpi) and facilitate the expression of ISG12 and CH25H at 24 hpi. However, the expression of the env gene of M180 strain was not significantly at 6 and 24 hpi. We conclude that there is no evidence of recombination between endogenous retrovirus ev21 and ALV-J strain M180 in late-feathering Chinese yellow chicken, and envelope protein of ev21 can affect the expression of host ISGs, but appears not to influence the replication of ALV-J strain M180. This is the first report of interaction among the endogenous retrovirus ev21, ALV-J and the late-feathering chicken.
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: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: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:Background:Henny feathering in chickens is determined by a dominant mutation that transforms male-specific plumage to female-like plumage. Previous studies indicated that this phenotype is caused by ectopic expression in skin of CYP19A1 encoding aromatase that converts androgens to estrogen and thereby inhibits the development of male-specific plumage. A long terminal repeat (LTR) from an uncharacterized endogenous retrovirus (ERV) insertion was found in an isoform of the CYP19A1 transcript from henny feathering chicken. However, the complete sequence and the genomic position of the insertion were not determined. Results:We used publicly available whole genome sequence data to determine the flanking sequences of the ERV, and then PCR amplified the entire insertion and sequenced it using Nanopore long reads and Sanger sequencing. The 7524?bp insertion contains an intact endogenous retrovirus that was not found in chickens representing 31 different breeds not showing henny feathering or in samples of the ancestral red junglefowl. The sequence shows over 99% sequence identity to the avian leukosis virus ev-1 and ev-21 strains, suggesting a recent integration. The ERV 3'LTR, containing a powerful transcriptional enhancer and core promoter with TATA box together with binding sites for EFIII and Ig/EBP inside the CYP19A1 5' untranslated region, was detected partially in an aromatase transcript, which present a plausible explanation for ectopic expression of aromatase in non-ovarian tissues underlying the henny feathering phenotype. Conclusions:We demonstrate that the henny feathering allele harbors an insertion of an intact avian leukosis virus at the 5'end of CYP19A1. The presence of this ERV showed complete concordance with the henny feathering phenotype both within a pedigree segregating for this phenotype and across breeds.
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:Subgroup J avian leukosis virus (ALV-J) is a recently identified avian oncogenic retrovirus responsible for severe economic losses worldwide. In contrast with the other ALV subgroups, ALV-J predominantly induces myeloid leukosis in meat-type chickens. Despite significant homology with the other ALV subgroups across most of the genome, the envelope protein of ALV-J (EnvJ) shares low homology with the others. Pathogenicity and myeloid leukosis induction map to the env gene of ALV-J. A chimeric protein composed of the surface domain of EnvJ fused to the constant region of a rabbit IgG and mass spectrometry were used to identify the chicken Na(+)/H(+) exchanger type 1 (chNHE1) as a binding protein for ALV-J. Flow cytometry analysis and coprecipitation experiments demonstrated a specific interaction between EnvJ and chNHE1. When introduced into nonpermissive human 293T cells and quail QT6 cells, chNHE1 conferred susceptibility to EnvJ-mediated infection. Furthermore, 293T cells expressing chNHE1 fused with 293T cells expressing EnvJ in a low-pH-dependent manner. Together, these data identify chNHE1 as a cellular receptor for the highly pathogenic ALV-J.
