UBE2J1 promotes ALV-A proviral DNA synthesis through the STAT3/IRF1 signaling pathway.

The ubiquitin-binding enzyme E2J1 is located on the endoplasmic reticulum membrane. It plays a role in transport throughout the process of ubiquitination. In mammals, UBE2J1 can promote RNA virus replication. However, the biological function of chicken UBE2J1 is unclear. In this study, chicken UBE2J1 was cloned for the first time, and UBE2J1 overexpression and shRNA knockdown plasmids were constructed. In chicken embryo fibroblasts, overexpression of UBE2J1 promoted the replication of subtype A avian leukosis virus, while knockdown of UBE2J1 inhibited the replication of ALV-A virus. In addition, we divided virus replication into virus adsorption and invasion into DF-1 cells, synthesis of proviral DNA, and release of viral particles. UBE2J1 promoted the replication of ALV-A virus by promoting the synthesis of proviral DNA. This result was caused by UBE2J1 inhibiting the production of interferon by inhibiting the STAT3/IRF1 pathway. We mutated ser at position 184 of UBE2J1 to Gly and found that this site plays a role as the phosphorylation site of UBE2J1. We confirmed that UBE2J1 promotes ALV-A replication in chicken embryo fibroblasts by inhibiting the STAT3/IRF1 pathway. This study provides new ideas and insights into ubiquitin-related proteins and antiviral immunity.

Critical role for ribonucleoside-diphosphate reductase subunit M2 in ALV-J-induced activation of Wnt/ß-catenin signaling via interaction with P27.

Avian leukemia virus subgroup J (ALV-J) causes various diseases associated with tumor formation and decreased fertility and induced immunosuppressive disease, resulting in significant economic losses in the poultry industry globally. Virus usually exploits the host cellular machinery for their replication. Although there are increasing evidences for the cellular proteins involving viral replication, the interaction between ALV-J and host proteins leading to the pivotal steps of viral life cycle are still unclear. Here, we reported that ribonucleoside-diphosphate reductase subunit M2 (RRM2) plays a critical role during ALV-J infection by interacting with capsid protein P27 and activating Wnt/ß-catenin signaling. We found that the expression of RRM2 is effectively increased during ALV-J infection, and that RRM2 facilitates ALV-J replication by interacting with viral capsid protein P27. Furthermore, ALV-J P27 activated Wnt/ß-catenin signaling by promoting ß-catenin entry into the nucleus, and RRM2 activated Wnt/ß-catenin signaling by enhancing its phosphorylation at Ser18 during ALV-J infection. These data suggest that the upregulation of RRM2 expression by ALV-J infection favors viral replication in host cells via activating Wnt/ß-catenin signaling. IMPORTANCE Our results revealed a novel mechanism by which RRM2 facilitates ALV-J growth. That is, the upregulation of RRM2 expression by ALV-J infection favors viral replication by interacting with capsid protein P27 and activating Wnt/ß-catenin pathway in host cells. Furthermore, the phosphorylation of serine at position 18 of RRM2 was verified to be the important factor regulating the activation of Wnt/ß-catenin signaling. This study provides insights for further studies of the molecular mechanism of ALV-J infection.

Residues L55 and W69 of Tva Mediate Entry of Subgroup A Avian Leukosis Virus.

