BackgroundThe pathogenesis of immunological tolerance caused by avian leukosis virus subgroup J (ALV-J), an oncogenic retrovirus, is largely unknown.ResultsIn this study, the development, differentiation, and immunological capability of B cells and their progenitors infected with ALV-J were studied both morphologically and functionally by using a model of ALV-J congenital infection. Compared with posthatch infection, congenital infection of ALV-J resulted in severe immunological tolerance, which was identified as the absence of detectable specific antivirus antibodies. In congenitally infected chickens, immune organs, particularly the bursa of Fabricius, were poorly developed. Moreover, IgM-and IgG-positive cells and total immunoglobulin levels were significantly decreased in these chickens. Large numbers of bursa follicles with no differentiation into cortex and medulla indicated that B cell development was arrested at the early stage. Flow cytometry analysis further confirmed that ALV-J blocked the differentiation of CD117+chB6+ B cell progenitors in the bursa of Fabricius. Furthermore, both the humoral immunity and the immunological capability of B cells and their progenitors were significantly suppressed, as assessed by (a) the antibody titres against sheep red blood cells and the Marek’s disease virus attenuated serotype 1 vaccine; (b) the proliferative response of B cells against thymus-independent antigen lipopolysaccharide (LPS) in the spleen germinal centres; and (c) the capacities for proliferation, differentiation and immunoglobulin gene class-switch recombination of B cell progenitors in response to LPS and interleukin-4(IL-4) in vitro.ConclusionsThese findings suggested that the anergy of B cells in congenitally infected chickens is caused by the developmental arrest and dysfunction of B cell progenitors, which is an important factor for the immunological tolerance induced by ALV-J.

BackgroundCo-infection with avian leukosis virus subgroup J and reticuloendotheliosis virus induces synergistic pathogenic effects and increases mortality. However, the role of exosomal miRNAs in the molecular mechanism of the synergistic infection of the two viruses remains unknown.ResultsIn this study, exosomal RNAs from CEF cells infected with ALV-J, REV or both at the optimal synergistic infection time were analysed by Illumina RNA deep sequencing. A total of 54 (23 upregulated and 31 downregulated) and 16 (7 upregulated and 9 downregulated) miRNAs were identified by comparing co-infection with two viruses, single-infected ALV-J and REV, respectively. Moreover, five key miRNAs, including miR-184-3p, miR-146a-3p, miR-146a-5p, miR-3538 and miR-155, were validated in both exosomes and CEF cells by qRT-PCR. GO annotation and KEGG pathway analysis of the miRNA target genes showed that the five differentially expressed miRNAs participated in virus-vector interaction, oxidative phosphorylation, energy metabolism and cell growth.ConclusionsWe demonstrated that REV and ALV-J synergistically increased the accumulation of exosomal miRNAs, which sheds light on the synergistic molecular mechanism of ALV-J and REV.Electronic supplementary materialThe online version of this article (10.1186/s12977-018-0427-0) contains supplementary material, which is available to authorized users.

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Congenital avian leukosis virus subgroup J (ALV-J) infection can induce persistent immunotolerance in chicken, however, the underlying mechanism remains unclear. Here, we demonstrate that congenital ALV-J infection induces the production of high-frequency and activated CD4+CD25+ Tregs that maintain persistent immunotolerance. A model of congenital infection by ALV-J was established in fertilized eggs, and hatched chicks showed persistent immunotolerance characterized by persistent viremia, immune organ dysplasia, severe imbalance of the ratio of CD4+/CD8+ T cells in blood and immune organs, and significant decrease in CD3+ T cells and Bu-1+ B cells in the spleen. Concurrently, the mRNA levels of IL-2, IL-10, and IFN-γ showed significant fluctuations in immune organs. Moreover, the frequency of CD4+CD25+ Tregs in blood and immune organs significantly increased, and the frequency of CD4+CD25+ Tregs was positively correlated with changes in ALV-J load in immune organs. Interestingly, CD4+CD25+ Tregs increased in the marginal zone of splenic nodules in ALV-J-infected chickens and dispersed to the germinal center. In addition, the proliferation and activation of B cells in splenic nodules was inhibited, and the number of IgM+ and IgG+ cells in the marginal zone significantly decreased. We further found that the mRNA levels of TGF- β and CTLA-4 in CD4+CD25+ Tregs of ALV-J-infected chickens significantly increased. Together, high-frequency and activated CD4+CD25+ Tregs inhibited B cells functions by expressing the inhibitory cytokine TGF-β and inhibitory surface receptor CTLA-4, thereby maintaining persistent immunotolerance in congenital ALV-J-infected chickens.

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22The tumorigenesis is the result of the accumulation of multiple oncogenes and tumor 23 suppressor genes changes. Co-infection of avian leucosis virus subgroup J (ALV-J) 24 and reticuloendotheliosis virus ( REV) , as two oncogenic retroviruses, showed 25 synergistic pathogenic effects characterized by enhanced tumor initiation and 26 progression. The molecular mechanism underlying synergistic effects of ALV-J and 27 REV on the neoplasia remains unclear. Here, we found co-infection of ALV-J and 28 REV enhanced the ability of virus infection, increased viral life cycle, maintained cell 29 survival and enhanced tumor formation. We combined the high-throughput proteomic 30 readout with a large-scale miRNA screening to identify which molecules are involved 31 in the synergism. Our results revealed co-infection of ALV-J and REV activated a 32 latent oncogene of KIAA1199 and inhibited the expression of tumor suppressor miR-33 147. Further, enhanced KIAA1199, down-regulated miR-147, activated NF-κB and 34 EGFR were demonstrated in co-infected tissues and tumor. Mechanistically, we 35 showed ALV-J and REV synergistically enhanced KIAA1199 by activation of NF-κB 36 and EGFR signalling pathway, and the suppression of tumor suppressor miR-147 37 was contributed to maintain the NF-κB/KIAA1199/EGFR pathway crosstalk by 38 targeting the 3'UTR region sequences of NF-κB p50 and KIAA1199. Our results 39 contributed to the understanding of the molecular mechanisms of viral synergistic 40 tumorgenesis, which provided the evidence that suggested the synergistic actions of 41 two retroviruses could result in activation of latent pro-oncogenes. 42 Author summary 3 43The tumorigenesis is the result of the accumulation of multiple oncogenes and tumor 44 suppressor genes changes. Co-infection with ALV-J and REV showed synergistic 45 pathogenic effects characterized by enhanced tumor progression, however, the 46 molecular mechanism on the neoplasia remains unclear. Our results revealed co-47 infection of ALV-J and REV promotes tumorigenesis by both induction of a latent 48 oncogene of KIAA1199 and suppression of the expression of tumor suppressor miR-49 147. Mechanistic studies revealed that ALV-J and REV synergistically enhance 50 KIAA1199 by activation of NF-κB and EGFR signalling pathway, and the suppression 51 of tumor suppressor miR-147 was contributed to maintain the NF-52 κB/KIAA1199/EGFR pathway crosstalk by targeting the 3'UTR region sequences of 53 NF-κB p50 and KIAA1199. These results provided the evidence that suggested the 54 synergistic actions of two retroviruses could result in activation of latent pro-55 oncogenes, indicating the potential preventive target and predictive factor for ALV-J 56 and REV induced tumorigenesis. 57 Introduction 58 Viral synergism occurs commonly in the nature when co-infection of two or more 59 unrelated viruses invades the same host. As two oncogenic retroviruses, avian 60 leukosis virus subgroup J (ALV-J) and reticuloendotheliosis virus (REV) are the 61 optimal model to study the synergisti...

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