Detection and molecular characterization of J subgroup avian leukosis virus in wild ducks in China.
ABSTRACT: To assess the status of avian leukosis virus subgroup J (ALV-J) in wild ducks in China, we examined samples from 528 wild ducks, representing 17 species, which were collected in China over the past 3 years. Virus isolation and PCR showed that 7 ALV-J strains were isolated from wild ducks. The env genes and the 3'UTRs from these isolates were cloned and sequenced. The env genes of all 7 wild duck isolates were significantly different from those in the prototype strain HPRS-103, American strains, broiler ALV-J isolates and Chinese local chicken isolates, but showed close homology with those found in some layer chicken ALV-J isolates and belonged to the same group. The 3'UTRs of 7 ALV-J wild ducks isolates showed close homology with the prototype strain HPRS-103 and no obvious deletion was found in the 3'UTR except for a 1 bp deletion in the E element that introduced a binding site for c-Ets-1. Our study demonstrated the presence of ALV-J in wild ducks and investigated the molecular characterization of ALV-J in wild ducks isolates.
Project description:A new subgroup of avian leukosis virus (ALV) that includes a unique env gene, designated J, was identified recently in England. Sequence analysis of prototype English isolate HPRS-103 revealed several other unique genetic characteristics of this strain and provided information that it arose by recombination between exogenous and endogenous virus sequences. In the past several years, ALV J type viruses (ALV-J) have been isolated from broiler breeder flocks in the United States. We were interested in determining the relationship between the U.S. and English isolates of ALV-J. Based on sequence data from two independently derived U.S. field isolates, we conclude that the U.S. and English isolates of ALV-J derive from a common ancestor and are not the result of independent recombination events.
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:The existence of novel endogenous retrovirus elements in the chicken genome, designated EAV-HP, with close sequence identity to the env gene of avian leukosis virus (ALV) subgroup J has been reported (L. M. Smith, A. A. Toye, K. Howes, N. Bumstead, L. N. Payne, and K. Venugopal, J. Gen. Virol. 80:261-268, 1999). To resolve the genome structure of these retroviral elements, we have determined the complete sequence of two proviral clones of EAV-HP from a line N chicken genomic DNA yeast artificial chromosome library and from a meat-type chicken line 21 lambda library. The EAV-HP sequences from the two lines were 98% identical and had a typical provirus structure. The two EAV-HP clones showed identical large deletions spanning part of the gag, the entire pol, and part of the env genes. The env region of the EAV-HP clones was 97% identical to the env sequence of HPRS-103, the prototype subgroup J ALV. The 5' region of EAV-HP comprising the R and U5 regions of the long terminal repeat (LTR), the untranslated leader, and the 5' end of the putative gag region were 97% identical to the avian retrotransposon sequence, ART-CH. The remaining gag sequence shared less than 60% identity with other ALV sequences. The U3 region of the LTR was distinct from those of other retroviruses but contained some of the conserved motifs required for functioning as a promoter. To examine the ability of this endogenous retroviral LTR to function as a transcriptional promoter, the EAV-HP and HPRS-103 LTR U3 regions were compared in a luciferase reporter gene assay. The low luciferase activity detected with the EAV-HP LTR U3 constructs, at levels close to those observed for a control vector lacking the promoter or enhancer elements, suggested that these elements function as a weak promoter, possibly accounting for their low expression levels in chicken embryos.
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:During the process of transmission and spread of avian leukosis virus subgroup J (ALV-J) in chickens worldwide, the viral genome is constantly changing. A comprehensive and systematic study of the evolutionary process of ALV-J in China is needed. In this study, we amplified the full-length viral cDNA sequences of 16 ALV-J isolates of Yellow-chicken origin and analyzed and compared these sequences with another 69 ALV-J strains isolated during the years 1988-2018. These isolates were then sorted into 2 clusters: cluster I included isolates that mainly originated from the layers and White-feather broilers from northern China; cluster II included isolates mainly from the Yellow-chicken, most of them being from southern China. According to the sequence homologies of the whole genome and gag, pol, gp85, and gp37 genes, the ALV-J strains are more likely to randomly change in different directions from the original strain HPRS-103 as time passes. The results of entropy analysis of the sequences of gag, pol, and env revealed that the env gene had the largest variation, and the gag gene nonconserved sites are mainly concentrated in p19, p10, and p12. In addition, 84.71% (72/85) of the isolates had the 205-nucleotide (nt) deletion in the 3'UTR region, and 30.59% (26/85) of the isolates had the 125-nt to 127-nt deletion in the E element. Our study provides evidence for the coexistence of 2 extremely different clusters of ALV-J prevailing in China and in some other countries during the period of 1988-2018 and implies that the clusters are highly dependent on the host genetic background and the geographic location.