The receptor of the subgroup A avian leukosis virus (ALV-A) in chicken is Tva, which is the homologous protein of human CD320 (huCD320), contains a low-density lipoprotein (LDL-A) module and is involved in the uptake of transcobalamin bound vitamin B12/cobalamin (Cbl). To map the functional determinants of Tva responsible for ALV-A receptor activity, a series of chimeric receptors were created by swapping the LDL-A module fragments between huCD320 and Tva. These chimeric receptors were then used for virus entry and binding assays to map the minimal ALV-A functional domain of Tva. The results showed that Tva residues 49 to 71 constituted the minimal functional domain that directly interacted with the ALV-A gp85 protein to mediate ALV-A entry. Single-residue substitution analysis revealed that L55 and W69, which were spatially adjacent on the surface of the Tva structure, were key residues that mediate ALV-A entry. Structural alignment results indicated that L55 and W69 substitutions did not affect the Tva protein structure but abolished the interaction force between Tva and gp85. Furthermore, substituting the corresponding residues of huCD320 with L55 and W69 of Tva converted huCD320 into a functional receptor of ALV-A. Importantly, soluble huCD320 harboring Tva L55 and W69 blocked ALV-A entry. Finally, we constructed a Tva gene-edited cell line with L55R and W69L substitutions that could fully resist ALV-A entry, while Cbl uptake was not affected. Collectively, our findings suggested that amino acids L55 and W69 of Tva were key for mediating virus entry. IMPORTANCE Retroviruses bind to cellular receptors through their envelope proteins, which is a crucial step in infection. While most retroviruses require two receptors for entry, ALV-A requires only one. Various Tva alleles conferring resistance to ALV-A, including Tvar1 (C40W substitution), Tvar2 (frame-shifting four-nucleotide insertion), Tvar3, Tvar4, Tvar5, and Tvar6 (deletion in the first intron), are known. However, the detailed entry mechanism of ALV-A in chickens remains to be explored. We demonstrated that Tva residues L55 and W69 were key for ALV-A entry and were important for correct interaction with ALV-A gp85. Soluble Tva and huCD320 harboring the Tva residues L55 and W69 effectively blocked ALV-A infection. Additionally, we constructed gene-edited cell lines targeting these two amino acids, which completely restricted ALV-A entry without affecting Cbl uptake. These findings contribute to a better understanding of the infection mechanism of ALV-A and provided novel insights into the prevention and control of ALV-A.

Glycosylation of ALV-J Envelope Protein at Sites 17 and 193 Is Pivotal in the Virus Infection.

Glycans on envelope glycoprotein (Env) of the subgroup J avian leukosis virus (ALV-J) play an essential role in the virion integrity and infection process. In this study, we found that, among the 13 predicted N-linked glycosylation sites (NGSs) in gp85 of Tibetan chicken strain TBC-J6, N17, and N193/N191 are pivotal for virus replication. Further research illustrated that a mutation at N193 weakened Env-receptor binding in a blocking assay of the viral entrance, coimmunoprecipitation, and ELISA. Our studies also showed that N17 was involved in Env protein processing and later virion incorporation based on the detection of p27 and Env protein in the supernatant and gp37 in the cell culture. This report is systematic research on clarifying the biological function of NGSs on ALV-J gp85, which would provide valuable insight into the role of gp85 in the ALV life cycle and anti-ALV-J strategies. IMPORTANCE ALV-J is a retrovirus that can cause multiple types of tumors in chickens. Among all the viral proteins, the heavily glycosylated envelope protein is especially crucial. Glycosylation plays a major role in Env protein function, including protein processing, receptor attachment, and immune evasion. Notably, viruses isolated recently seem to lose their 6th and 11th NGS, which proved to be important in receptor binding. In our study, the 1st (N17) and 8th (N193) NGS of gp85 of the strain TBC-J6 can largely influence the titer of this virus. Deglycosylation at N193 weakened Env-receptor binding while mutation at N17 influenced Env protein processing. This study systemically analyzed the function of NGSs in ALV-J in different aspects, which may help us to understand the life cycle of ALV-J and provide antiviral targets for the control of ALV-J.

Regulation of Avian Leukosis Virus Subgroup J Replication by Wnt/ß-Catenin Signaling Pathway.

