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Avian leukosis virus subgroup J (ALV-J) is an oncogenic virus that causes serious economic loss in the poultry industry. Currently, no effective vaccine or drug is available against this virus. Therefore, it is imperative to explore and understand the molecular regulatory mechanisms underlying ALV-J infection. In this study, blood samples from 21 ALV-J-infected and 22 ALV-J-uninfected (DZ) chickens (JZ) were analyzed by whole-genome resequencing (WGR). By combining the fixation index (FST) with the nucleotide diversity (π) ratio based on WGR data, 425 candidate genes were identified. Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis revealed the top 20 enriched pathways, among which 9 pathways were significantly associated with diseases, including endometrial cancer, Chagas disease, PD-L1 expression and PD-1 checkpoint pathway in cancer, colorectal cancer, endocrine resistance, fluid shear stress, atherosclerosis, basal cell carcinoma, non-small cell lung cancer, and melanoma. Fourteen single nucleotide polymorphisms related to twelve genes showed a notable difference between DZ and JZ group chickens. The genes included COMMD3, PPP1CB, VEGFA, GTF2H1, NOTCH2, ITPR1, FGFR4, GNAS, NECTIN1, WNT2B, PPP1CC, and MRC2. These findings may provide a valuable foundation for further exploration of the pathogenesis of ALV-J in chickens.

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In recent years, superinfections of avian leukosis virus subgroup J (ALV-J) and infectious bursal disease virus (IBDV) have been frequently observed in nature, which has led to the increasing virulence in infected chickens. However, the reason for the enhanced pathogenicity has remained unclear. In this study, we demonstrated an effective candidate model for studying the outcome of superinfections with ALV-J and IBDV in cells and specific-pathogen-free (SPF) chicks. Through in vitro experiments, we found that ALV-J and IBDV can establish the superinfection models and synergistically promote the expression of IL-6, IL-10, IFN-α, and IFN-γ in DF-1 and CEF cells. In vivo, the weight loss, survival rate, and histopathological observations showed that more severe pathogenicity was present in the superinfected chickens. In addition, we found that superinfections of ALV-J and IBDV synergistically increased the viral replication of the two viruses and inflammatory mediator secretions in vitro and in vivo. Moreover, by measuring the immune organ indexes and blood proportions of CD3+, CD4+, and CD8α+ cells, our results showed that the more severe instances of immunosuppression were observed in the superinfected chickens. In the present study, we concluded that the more severe immunosuppression induced by the synergistic viral replication of ALV-J and IBDV is responsible for the enhanced pathogenicity.

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Exploration of the abnormal expression of exosomal molecules during the infection of avian leukosis virus subgroup J (ALV-J) is essential to provide a deeper understanding of the exosome's role in the viral pathogenesis involved. The study aimed to investigate the differentially expressed proteins and miRNAs of the exosomes derived from DF-1 cells infected by ALV-J, their gene function and involved signal pathways. We isolated exosomes from DF-1 cells infected by ALV-J. The differentially expressed proteins and miRNAs of the exosomes were determined by proteomics and transcription detection technology. A Gene Ontology (GO) analysis and a Kyoto Encyclopedia of Genes and Genomes (KEGG) signal pathway analysis identified the miRNAs target genes and the signal pathways regulated by the different proteins or/and miRNAs. A total of 116 proteins (58 upregulated and 58 downregulated) and 3 miRNAs (all upregulated) were determined. These proteins were involved in 155 signal pathways, in which the highest number of proteins involved in the cancer pathway was (up to) seven. The target genes of the miRNAs were involved in 3 signal pathways. Both the proteins and target genes of the miRNAs were involved in the Ribosome pathway and ECM-receptor interaction pathway. The results suggested that the ALV-J infection changed the proteins and miRNAs of the exosomes significantly.

