Complete genome sequence of an avian leukosis virus isolate associated with hemangioma and myeloid leukosis in egg-type and meat-type chickens.
ABSTRACT: 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: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:Subgroup J avian leukosis virus (ALV-J) is a recently identified avian oncogenic retrovirus responsible for severe economic losses worldwide. In contrast with the other ALV subgroups, ALV-J predominantly induces myeloid leukosis in meat-type chickens. Despite significant homology with the other ALV subgroups across most of the genome, the envelope protein of ALV-J (EnvJ) shares low homology with the others. Pathogenicity and myeloid leukosis induction map to the env gene of ALV-J. A chimeric protein composed of the surface domain of EnvJ fused to the constant region of a rabbit IgG and mass spectrometry were used to identify the chicken Na(+)/H(+) exchanger type 1 (chNHE1) as a binding protein for ALV-J. Flow cytometry analysis and coprecipitation experiments demonstrated a specific interaction between EnvJ and chNHE1. When introduced into nonpermissive human 293T cells and quail QT6 cells, chNHE1 conferred susceptibility to EnvJ-mediated infection. Furthermore, 293T cells expressing chNHE1 fused with 293T cells expressing EnvJ in a low-pH-dependent manner. Together, these data identify chNHE1 as a cellular receptor for the highly pathogenic ALV-J.
Project description:Subgroup J avian leukosis virus (ALV-J) was first isolated from meat-type chickens that had developed myeloid leukosis and since 2008, ALV-J infections in chickens have become widespread in China. A comparison of the sequence of ALV-J epidemic isolates with HPRS-103, the ALV-J prototype virus, revealed several distinct features, one of which is a 19-nucleotide (nt) insertion in the leader sequence. To determine the role of the 19-nt insertion in ALV-J pathogenicity, a pair of viruses were constructed and rescued. The first virus was an ALV-J Chinese isolate (designated rSD1009) containing the 19-nt insertion in its leader sequence. The second virus was a clone, in which the leader sequence had a deleted 19-nt sequence (designated rSD1009?19). Compared with rSD1009?19, rSD1009 displayed a moderate growth advantage in vitro. However, no differences were demonstrated in either viral replication or oncogenicity between the two rescued viruses in chickens. These results indicated that the 19-nt insertion contributed to ALV-J replication in vitro but was not related to its pathogenicity in vivo.
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:Avian leukosis virus subgroup J (ALV-J) was first isolated from meat-type chickens in 1988. No field cases of ALV-J infection or tumors in layer chickens were observed worldwide until 2004. However, layer flocks in China have experienced outbreaks of this virus in recent years. The molecular epidemiology of ALV-J strains isolated from layer flocks was investigated. The env genes of 77.8% (21/27) of the ALV-J layer isolates with a high degree of genetic variation were significantly different from the env genes of the prototype strain of ALV-J (HPRS-103) and American and Chinese strains from meat-type chickens (designated ALV-J broiler isolates). A total of 205 nucleotides were deleted from the 3' untranslated region of 89.5% (17/19) of the ALV-J layer isolates. Approximately 94.7% (16/17) of the layer isolates contained a complete E element of 146 to 149 residues. The U3 sequences of 84.2% (16/19) of the ALV-J layer isolates displayed less than 92.5% sequence homology to those of the ALV-J broiler isolates, although the transcriptional regulatory elements that are typical of avian retroviruses were highly conserved. Several unique nucleotide substitutions in the env gene, the U3 region, and the E element of most of the ALV-J layer isolates were detected. These results suggested that the env gene, E element, and U3 region in the ALV-J layer isolates have evolved rapidly and were significantly different from those of the ALV-J broiler isolates. These findings will contribute to a better understanding of the pathogenic mechanism of layer tumor diseases induced by ALV-J.
Project description:In spite of the purification of the laying hens and broilers of avian leukosis virus (ALV) has made remarkable achievements, the infection of ALV was still serious in Chinese indigenous chickens.In order to assess the epidemic state of avian leukosis virus in indigenous chickens in China, 10 novel strains of ALV subgroup J (ALV-J), named JS16JH01 to JS16JH10, were isolated and identified by virus isolation and immunofluorescence antibody assays from a Chinese local breed farm with a sporadic incidence of tumors. To understand their virological characteristics further, the proviral genome of ENV-LTR was sequenced and compared with the reference strains.The homology of the gp85 gene between the ten ALV-J strains and NX0101 was in the range from 89.7-94.8% at the nuclear acid level. In addition, their gp85 genes were quite varied, with identities of 92-98% with themselves at the nuclear acid level. There were several snp and indel sites in the amino acid sequence of gp85 genes after comparison with other reference strains of ALV. Interestingly, a novel insertion in the gp85 region was found in two strains, JS16JH01 and JS16JH07, compared with NX0101 and HPRS-103.At present, owing to the large-scale purification of ALV in China, laying hens and broiler chickens with ALV infection are rarely detected, but ALVs are still frequently detected in the local chickens, which suggests that more efforts should be applied to the purification of ALV from indigenous chickens.
Project description:BACKGROUND: Avian Leukosis virus (ALV) of subgroup J (ALV-J) belong to retroviruses, which could induce tumors in domestic and wild birds. Myelocytomatosis was the most common neoplasma observed in infected flocks; however, few cases of hemangioma caused by ALV-J were reported in recent year. RESULTS: An ALV-J strain SCDY1 associated with hemangioma was isolated and its proviral genomic sequences were determined. The full proviral sequence of SCDY1 was 7489 nt long. Homology analysis of the env, pol and gag gene between SCDY1 and other strains in GenBank were 90.3-94.2%, 96.6-97.6%, and 94.3-96.5% at nucleotide level, respectively; while 85.1-90.7%, 97.4-98.7%, and 96.2-98.4% at amino acid level, respectively. Alignment analysis of the genomic sequence of ALV-J strains by using HPRS-103 as reference showed that a special 11 bp deletion was observed in U3 region of 3'UTR of SCDY1 and another ALV-J strain NHH isolated from case of hemangioma, and the non-functional TM and E element were absent in the genome of SCDY1, but the transcriptional regulatory elements including C/EBP, E2BP, NFAP-1, CArG box and Y box were highly conserved. Phylogenetic analysis revealed that all analyzed ALV-J strains could be separated into four groups, and SCDY1 as well as another strain NHH were included in the same cluster. CONCLUSION: The variation in envelope glycoprotein was higher than other genes. The genome sequence of SCDY1 has a close relationship with that of another ALV-J strain NHH isolated from case of hemangioma. A 11 bp deletion observed in U3 region of 3'UTR of genome of ALV-J isolated from case of hemangioma is interesting, which may be associated with the occurrence of hemangioma.
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:We report the complete genome sequence of avian leukosis virus subgroup J (ALV-J) isolate PDRC-59831, which causes myeloid leukosis and hemangiomas in chickens. This is an American ALV-J isolate, which was found in a 38-week-old broiler breeder chicken on a farm in Georgia in 2007.