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Differentially expressed genes in a flock of Chinese local-breed chickens infected with a subgroup J avian leukosis virus using suppression subtractive hybridization.
ABSTRACT: Avian leukosis virus subgroup J (ALV-J) is a new type of virus that mainly induces myeloid leukosis (ML) in chickens. To further elucidate the pathogenesis of ALV-J infection and tumor development, expression profiles from the bone marrow tissue of 15 infected and 18 non-infected birds from a local-breed poultry-farm under naturally infected conditions, were analyzed by suppression-subtractive hybridization. The birds were diagnosed as ML+ (or ML-) by specific ALV-J detection methods, involving serological tests for antigens and antibodies, and RT-PCR to detect viral RNA. A total of 59 partial gene sequences were revealed by differential screening of 496 forward and 384 reverse subtracted cDNA clones. Of these, 22 identified genes, including 8 up-regulated and 14 down-regulated, were related to immune functions, these genes being, MHC B-G antigen, translationally-controlled tumor protein (TPT1/TPTC), transferrin and ferritin, hemoglobin and Carbonic anhydrase. Four of the down-regulated genes were selected for further analysis, in view of their predicted roles in infection and immunity by real-time qRT-PCR, using RNA collected from the same birds as those used for SSH. The four genes were expressed at significantly lower levels (p < 0.001) in ALV-J infected birds than in non-infected ones.
Project description:Subgroup J avian leukosis virus (ALV-J) was first isolated from meat-type chickens that developed myeloid leukosis (ML). In recent years, field cases of hemangioma (HE) or HE and ML, rather than ML alone, have been reported in commercial layer flocks exposed to ALV-J with a high incidence in China. Here we report the complete genomic sequence of an ALV-J isolate that caused both HE and ML in egg-type and meat-type chickens in China. These findings will provide additional insights into the molecular characteristics in genomes, host range, and pathogenicity of ALV-J.
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:BACKGROUND: Avian leukosis virus subgroup J (ALV-J) preferentially induces myeloid leukosis (ML) in meat-type birds. Since 2008, many clinical cases of hemangioma rather than ML have frequently been reported in association with ALV-J infection in Chinese layer flocks. RESULTS: Three ALV-J strains associated with hemangioma were isolated and their proviral genomic sequences were determined. The three isolates, JL093-1, SD09DP03 and HLJ09MDJ-1, were 7,670, 7,670, and 7,633 nt in length. Their gag and pol genes were well conserved, with identities of 94.5-98.6% and 97.1-99.5%, respectively, with other ALV-J strains at the amino acid level (aa), while the env genes of the three isolates shared a higher aa identity with the env genes of other hemangioma strains than with those of ML strains. Interestingly, two novel 19-bp insertions in the U3 region in the LTR and 5' UTR, most likely derived from other retroviruses, were found in all the three isolates, thereby separately introducing one E2BP binding site in the U3 region in the LTR and RNA polymerase II transcription factor IIB and core promoter motif ten elements in the 5' UTR. Meanwhile, two binding sites in the U3 LTRs of the three isolates for NFAP-1 and AIB REP1 were lost, and a 1-base deletion in the E element of the 3' UTR of JL093-1 and SD09DP03 introduced a binding site for c-Ets-1. In addition to the changes listed above, the rTM of the 3' UTR was deleted in each of the three isolates. CONCLUSION: Our study is the first to discovery the coexistence of two novel insertions in the U3 region in the LTR and the 5' UTR of ALV-J associated with hemangioma symptoms, and the transcriptional regulatory elements introduced should be taken into consideration in the occurrence of hemangioma.