Project description:The avian leukosis virus subgroup J (ALV-J) belongs to the chicken retrovirus that causes enormous economic losses in the poultry industry. Interferon-stimulated genes (ISGs) are critical for controlling virus infections. Here, we identified 897 type I ISGs induced by interferon-? (IFN-?) in chicken peripheral blood mononuclear cell (PBMC) by RNA-Seq. In addition, we further identified 152 potential anti-ALV-J chicken type I ISGs. Among these potential anti-ALV-J ISGs, chicken cholesterol 25-hydroxylase (chCH25H) was selected for further antiviral mechanism studies in chicken embryo fibroblast cell lines (DF1). The gene chCH25H is located on chromosome 6 and clustered in a distinct group with mammals CH25H in the phylogenetic tree. The core promoter region of chCH25H was located within -75/-1 sequence. We found that chCH25H was induced by chicken IFN-? and ALV-J in DF1 cells. The overexpression of chCH25H significantly inhibited ALV-J replication in DF1 cells at 48 h post infection (hpi). In addition, ALV-J replication was significantly enhanced in the chCH25H- knockout DF1 cells. Furthermore, we demonstrated that chCH25H restricted ALV-J infection through the production of 25-hydroxycholesterol (25HC), rather than type I and II interferon. Our results identified 152 potential anti-ALV-J chicken type I ISGs and revealed that 25HC, the product of chCH25H, could be used as a natural antiviral agent to control ALV-J infection.
Project description:Avian leukosis virus subgroup J (ALV-J) infection can cause tumors and immunosuppression in infected chickens. Macrophages play a central role in host defense against invading pathogens. In this study, we discovered an interesting phenomenon: ALV-J replication is weakened from 3 hours post-infection (hpi) to 36 hpi, which was verified using Western blotting and RT-PCR. To further investigate the interaction between ALV-J and macrophages, transcriptome analysis was performed to analyze the host genes' function in chicken primary monocyte-derived macrophages (MDM). Compared to the uninfected control, 624 up-regulated differentially expressed genes (DEG) and 341 down-regulated DEG at 3 hpi, and 174 up-regulated DEG and 87 down-regulated DEG at 36 hpi were identified in chicken MDM, respectively. ALV-J infection induced strong innate immune responses in chicken MDM at 3 hpi, instead of 36 hpi, according to the analysis results of Gene Ontology and KEGG pathway. Importantly, the host factors, such as up-regulated MIP-3?, IL-1?, iNOS, K60, IRG1, CH25H, NFKBIZ, lysozyme and OASL were involved in the host defense response during the course of ALV-J infection. On the contrary, up-regulated EX-FABP, IL4I1, COX-2, NFKBIA, TNFAIP3 and the Jak STAT pathway inhibitors including CISH, SOCS1 and SOCS3 are beneficial to ALV-J survival in chicken macrophages. We speculated that ALV-J tropism for macrophages helps to establish a latent infection in chicken MDM from 6 to 36 hpi. The present study provides a comprehensive view of the interactions between macrophages and ALV-J. It suggests the mechanisms of defense of chicken macrophages against ALV-J invasion and how ALV-J escape the host innate immune responses.
Project description:<h4>Background</h4>As a typical retrovirus, the evolution of Avian leukosis virus subgroup J (ALV-J) in different infectious ecosystems is not characterized, what we know is there are a cloud of diverse variants, namely quasispecies with considerable genetic diversity. This study is to explore the selection of infectious ecosystems on dominant variants and their evolutionary dynamics of ALV-J between DF1 cells and specific-pathogen-free (SPF) chickens. High-throughput sequencing platforms provide an approach for detecting quasispecies diversity more fully.<h4>Results</h4>An average of about 20,000 valid reads were obtained from two variable regions of gp85 gene and LTR-U3 region from each sample in different infectious ecosystems. The top 10 dominant variants among ALV-J from chicken plasmas, DF1 cells and liver tumor were completely different from each other. Also there was a difference of shannon entropy and global selection pressure values (?) in different infectious ecosystems. In the plasmas of two chickens, a large portion of quasispecies contained a 3-peptides "LSD" repeat insertion that was only less than 0.01% in DF1 cell culture supernatants. In parallel studies, the LTR-U3 region of ALV-J from the chicken plasmas demonstrated more variants with mutations in their transcription regulatory elements than those from DF1 cells.<h4>Conclusions</h4>Our data taken together suggest that the molecular epidemiology based on isolated ALV-J in cell culture may not represent the true evolution of virus in chicken flocks in the field. The biological significance of the "LSD" insert and mutations in LTR-U3 needs to be further studied.
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.