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A sensitive sandwich-type electrochemical immunosensor was developed for the detection of avian leukosis virus subgroup J (ALV-J), which benefitted from multiple signal amplification involving graphene-perylene-3,4,9,10-tetracarboxylic acid nanocomposites (GR-PTCA), nanocellulose-Au NP composites (NC-Au) and the alkaline phosphatase (ALP) catalytic reaction. GR-PTCA nanocomposites on glassy carbon electrodes served as the immunosensor platform. Due to their excellent electrical conductivity and abundant polycarboxylic sites, the GR-PTCA nanocomposites allowed fast electron transfer and good immobilization of primary antibodies, thereby affording a strong immunosensor signal in the presence of ALV-J. The detected signal could be further amplified by the introduction of NC-Au composites as a carrier of secondary antibodies (Ab2) and by harnessing the catalytic properties of Au and ALP. Under optimized testing conditions, the electrochemical immunosensor displayed excellent analytical performance for the detection of ALV-J, showing a linear current response from 102.08 to 104.0 TCID50/mL (TCID50: 50% tissue culture infective dose) with a low detection limit of 101.98 TCID50/mL (S/N = 3). In addition to high sensitivity, the immunosensor showed very good selectivity, reproducibility and operational stability, demonstrating potential application for the quantitative detection of ALV-J in clinical diagnosis.

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Avian leukosis virus subgroup J (ALV-J) is an oncogenic retrovirus that has a similar replication cycle to multiple viruses and therefore can be used as a model system for viral entry into host cells. However, there are few reports on the genes or microRNAs (miRNAs) that are responsible for the replication of ALV-J. Our previous miRNA and RNA sequencing data showed that the expression of miR-34b-5p was significantly upregulated in ALV-J-infected chicken spleens compared to non-infected chicken spleens, but melanoma differentiation-associated gene 5 (MDA5) had the opposite expression pattern. In this study, a dual-luciferase reporter assay showed that MDA5 is a direct target of miR-34b-5p. In vitro, overexpression of miR-34b-5p accelerated the proliferation of ALV-J-infected cells by inducing the progression from G2 to S phase and it promoted cell migration. Ectopic expression of MDA5 inhibited ALV-J-infected cell proliferation, the cell cycle and cell migration, and knockdown of MDA5 promoted proliferation, the cell cycle and migration. In addition, during ALV-J infections, MDA5 can detect virus invasion and it triggers the MDA5 signaling pathway. MDA5 overexpression can activate the MDA5 signaling pathway, and thus it can inhibit the mRNA and protein expression of the ALV-J env gene and it can suppress virion secretion. In contrast, in response to the knockdown of MDA5 by small interfering RNA (siRNA) or an miR-34b-5p mimic, genes in the MDA5 signaling pathway were significantly downregulated (P < 0.05), but the mRNA and protein expression of ALV-J env and the sample-to-positive ratio of virion in the supernatants were increased. This indicates that miR-34b-5p is able to trigger the MDA5 signaling pathway and affect ALV-J infections. Together, these results suggest that miR-34b-5p targets MDA5 to accelerate the proliferation and migration of ALV-J-infected cells, and it promotes ALV-J replication, via the MDA5 signaling pathway.

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Avian leukosis virus subgroup J (ALV-J), an oncogenic retrovirus, induces growth retardation and neoplasia in chickens, leading to enormous economic losses in poultry industry. Increasing evidences showed several signal pathways involved in ALV-J infection. However, what signaling pathway involved in growth retardation is largely unknown. To explore the possible signaling pathway, we tested the cell proliferation and associated miRNAs in ALV-J infected CEF cells by CCK-8 and Hiseq, respectively. The results showed that cell proliferation was significantly inhibited by ALV-J and three associated miRNAs were identified to target Wnt/ß-catenin pathway. To verify the Wnt/ß-catenin pathway involved in cell growth retardation, we analyzed the key molecules of Wnt pathway in ALV-J infected CEF cells. Our data demonstrated that protein expression of ß-catenin was decreased significantly post ALV-J infection compared with the normal (P < 0.05). The impact of this down-regulation caused low expression of known target genes (Axin2, CyclinD1, Tcf4 and Lef1). Further, to obtain in vivo evidence, we set up an ALV-J infection model. Post 7 weeks infection, ALV-J infected chickens showed significant growth retardation. Subsequent tests showed that the expression of ß-catenin, Tcf1, Tcf4, Lef1, Axin2 and CyclinD1 were down-regulated in muscles of growth retardation chickens. Taken together, all data demonstrated that chicken growth retardation caused by ALV-J associated with down-regulated Wnt/ß-catenin signaling pathway.

