Project description:Purpose: The goals of this study are to investigate the differentially expressed miRNAs between ALV-J infected (WRR+) and uninfected (WRR-)chickens spleens by Illumina deep sequencing. Methods: 140-day-old female chickens of White Recessive Rock (WRR) were confirmed as J subgroup avian leukosis virus (ALV-J) infection. Total RNA from three ALV-J-infected spleens (designated: WRR1+, WRR2+, WRR3+) and three uninfected normal spleen samples (designated: WRR1-, WRR2-, WRR3-) was isolated by TRIzol following the manufacturer’s instruction (Invitrogen, CA, USA). RNA samples of three individuals within each group were pooled with equal amounts, and then were subjected to Illumina deep sequencing by Illumina Hiseq 2500. Results: After raw data filtered, 12,150,275 and 15,227,930 reads of 18-32 bp, representing 569,847 and 543,062 unique sequences, were obtained for WRR- and WRR+ libraries, respectively. Through blasting with the chicken reference genome, 360,180 WRR- sequences and 327,391 WRR+ sequences, which accounted for more than 60% of the unique sequences, were perfectly matched.To analyze the miRNA detection efficiency of Illimuna deep sequencing, all the clean reads were blasted with the Rfam data base 10.1, annotated and then removed rRNA, tRNA, snoRNA and other snRNAs. The annotation results revealed that miRNAs accounted for more than 68% of all clean reads in the WRR− and WRR+ libraries. In this study, a total of 476 miRNAs were identified after compared the unique sequences against the chicken miRNAs precursors in miRBase 18.0. Base on unique sequences matched counts, 167 differential expression miRNAs were identified by DEGseq package using Benjamini-q-value of 0.001 as a cut-off. In ALV-J infected spleens, 83 miRNAs showed up-regulated expression and 84 were down-regulated when compared to uninfected samples. Conclusions: Our study represents the first time to analysis of miRNA Expression in Spleen of J Subgroup Avian Leukosis Virus (ALV-J) Infected (WRR+) and Uninfected (WRR-) Broilers. A total of 167 miRNAs were found to be differentially expressed in ALV-J infected spleens when compared to uninfected chickens. These miRNAs can be considered as candidates for further study ALV-J invasion. Overall design: Spleen miRNA profiles of 140-day-old ALV-J infected (WRR+) and uninfected (WRR-) female chickens of White Recessive Rock were generated by deep sequencing, using Illumina Hiseq 2500.
Project description:Avian leukosis virus subgroup J (ALV-J) is an avian oncogenic retrovirus that induces myeloid tumors and hemangiomas in chickens and causes severe economic losses with commercial layer chickens and meat-type chickens. High-throughput sequencing followed by quantitative real-time polymerase chain reaction and bioinformatics analyses were performed to advance the understanding of regulatory networks associated with differentially expressed non-coding RNAs and mRNAs that facilitate ALV-J infection. We examined the expression of mRNAs, long non-coding RNAs (lncRNAs), and miRNAs in the spleens of 20-week-old chickens infected with ALV-J and uninfected chickens. We found that 1723 mRNAs, 7,883 lncRNAs and 13 miRNAs in the spleen were differentially expressed between the uninfected and infected groups (P < 0.05). Transcriptome analysis showed that, compared to mRNA, chicken lncRNAs shared relatively fewer exon numbers and shorter transcripts. Through competing endogenous RNA and co-expression network analyses, we identified several tumor-associated or immune-related genes and lncRNAs. Along transcripts whose expression levels significantly decreased in both ALV-J infected spleen and tumor tissues, BCL11B showed the greatest change. These results suggest that BCL11B may be mechanistically involved in tumorigenesis in chicken and neoplastic diseases, may be related to immune response, and potentially be novel biomarker for ALV-J infection. Our results provide new insight into the pathology of ALV-J infection and high-quality transcriptome resource for in-depth study of epigenetic influences on disease resistance and immune system.