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:The integration of retroviruses into the host genome following nonrandom genome-wide patterns may lead to the deregulation of gene expression and oncogene activation near the integration sites. Slow-transforming retroviruses have been widely used to perform genetic screens for the identification of genes involved in cancer. To investigate the involvement of avian leukosis virus subgroup J (ALV-J) integration in myeloid leukosis (ML) in chickens, we utilized an ALV-J insertional identification platform based on hybrid capture target enrichment and next-generation sequencing (NGS). Using high-definition mapping of the viral integration sites in the chicken genome, 241 unique insertion sites were obtained from six different ALV-J-induced ML samples. On the basis of previous statistical definitions, MYC, TERT, and ZIC1 genes were identified as common insertion sites (CIS) of provirus integration in tumor cells; these three genes have previously been shown to be involved in the malignant transformation of different human cell types. Compared to control samples, the expression levels of all three CIS genes were significantly upregulated in chicken ML samples. Furthermore, they were frequently, but not in all field ML cases, deregulated at the mRNA level as a result of ALV-J infection. Our findings contribute to the understanding of the relationship between multipathotypes associated with ALV-J infection and the molecular background of tumorigenesis.ALV-Js have been successfully eradicated from chicken breeding flocks in the poultry industries of developed countries, and the control and eradication of ALV-J in China are now progressing steadily. To further study the pathogenesis of ALV-J infections, it will be necessary to elucidate the in vivo viral integration and tumorigenesis mechanism. In this study, 241 unique insertion sites were obtained from six different ALV-J-induced ML samples. In addition, MYC, TERT, and ZIC1 genes were identified as the CIS of ALV-J in tumor cells, which might be a putative "driver" for the activation of the oncogene. In addition, the CIS genes showed deregulated expression compared to nontumor samples. These results have potentially important implications for the mechanism of viral carcinogenesis.
Project description:Hens of a commercial Hy-line brown layer flock in China exhibited increased mortality and decreased egg production at 47 wk of age. From 47 to 57 wk, average weekly mortality increased from 0.11 to 3.0%, and egg production decreased from 10 to 30%, with a peak mortality rate (3.0%) observed at 54 wk of age. Necropsy of 11 birds demonstrated tissue damage that included hepatitis, liver hemorrhage, rupture, and/or enlarged livers. Microscopic liver lesions exhibited hepatocytic necrosis, lymphocytic periphlebitis, and myeloid leukosis. While no bacteria were recovered from liver and spleen samples, avian hepatitis E virus (HEV) RNA was detected in all 11 tested hens by nested reverse transcription-polymerase chain reaction. Of these, subgroup J avian leukosis virus (ALV-J) proviral DNA was detected in 5 hens by PCR. Alignments of partial ORF2 gene sequences obtained here demonstrated shared identity (76 to 97%) with corresponding sequences of other known avian HEV isolates. Env sequences of ALV-J isolates obtained here shared 50.1 to 55% identity with other ALV subgroups and 91.8 to 95.5% identity with other known ALV-J isolates. Phylogenetic tree analysis of selected sequences obtained here grouped an avian HEV sequence with genotype 3 HEV and assigned an ALV-J sequence to a branch separate from known ALV-J subgroups. Immunohistochemical results confirmed the presence of avian HEV and ALV-J in livers. Therefore, these results suggest that avian HEV and ALV-J co-infection caused the outbreak of hepatitis and liver hemorrhagic syndrome observed in the layer hen flock analyzed in this study.
Project description:In 2010, sporadic cases of avian leukosis virus (ALV)-like bursal lymphoma, also known as spontaneous lymphoid leukosis (LL)-like tumors, were identified in two commercial broiler breeder flocks in the absence of exogenous ALV infection. Two individual ALV subgroup E (ALV-E) field strains, designated AF227 and AF229, were isolated from two different breeder farms. The role of these ALV-E field isolates in development of and the potential joint impact in conjunction with a Marek's disease virus (MDV) vaccine (SB-1) were further characterized in chickens of an experimental line and commercial broiler breeders. The experimental line 0.TVB*S1, commonly known as the rapid feathering-susceptible (RFS) line, of chickens lacks all endogenous ALV and is fully susceptible to all subgroups of ALV, including ALV-E. Spontaneous LL-like tumors occurred following infection with AF227, AF229, and a reference ALV-E strain, RAV60, in RFS chickens. Vaccination with serotype 2 MDV, SB-1, in