Project description:Compared to other avian leukosis viruses (ALV), ALV-J primarily induces myeloid leukemia and hemangioma and causes significant economic loss for the poultry industry. The ALV-J Env protein is hypothesized to be related to its unique pathogenesis. However, the molecular determinants of Env for ALV-J pathogenesis are unclear. In this study, we compared and analyzed GP37 of ALV-J Env and the EAV-HP sequence, which has high homology to that of ALV-J Env. Phylogenetic analysis revealed five groups of ALV-J GP37 and two novel ALV-J Envs with endemic GP85 and EAV-HP-like GP37. Furthermore, at least 15 virus-adapted mutations were detected in GP37 compared to the EAV-HP sequence. Further analysis demonstrated that three tyrosine-based motifs (YxxM, ITIM (immune tyrosine-based inhibitory motif) and ITAM-like (immune tyrosine-based active motif like)) associated with immune disease and oncogenesis were found in the cytoplasmic tail of GP37. Based on the potential function and distribution of these motifs in GP37, ALV-J Env was grouped into three species, inhibitory Env, bifunctional Env and active Env. Accordingly, 36.91%, 61.74% and 1.34% of ALV-J Env sequences from GenBank are classified as inhibitory, bifunctional and active Env, respectively. Additionally, the Env of the ALV-J prototype strain, HPRS-103, and 17 of 18 EAV-HP sequences belong to the inhibitory Env. And models for signal transduction of the three ALV-J Env species were predicted. Our findings and models provide novel insights for identifying the roles and molecular mechanism of ALV-J Env in the unique pathogenesis of ALV-J.
Project description:The avian leukosis and sarcoma virus (ALSV) group comprises eight subgroups based on envelope properties. HPRS-103, an exogenous retrovirus recently isolated from meat-type chicken lines, is similar to the viruses of these subgroups in group antigen but differs from them in envelope properties and has been assigned to a new subgroup, J. HPRS-103 has a wide host range in birds, and unlike other nontransforming ALSVs which cause late-onset B-cell lymphomas, HPRS-103 causes late-onset myelocytomas. Analysis of the sequence of an infectious clone of the complete proviral genome indicates that HPRS-103 is a multiple recombinant of at least five ALSV sequences and one EAV (endogenous avian retroviral) sequence. The HPRS-103 env is most closely related to the env gene of the defective EAV-E51 but divergent from those of other ALSV subgroups. Probing of restriction digests of line 0 chicken genomic DNA has identified a novel group of endogenous sequences (EAV-HP) homologous to that of the HPRS-103 env gene but different from sequences homologous to EAV and E51. Unlike other replication-competent nontransforming ALSVs, HPRS-103 has an E element in its 3' noncoding region, as found in many transforming ALSVs. A deletion found in the HPRS-103 U3 EFII enhancer factor-binding site is also found in all replication-defective transforming ALSVs (including MC29, which causes rapid-onset myelocytomas).
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:J subgroup avian leukosis virus (ALV-J) infects domestic chicken, jungle fowl, and turkey and enters the host cell through a receptor encoded by tvj locus and identified as Na+/H+ exchanger 1 (NHE1). The resistance to ALV-J in a great majority of examined galliform species was explained by deletions or substitutions of the critical tryptophan 38 in the first extracellular loop of NHE1, and genetic polymorphisms around this site predict the susceptibility or resistance of a given species or individual. In this study, we examined the NHE1 polymorphism in domestic chicken breeds and documented quantitative differences in their susceptibility to ALV-J in vitro. In a panel of chicken breeds assembled with the aim to cover the maximum variability encountered in domestic chickens, we found a completely uniform sequence of NHE1 extracellular loop 1 (ECL1) without any source of genetic variation for the selection of ALV-J-resistant poultry. In parallel, we studied the natural polymorphisms of NHE1 in wild ducks and geese because of recent reports on ALV-J positivity in feral Asian species. In anseriform species, we demonstrate a specific and highly conserved critical ECL1 sequence without any homologue of tryptophan 38 in accordance with the resistance of duck cells to prototype ALV-J. Last, we demonstrated that the new Asian strains of ALV-J have not evolved their envelope glycoprotein to the entry the duck cells. Our results contribute substantially to the current discussion of possible heterotransmission of ALV-J and its spill-over into the wild ducks and geese.
Project description:Aim:This study aimed to determine the prevalence of layer flock tumor disease in Lower Egypt during the period of 2018-2019 and to undertake molecular characterization and determine the genetic diversity of all identified viruses. Materials and Methods:Forty samples were collected from layer chicken located in six governorates of Lower Egypt during the period of 2018-2019. Samples were taken from tumors in different organs. Tumor tissues were identified by histopathological sectioning and then further confirmed by a reverse-transcription polymerase chain reaction. Finally, genetic evolution of Avian leukosis virus (ALV-J) gp85 gene was studied. Results:All the study samples were negative for Marek's disease virus, reticuloendotheliosis virus, ALV (A,B,C and D) and 20 samples were positive for ALV-J in backyard in six governrates. Sequencing of ALV-J gp85 gene was performed for six representative samples (one from each governorate), and they were found to be genetically related to prototype virus HPRS-1003 (identity percentage: 91.2-91.8%), but they were from a different group that was similar to the AF88-USA strain (first detected in 2000) with specific mutations, and they differed from a strain that was previously isolated in Egypt in 2005, forming two different subgroups (I and II) that had mutations in the hr1domain (V128F, R136A) and hr2 domain (S197G, E202K). Conclusion:The ALV-J virus was the main cause of neoplastic disease in layer chickens from Lower Egypt in the period of 2018-2019. We found that the genetic evolution of ALV-J gp85 gene was related to prototype virus HPRS-1003 but in a different group with a specific mutation. Further studies are needed to evaluate the antigenicity and pathogenicity of recently detected ALV-J strains.