Wnt/ß-catenin signaling is a highly conserved pathway related to a variety of biological processes in different cells. The regulation of replication of various viruses by Wnt/ß-catenin signaling pathway has been reported. However, the interaction between the Wnt/ß-catenin pathway and avian leukosis virus is unknown. In the present study, we investigated the effect of modulating the Wnt/ß-catenin pathway during avian leukosis virus subgroup J (ALV-J) infection. The activation of the Wnt/ß-catenin pathway by GSK-3 inhibitor increased ALV-J mRNA, viral protein expression, and virus production in CEF cells. This increase was suppressed by iCRT14, one of the specific inhibitors of the Wnt/ß-catenin signaling pathway. Moreover, treatment with iCRT14 reduced virus titer and viral gene expression significantly in CEF and LMH cells in a dose-dependent manner. Inhibition Wnt/ß-catenin signaling pathway by knockdown of ß-catenin reduced virus proliferation in CEF cells also. Collectively, these results suggested that the status of Wnt/ß-catenin signaling pathway modulated ALV-J replication. These studies extend our understanding of the role of Wnt/ß-catenin signaling pathway in ALV-J replication and make a new contribution to understanding the virus-host interactions of avian leukosis virus.

ALV-J inhibits autophagy through the GADD45ß/MEKK4/P38MAPK signaling pathway and mediates apoptosis following autophagy.

Autophagy and apoptosis, which are important processes for host immunity, are commonly exploited by viruses to facilitate their survival. However, to the best of our knowledge, very few studies have researched the mechanisms of action of the autophagic and apoptotic signaling pathways following viral infection. Thus, the present study aimed to investigate the mechanisms of action of growth arrest and DNA-damage-inducible ß (GADD45ß), an important resistance gene involved in the host resistance to ALV-J. Both ALV-J infection and the overexpression of GADD45ß inhibited autophagy during the early stages, which prevented the autophagosomes from binding to the lysosomes and resulted in an incomplete autophagic flux. Notably, GADD45ß was discovered to interact with MEKK4 in DF-1 cells. The genetic knockdown of GADD45ß and MEKK4 using small interfering RNA-affected ALV-J infection, which suggested that ALV-J may promote the binding of GADD45ß to MEKK4 to activate the p38MAPK signaling pathway, which subsequently inhibits autophagy. Furthermore, ALV-J was revealed to affect the autophagic pathway prior to affecting the apoptotic pathway. In conclusion, to the best of our knowledge, the present study was the first to investigate the combined effects of ALV-J infection on autophagy and apoptosis, and to suggest that ALV-J inhibits autophagy via the GADD45ß/MEKK4/p38MAPK signaling pathway.

Gp37 Regulates the Pathogenesis of Avian Leukosis Virus Subgroup J via Its C Terminus.

Different from other subgroups of avian leukosis viruses (ALVs), ALV-J is highly pathogenic. It is the main culprit causing myeloid leukemia and hemangioma in chickens. The distinctiveness of the env gene of ALV-J, with low homology to those of other ALVs, is linked to its unique pathogenesis, but the underlying mechanism remains unclear. Previous studies show that env of ALV-J can be grouped into three species based on the tyrosine motifs in the cytoplasmic domain (CTD) of Gp37, i.e., the inhibitory, bifunctional, and active groups. To explore whether the C terminus or the tyrosine motifs in the CTD of Gp37 affect the pathogenicity of ALV-J, a set of ALV-J infectious clones containing different C termini of Gp37 or the mutants at the tyrosine sites were tested in vitro and in vivo Viral growth kinetics indicated not only that ALV-J with active env is the fastest in replication and ALV-J with inhibitory env is the lowest but also that the tyrosine sites essentially affected the replication of ALV-J. Moreover, in vivo studies demonstrated that chickens infected by ALV-J with active or bifunctional env showed higher viremia, cloacal viral shedding, and viral tissue load than those infected by ALV-J with inhibitory env Notably, the chickens infected by ALV-J with active or bifunctional env showed significant loss of body weight compared with the control chickens. Taken together, these findings reveal that the C terminus of Gp37 plays a vital role in ALV-J pathogenesis, and change from inhibitory env to bifunctional or active env increases the pathogenesis of ALV-J.IMPORTANCE ALV-J can cause severe immunosuppression and myeloid leukemia in infected chickens. However, no vaccine or antiviral drug is available against ALV-J, and the mechanism for ALV-J pathogenesis needs to be elucidated. It is generally believed that gp85 and LTR of ALV contribute to its pathogenesis. Here, we found that the C terminus and the tyrosine motifs (YxxM, ITIM, and ITAM-like) in the CTD of Gp37 of ALV-J could affect the pathogenicity of ALV-J in vitro and in vivo The pathogenicity of ALV-J with Gp37 containing ITIM only was significantly less than ALV-J with Gp37 containing both YxxM and ITIM and ALV-J with Gp37 containing both YxxM and ITAM-like. This study highlights the vital role of the C terminus of Gp37 in the pathogenesis of ALV-J and thus provides a new perspective to elucidate the interaction between ALV-J and its host and a molecular basis to develop efficient strategies against ALV-J.