Project description:Avian leukosis virus subgroup J (ALV-J) can cause several different leukemia-like proliferative diseases in the hemopoietic system of chickens. Here, we investigated the transcriptome profiles and miRNA expression profiles of ALV-J-infected and uninfected chicken spleens to identify the genes and miRNAs related to ALV-J invasion. In total, 252 genes and 167 miRNAs were differentially expressed in ALV-J-infected spleens compared to control uninfected spleens. miR-23b expression was up-regulated in ALV-J-infected spleens compared with the control spleens, and transcriptome analysis revealed that the expression of interferon regulatory factor 1 (IRF1) was down-regulated in ALV-J-infected spleens compared to uninfected spleens. A dual-luciferase reporter assay showed that IRF1 was a direct target of miR-23b. miR-23b overexpression significantly (P = 0.0022) decreased IRF1 mRNA levels and repressed IRF1-3'-UTR reporter activity. In vitro experiments revealed that miR-23b overexpression strengthened ALV-J replication, whereas miR-23b loss of function inhibited ALV-J replication. IRF1 overexpression inhibited ALV-J replication, and IRF1 knockdown enhanced ALV-J replication. Moreover, IRF1 overexpression significantly (P = 0.0014) increased IFN-β expression. In conclusion, these results suggested that miR-23b may play an important role in ALV-J replication by targeting IRF1.
Project description:Avian leukosis is a neoplastic disease caused in part by subgroup J avian leukosis virus J (ALV-J). Micro ribonucleic acids (miRNAs) play pivotal oncogenic and tumour-suppressor roles in tumour development and progression. However, little is known about the potential role of miRNAs in avian leukosis tumours. We have found a novel tumour-suppressor miRNA, gga-miR-375, associated with avian leukosis tumorigenesis by miRNA microarray in a previous report. We have also previously studied the biological function of gga-miR-375; Overexpression of gga-miR-375 significantly inhibited DF-1 cell proliferation, and significantly reduced the expression of yes-associated protein 1 (YAP1) by repressing the activity of a luciferase reporter carrying the 3'-untranslated region of YAP1. This indicates that gga-miR-375 is frequently downregulated in avian leukosis by inhibiting cell proliferation through YAP1 oncogene targeting. Overexpression of gga-miR-375 markedly promoted serum starvation induced apoptosis, and there may be the reason why the tumour cycle is so long in the infected chickens. In vivo assays, gga-miR-375 was significantly downregulated in chicken livers 20 days after infection with ALV-J, and YAP1 was significantly upregulated 20 days after ALV-J infection (P<0.05). We also found that expression of cyclin E, an important regulator of cell cycle progression, was significantly upregulated (P<0.05). Drosophila inhibitor of apoptosis protein 1 (DIAP1), which is related to caspase-dependent apoptosis, was also significantly upregulated after infection. Our data suggests that gga-miR-375 may function as a tumour suppressor thereby regulating cancer cell proliferation and it plays a key role in avian leukosis tumorigenesis.
Project description:Co-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.In 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.We 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.
Project description:Purpose: The goals of this study are to investigate the differentially expressed genes between ALV-J infected (WRR+) and uninfected (WRR-)chickens spleens by Illumina deep sequencing. Methods: 140-day-old female chickens of White Recessive Rock (WRR) were confirmed as J subgroup avian leukosis virus (ALV-J) infection. Total RNA from three ALV-J-infected spleens (designated: WRR1+, WRR2+, WRR3+) and three uninfected normal spleen samples (designated: WRR1-, WRR2-, WRR3-) was isolated by TRIzol following the manufacturer’s instruction (Invitrogen, CA, USA). RNA samples of three individuals within each group were pooled with equal amounts, and then were subjected to Illumina deep sequencing by Illumina Genome Analyzer IIx. Results: Through raw data processed, 49,979,648 and 43,704,401 clean reads with an average length of 101 bp, which represented total residues of 4,859,084,087 and 4,238,826,168 bp, were obtained for WRR- and WRR+ libraries, respectively. Subsequently, the clean reads in the two libraries were assembled. Altogether, 121,493 contigs were assembled with an average length of 927 bp (ranged from 300 bp to 23,402 bp), leading to generation of 82,829 unigenes. The length of unigenes varied from 351 bp to 28,928 bp, with an average length of 1,155 bp. Based on the FPKM value of each gene, 252 DEGs were identified by DEGseq package using Benjamini-q-value of 0.05 as a cut-off. In ALV-J infected spleens, 90 genes showed up-regulated and 162 showed down-regulated expression when compared to uninfected samples. Conclusions: Our study represents the first time to elucidate the ALV-J infected chickens’spleens at the transcription level by RNA-seq technology. A total of 252 genes were found to be differentially expressed in ALV-J infected spleens when compared to uninfected chickens. These genes can be considered as candidates for further study ALV-J invasion. Spleen mRNA profiles of 140-day-old ALV-J infected (WRR+) and uninfected (WRR-) female chickens of White Recessive Rock were generated by deep sequencing, using Illumina Genome Analyzer IIx.
