Deep Sequencing Analysis of miRNAs Expression in Spleen of J subgroup Avian Leukosis Virus Infected and Uninfected Broilers.
ABSTRACT: 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: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:Avian leukosis virus (ALV) is detrimental to poultry health and causes substantial economic losses from mortality and decreased performance. Because tumorigenesis is a complex mechanism, the regulatory architecture of the immune system is likely to include the added dimensions of modulation by miRNAs and long-noncoding RNA (lncRNA). To characterize the response to ALV challenge, we developed a novel methodology that combines four datasets: mRNA expression and the associated regulatory factors of miRNA and lncRNA, and ALV gene expression. Specific Pathogen-Free (SPF) layer chickens were infected with ALV-J or maintained as non-injected controls. Spleen samples were collected at 40 days post injection (dpi), and sequenced. There were 864 genes, 7 miRNAs and 17 lncRNAs differentially expressed between infected and non-infected birds. The combined analysis of the 4 RNA expression datasets revealed that ALV infection is detected by pattern-recognition receptors (TLR9 and TLR3) leading to a type-I IFN mediated innate immune response that is modulated by IRF7 and IRF1. Co-expression network analysis of mRNA with miRNA, lncRNA and virus genes identified key elements within the complex networks utilized during ALV response. The integration of information from the host transcriptomic, epigenetic and virus response also has the potential to provide deeper insights into other host-pathogen interactions.
Project description:Avian leukosis virus (ALV) causes substantial economic losses from mortality and decreased performance in poultry industry. To characterize the response to ALV challenge, we developed a novel methodology that combines four datasets: mRNA expression and their associated regulatory factors of miRNA and lncRNA, and ALV gene expression. Specific Pathogen-Free (SPF) layer chickens were assigned to the ALV-infected or control group. Spleen samples (n=6) were collected at 40 days post injection (dpi), and sequenced. Comparing the infected and non-infected groups, 864 genes, 7 miRNAs and 17 lncRNAs were differentially expressed.
Project description:Gross lesions characterized by swollen livers and spleens accompanied by diffuse white miliary spots, which resembled those of Marek's disease, were detected in two flocks of local meat-type chickens at a Japanese poultry processing plant in June and August 2010. The microscopic examinations revealed proliferative foci consisting of spindle or polymorphic cells in the interstitium of livers, splenic follicles and the interstitium of kidneys. These cells were positive immunohistochemically with Iba1 antibody, indicating they were histiocytic cells. Some of them contained antigens of avian leukosis virus (ALV) by immunohistochemistry,and the env gene of ALV subgroup J was detected from the spleens by polymerase chain reaction (PCR). Phylogenetic analysis of the PCR product indicated that the env gene might be descended from the American ADOL-7501 strain of ALV-J. These results suggest that the swollen livers and spleens of the meat-type chickens may come from histiocytic proliferation caused by ALV-J infection.