Expression patterns of novel circular RNAs in chicken cells after avian leukosis virus subgroup J infection.

Avian leukosis virus subgroup J (ALV-J) is an oncogenic retrovirus that causes severe economic losses to the poultry industry worldwide. Circular RNAs (circRNAs) are a class of non-coding RNAs that has been described in various biological systems and pathogenic processes. However, the immune mechanisms in response to circRNAs remain unknown. In this study, high-throughput transcriptome sequencing was used to detect circRNAs present in chicken macrophage (HD11) and chick embryo fibroblast (CEF) cells infected with ALV-J. We identified 7684 circRNAs from diverse genomic locations in CEF and HD11 after ALV-J infection, these RNAs showed complex expression patterns that differed based on the cells type and infection time. In total, 302 differentially expressed (DE) circRNAs and 164 DE circRNAs were identified in CEF and HD11 after ALV-J infection, respectively. CircRNA7419-associated with KDM4C- and circRNA6679 and circRNA6680-associated with TNFAIP6- were involved in the immune response upon ALV-J infection in CEF. Host genes were analyzed through further bioinformatics analysis. The result confirmed that a large number of DE circRNAs corresponded to several immune-associated or tumor-associated terms and pathways, such as Mucin type O-Glycan biosynthesis, MAPK signaling pathway, B cell receptor signaling, and Wnt signaling pathway in CEF, as well as Jak-STAT signaling pathway, apoptosis, and MAPK signaling pathway in HD11. CircRNAs related to the B cell receptor signaling pathway in CEF, and the Jak-STAT signaling pathway in HD11, were selected for circRNA-miRNA interaction network analyses. Our study indicates that circRNAs expression was altered by ALV-J infection in both CEF and HD11, and may play a key role in the progression of ALV-J infection.

Naturally Occurring Frameshift Mutations in the tvb Receptor Gene Are Responsible for Decreased Susceptibility of Chicken to Infection with Avian Leukosis Virus Subgroups B, D, and E.

The group of highly related avian leukosis viruses (ALVs) in chickens are thought to have evolved from a common retroviral ancestor into six subgroups, A to E and J. These ALV subgroups use diverse cellular proteins encoded by four genetic loci in chickens as receptors to gain entry into host cells. Hosts exposed to ALVs might be under selective pressure to develop resistance to ALV infection. Indeed, resistance alleles have previously been identified in all four receptor loci in chickens. The tvb gene encodes a receptor, which determines the susceptibility of host cells to ALV subgroup B (ALV-B), ALV-D, and ALV-E. Here we describe the identification of two novel alleles of the tvb receptor gene, which possess independent insertions each within exon 4. The insertions resulted in frameshift mutations that reveal a premature stop codon that causes nonsense-mediated decay of the mutant mRNA and the production of truncated Tvb protein. As a result, we observed that the frameshift mutations in the tvb gene significantly lower the binding affinity of the truncated Tvb receptors for the ALV-B, ALV-D, and ALV-E envelope glycoproteins and significantly reduce susceptibility to infection by ALV-B, ALV-D and ALV-E in vitro and in vivo Taken together, these findings suggest that frameshift mutation can be a molecular mechanism of reducing susceptibility to ALV and enhance our understanding of virus-host coevolution.IMPORTANCE Avian leukosis virus (ALV) once caused devastating economic loss to the U.S. poultry industry prior the current eradication schemes in place, and it continues to cause severe calamity to the poultry industry in China and Southeast Asia, where deployment of a complete eradication scheme remains a challenge. The tvb gene encodes the cellular receptor necessary for subgroup B, D, and E ALV infection. Two tvb allelic variants that resulted from frameshift mutations have been identified in this study, which have been shown to have significantly reduced functionality in mediating subgroup B, D, and E ALV infection. Unlike the control of herpesvirus-induced diseases by vaccination, the control of avian leukosis in chickens has relied totally on virus eradication measures and host genetic resistance. This finding enriches the allelic pool of the tvb gene and expands the potential for genetic improvement of ALV resistance in varied chicken populations by selection.