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To compare the genetic diversity and quasispecies evolution of avian leukosis virus (ALV) among different individuals, 5 chickens, raised in Shandong Provice of China, were randomly selected from a local chicken flock associated with serious tumor cases. Blood samples were collected and inoculated into chicken embryo fibroblast and DF-1 cell lines for virus isolation and identification, respectively, of Marek's disease virus (MDV), reticuloendotheliosis virus (REV), and ALV. Five strains of ALV subgroup J (ALV-J) were identified, and the gp85 gene from each strain was amplified and cloned. For each strain, about 20 positive clones of gp85 gene were selected for sequence analyses and the variability of the quasispecies of the 5 strains was compared. The results showed that the nuclear acid length of gp85 gene of 5 ALV-J isolates is 921 bp, 921 bp, 924 bp, 918 bp, and 912 bp respectively, and amino acid homologies of different gp85 clones from the 5 ALV-J strains were 99.3 to 100%, 99.3 to 100%, 99.4 to 100%, 98.4 to 100%, 99.0 to 100%, respectively. The proportions of dominant quasispecies were 65.0%, 85.0%, 85.0%, 50.0%, 84.2%, respectively, and homology of the gp85 among these dominant quasispecies was 89.2 to 92.5%. These data demonstrated the composition of the ALV-J quasispecies varied among infected individuals even within the same flock, and the dominant quasispecies continued to evolve both for their proportion and gene mutation.

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BACKGROUND: Avian leukosis virus subgroup J (ALV-J) is an oncogenic retrovirus which causes immunosuppression and neoplasia in meat-type and egg-type chickens. ALV-J infects host cells via specific interaction between the viral Env and the cell surface receptor -chicken sodium hydrogen exchanger type 1 (chNHE1). NHE1 involved in altering the cellular pH and playing a critical role in tumorigenesis. However, little is known about the other relationship between ALV-J and chNHE1. METHODS AND RESULTS: In ALV-J infected DF-1 cells, the mRNA level of chNHE1 was up-regulated with time-dependent manner tested by real time PCR, and accordingly, intracellular pH was increased tested by spectrofluorometer. In vivo, the mRNA level of chNHE1 was determined by real time PCR in ALV-J infected experimental chickens and field cases. The result showed that the mRNA level of chNHE1 was up-regulated after virus shedding, especially in continuous viremic shedders (CS group). However, no significant difference was found between non-shedding group (NS group) and control group. In field cases, mRNA level of chNHE1 was positively correlated with increasing ALV-J load in tumor bearing and immune tolerance chickens. Furthermore, immunohistochemistry results showed that the protein expression of chNHE1 was up-regulated in different organs of both experimental chickens and tumor bearing chickens compared with the control. CONCLUSION: Taken together, we conclude that ALV-J induces chNHE1 up-regulation in viremia and neoplasia chickens.

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Cases of myeloid leukosis and hemangioma associated with avian leukosis virus subgroup J (ALV-J) are becoming more frequent in China in commercial layer chickens and breeders of egg-type chickens. In this study, two strains of ALV-J (SCAU11-H and SCAU11-XG) associated with hemangioma and myelocytoma were isolated from commercial broiler breeder animals in 2011. Their full-length proviral sequences were analyzed, revealing several unique genetic differences between the two isolates, and suggesting that the two viruses were derived from two distinct lineages. Strain SCAU11-H showed high sequence homology to early Chinese isolates associated with hemangioma, while strain SCAU11-XG was genetically closer to the prototype strain, HPRS-103. The complete genomic nucleotide sequences of SCAU11-H and SCAU11-XG were 7471 bp and 7727 bp in length, respectively. They shared 94.8% identity with each other, and had 94.0-96.8% nucleotide identity to ALV-J reference isolates. Homology analysis of the env, pol, and gag genes of the two isolates and other references strains showed that the gag and pol genes of the two viruses were more conserved than the env gene. In addition, the two isolates had significant deletions and substitutions in their 3'-UTR regions, compared to HPRS-103. These results suggest that the env gene and the 3'-UTR regions in these ALV-J isolates have evolved rapidly, and might be involved in the oncogenic spectrum of ALV-J. The results of this study contribute to our further study of the relationship between ALV integration patterns and multi-pathotypes associated with ALV-J.

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