Project description:BACKGROUND: Five isolates (JS09GY2, JS09GY3, JS09GY4, JS09GY5, and JS09GY6) of avian leukosis virus subgroup J (ALV-J) were isolated from six infected commercial layer flocks displaying both hemangioma and myeloid leukosis (ML), which shared the same parental line, in China in 2009. RESULTS: All six of the commercial layer chickens examined showed hemangiomas on their body surface or feet. Some developed hemangiomas in their internal organs, causing hepatorrhexis and blood loss. Histopathologically different stages of hemangiomas with ML in the liver, heart, and spleen, were observed. Five viral isolates were obtained from infected DF1 cells incubated with the spleen tissue or serum of the birds from the six flocks. By full genome sequences analysis, a 19-nucleotide repeat sequence was identified in the primer binding site (PBS)-leader region of isolates JS09GY3 and JS09GY6, located between sites 249 and 250 according to the sequence of reference strain HPRS103, and also present in Rous sarcoma virus strain Schmidt-Ruppin B (RSV-SRB), Rous associated virus type 1 (RAV-1), and Rous associated virus type 2 (RAV-2). The predicted Gp85 proteins of isolates JS09GY2, JS09GY3, JS09GY5, and JS09GY6 were highly variable. Interestingly, the E elements of these four examined isolates showed a key deletion at site 30, which produced a new c-Ets-1 binding site. An 11-bp insertion was also found in the E element of isolate JS09GY3 located between bp 66 and 67 according to the sequence of reference strain HPRS103, while almost all previously reported Chinese strains showed an almost identical deletion of 127 bp in the same region. CONCLUSIONS: Five ALV-J isolates were obtained from six field infected commercial layer chickens. Coexistence of hemangioma and ML were observed in these infected cases both macro- and microscopically. Complete proviral genome sequences of two isolates (JS09GY3 and JS09GY6) and the partial sequences of the other two isolates (JS09GY2 and JS09GY5) were determined. The isolates were found to be recombinants of ALV-J with a PBS-leader sequence originating from other retroviruses. The Gp85 protein with an amino acid deletion, a contiguous 11-bp insertion mutation in the E element, and a novel binding site, were noted in the proviral genomes.
Project description:Avian leukosis virus subgroup J (ALV-J) can induce myeloid tumors and hemangiomas in chickens and causes severe economic losses with commercial layer chickens and meat-type chickens. Here, we generated ribominus RNA sequencing data from three normal chicken spleen tissues and three ALV-J-infected chicken spleen tissues. Structure analysis of transcripts showed that, compared to mRNAs and lncRNAs, chicken circRNAs shared relatively shorter transcripts and similar GC content. Differentially expression analysis showed 152 differentially expressed circRNAs with 106 circRNAs up regulated and 46 circRNAs down regulated. Through comparing differentially expressed circRNA host genes and mRNAs and performed ceRNA network analysis, we found several tumor or immune-related genes, in which, there were four genes existed in both differentially expressed mRNAs and circRNA host genes (Dock4, Fmr1, Zfhx3, Ralb) and two genes (Mll, Aoc3) involved in ceRNA network. We further characterized one exon-intron circRNA derived from HRH4 gene in the ceRNA network, termed circHRH4, which is an abundant and stable circRNA expressed in various tissues and cells in chicken and localizes in cytoplasm. Our results provide new insight into the pathology of ALV-J infection and circRNAs may also mediate tumorigenesis in chicken.
Project description:Subgroup A of the avian leukosis virus (ALV-A) can cause severe pathological lesions and death in infected chickens, and its reported hosts have increased recently. To assess the susceptibility of adult chickens, quails, and pigeons to ALV-A, three sets of 250-day-old birds were intraperitoneally inoculated with ALV-A. Viremia and cloacal virus shedding were dynamically detected using an immunofluorescence assay (IFA), ALV-P27 antigen ELISA or RT-PCR; pathological lesions were assessed using tissue sections; ALV-A in tissues was detected by IFA; and ALV-A antibody responses were detected using antibody ELISA kits and an immune diffusion test. The results indicated that persistent viremia occurred in 80% (8/10) of infected chickens, and transient viremia occurred in 17% (2/12) of infected quails, but no viremia occurred in infected pigeons. Cloacal virus shedding occurred intermittently in 80% (8/10) of infected chickens and in 8% (1/12) of infected quails but did not occur in infected pigeons. Severe inflammatory pathological lesions occurred in the visceral tissues of most infected chickens, and mild lesions occurred in a few of the infected quails, but no pathological lesions occurred in the infected pigeons. The ALV-A virus was detected in the visceral tissues of most infected chickens but not in the infected quails and pigeons. Obviously different ALV-A antibody responses occurred in the infected chickens, quails and pigeons. It can be concluded that adult chickens, quails and pigeons have dramatically different susceptibilities to ALV-A. This is the first report on artificial infection by ALV-A in different birds.
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.