addition to AF227 or AF229 inoculation, significantly enhanced the spontaneous LL-like tumor incidence in the RFS chickens. The spontaneous LL-like tumor incidence jumped from 14% by AF227 alone to 42 to 43% by AF227 in combination with SB-1 in the RFS chickens under controlled conditions. RNA-sequencing analysis of the LL-like lymphomas and nonmalignant bursa tissues of the RFS line of birds identified hundreds of differentially expressed genes that are reportedly involved in key biological processes and pathways, including signaling and signal transduction pathways. The data from this study suggested that both ALV-E and MDV-2 play an important role in enhancement of the spontaneous LL-like tumors in susceptible chickens. The underlying mechanism may be complex and involved in many chicken genes and pathways, including signal transduction pathways and immune system processes, in addition to reported viral genes.IMPORTANCE Lymphoid leukosis (LL)-like lymphoma is a low-incidence yet costly and poorly understood disease of domestic chickens. The observed unique characteristics of LL-like lymphomas are that the incidence of the disease is chicken line dependent; pathologically, it appeared to mimic avian leukosis but is free of exogenous ALV infection; inoculation of the nonpathogenic ALV-E or MDV-2 (SB-1) boosts the incidence of the disease; and inoculation of both the nonpathogenic ALV-E and SB-1 escalates it to much higher levels. This study was designed to test the impact of two new ALV-E isolates, recently derived from commercial broiler breeder flocks, in combination with the nonpathogenic SB-1 on LL-like lymphoma incidences in both an experimental egg layer line of chickens and a commercial broiler breeder line of chickens under a controlled condition. Data from this study provided an additional piece of experimental evidence on the potency of nonpathogenic ALV-E, MDV-2, and ALV-E plus MDV-2 in boosting the incidence of LL-like lymphomas in susceptible chickens. This study also generated the first piece of genomic evidence that suggests host transcriptomic variation plays an important role in modulating LL-like lymphoma formation.
Project description:Subgroup J avian leukosis virus (ALV-J), an oncogenic retrovirus, causes hemangiomas and myeloid tumors in chickens. We previously showed that miR-125b is down-regulated in ALV-J-induced tumors. This study aimed to investigate the possible role of miR-125b in ALV-J-mediated infection and tumorigenesis. Knockdown of miR-125b expression in HP45 cells reduced, whereas over-expression induced late-stage apoptosis. Bioinformatics analysis and luciferase activity assays indicate that miR-125b targets Semaphorin 4D/CD100 (Sema4D) by binding the 3'-untranslated region of messenger RNA (mRNA). Up-regulation of miR-125b in the DF1 cell line suppressed Sema4D expression, whereas miR-125 down-regulation increased Sema4D expression levels. To uncover the function of Sema4D during ALV-J infection, animal infection experiments and in vitro assays were performed and show that Sema4D mRNA levels were up-regulated in ALV-J-infected tissues and cells. Finally, functional experiments show that miR-125 down-regulation and Sema4D over-expression inhibited apoptosis in HP45 cells. These results suggest that miR-125b and its target Sema4D might play an important role in the aggressive growth of HP45 cells induced by avian leukosis viruses (ALVs). These findings improve our understanding of the underlying mechanism of ALV-J infection and tumorigenesis.
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:Avian leukosis virus subgroup J (ALV-J) is an immunosuppressive virus that causes considerable economic losses to the chicken industry in China. However, there is currently no effective vaccine to prevent ALV-J infection. In order to reduce the losses caused by ALV-J, we constructed two effective ALV-J vaccines by inserting the ALV-J (strain JL093-1) env or gag+env genes into the US2 gene of the Marek's disease herpesviruses (MDV) by transfection of overlapping fosmid DNAs, creating two recombinant MDVs, rMDV/ALV-gag+env and rMDV/ALV-env. Analysis of cultured chicken embryo fibroblasts infected with the rMDVs revealed that Env and Gag were successfully expressed and that there was no difference in growth kinetics in cells infected with rMDVs compared with that of cells infected with the parent MDV. Chickens vaccinated with either rMDV revealed that positive serum antibodies were induced. Both rMDVs also effectively reduced the rate of positive viremia in chicken flocks challenged with ALV-J. The protective effect provided by rMDV/ALV-env inoculation was slightly stronger than that provided by rMDV/ALV-gag+env. This represents the first study where a potential rMDV vaccine, expressing ALV-J antigenic genes, has been shown to be effective in the prevention of ALV-J. Our study also opens new avenues for the control of MDV and ALV-J co-infection.