Project description:Subgroup J avian leukosis virus (ALV-J) is an oncogenic retrovirus that causes immunosuppression and enhances susceptibility to secondary infection. The innate immune system is the first line of defense in preventing bacterial and viral infections, and dendritic cells (DCs) play important roles in innate immunity. Because bone marrow is an organ that is susceptible to ALV-J, the virus may influence the generation of bone marrow-derived DCs. In this study, DCs cultured in vitro were used to investigate the effects of ALV infection. The results revealed that ALV-J could infect these cells during the early stages of differentiation, and infection of DCs with ALV-J resulted in apoptosis. miRNA sequencing data of uninfected and infected DCs revealed 122 differentially expressed miRNAs, with 115 demonstrating upregulation after ALV-J infection and the other 7 showing significant downregulation. The miRNAs that exhibited the highest levels of upregulation may suppress nutrient processing and metabolic function. These results indicated that ALV-J infection of chicken DCs could induce apoptosis via aberrant microRNA expression. These results provide a solid foundation for the further study of epigenetic influences on ALV-J-induced immunosuppression.
Project description:We have adapted the avian leukosis virus RCAS (replication-competent avian sarcoma-leukosis virus LTR splice acceptor)-mediated somatic gene transfer technique to introduce oncogenes into mammary cells in mice transgenic for the avian subgroup A receptor gene, tva, under control of the mouse mammary tumor virus (MMTV) promoter. Intraductal instillation of an RCAS vector carrying the polyoma middle T antigen (PyMT) gene (RCAS-PyMT) induced multiple, oligoclonal tumors within 3 weeks in infected mammary glands of MMTV-tva transgenic mice. The rapid appearance of these tumors from a relatively small pool of infected cells (estimated to be approximately 2 x 10(3) cells per gland by infection with RCAS carrying a GFP gene; RCAS-GFP) was accompanied by a high fraction of cells positive for Ki67, Cyclin D1, and c-Myc, implying strong proliferation competence. Furthermore, the tumors displayed greater cellular heterogeneity than did tumors arising in MMTV-PyMT mice, suggesting that RCAS-PyMT transforms a relatively immature cell type. Infection of mice transgenic for both MMTV-Wnt-1 and MMTV-tva with RCAS virus carrying an activated Neu oncogene dramatically enhanced tumor formation over what is observed in uninfected bitransgenic animals. We conclude that infection of mammary glands with retrovirus vectors is an efficient means to screen candidate oncogenes for their capacity to initiate or promote mammary carcinogenesis in the mouse.
Project description:Cordyceps militaris is a well-known Chinese medicinal fungus that has been used as a nutraceutical food in several Asian countries. Cordycepin (3'-deoxyadenosine), a secondary metabolite produced from Cordyceps militaris, has been demonstrated to exert a wide spectrum of pharmacological activities, such as anti-microbial and antitumor activities. However, the effect of cordycepin on immunomodulation in broilers is poorly investigated. In the current study, we investigated the effect of cordycepin (9.69, 19.38, and 38.76 mg) from Cordyceps militaris hot water extract (CMHW) on growth performance and immunocompetence in broilers. Results showed that CMHW significantly decreased inducible nitric oxide synthase (iNOS) mRNA levels in the bursa of Fabricius after 4 weeks of feeding (P<0.05). CMHW treatment reduced cyclooxygenase-2 (COX-2) mRNA levels in the spleen and bursa of Fabricius after 4 weeks of feeding (P<0.05). Supplementation of CMHW for 3 days after vaccination reduced iNOS mRNA level in the spleen of 14 and 28 day-old broilers (P<0.05). Prior to vaccination, CMHW pretreatment significantly down-regulated COX-2 mRNA levels in the spleen and bursa of Fabricius of 14-day-old broilers (P<0.05). Furthermore, CMHW significantly reduced lipopolysaccharide (LPS)-induced iNOS and COX-2 mRNA levels in the spleen and bursa of Fabricius (P<0.05). CMHW treatment attenuated LPS-induced IFN-? expression in the spleen and bursa of Fabricius, whereas CMHW induced IL-4 expression in these organs in response to LPS challenge (P<0.05). Taken together, these observations demonstrate that CMHW exerts an immunomodulatory role in broilers. CMHW is a potential novel feed additive with applications in inflammation-related diseases and bacterial infection in broilers.