Residues 28 to 39 of the Extracellular Loop 1 of Chicken Na+/H+ Exchanger Type I Mediate Cell Binding and Entry of Subgroup J Avian Leukosis Virus.

Chicken Na+/H+ exchanger type I (chNHE1), a multispan transmembrane protein, is a cellular receptor of the subgroup J avian leukosis virus (ALV-J). To identify the functional determinants of chNHE1 responsible for the ALV-J receptor activity, a series of chimeric receptors was created by exchanging the extracellular loops (ECL) of human NHE1 (huNHE1) and chNHE1 and by ECL replacement with a hemagglutinin (HA) tag. These chimeric receptors then were used in binding and entry assays to map the minimal ALV-J gp85-binding domain of chNHE1. We show that ECL1 of chNHE1 (chECL1) is the critical functional ECL that interacts directly with ALV-J gp85; ECL3 is also involved in ALV-J gp85 binding. Amino acid residues 28 to 39 of the N-terminal membrane-proximal region of chECL1 constitute the minimal domain required for chNHE1 binding of ALV-J gp85. These residues are sufficient to mediate viral entry into ALV-J nonpermissive cells. Point mutation analysis revealed that A30, V33, W38, and E39 of chECL1 are the key residues mediating the binding between chNHE1 and ALV-J gp85. Further, the replacement of residues 28 to 39 of huNHE1 with the corresponding chNHE1 residues converted the nonfunctional ALV-J receptor huNHE1 to a functional one. Importantly, soluble chECL1 and huECL1 harboring chNHE1 residues 28 to 39 both could effectively block ALV-J infection. Collectively, our findings indicate that residues 28 to 39 of chNHE1 constitute a domain that is critical for receptor function and mediate ALV-J entry.IMPORTANCE chNHE1 is a cellular receptor of ALV-J, a retrovirus that causes infections in chickens and serious economic losses in the poultry industry. Until now, the domains determining the chNHE1 receptor function remained unknown. We demonstrate that chECL1 is critical for receptor function, with residues 28 to 39 constituting the minimal functional domain responsible for chNHE1 binding of ALV-J gp85 and efficiently mediating ALV-J cell entry. These residues are located in the membrane-proximal region of the N terminus of chECL1, suggesting that the binding site of ALV-J gp85 on chNHE1 is probably located on the apex of the molecule; the receptor-binding mode might be different from that of retroviruses. We also found that soluble chECL1, as well as huECL1 harboring chNHE1 residues 28 to 39, effectively blocked ALV-J infection. These findings contribute to a better understanding of the ALV-J infection mechanism and also provide new insights into the control strategies for ALV-J infection.

Transcription analysis of the interaction between chicken thymus and recombinant avian leukosis virus isolate FJ15HT0.

Avian leukosis virus (ALV) induces multiple avian tumors, growth decrease and immune suppression. Previously, a novel natural recombinant ALV isolate FJ15HT0 was proven to be associated with significant body weight decrease, immune suppression and lymphocytoma in infected SPF chickens. In order to uncover the interaction between virus and host, we compared differences in the transcriptomes of the thymuses from the mock chickens and simulated congenitally infected chickens at 5days (d), 13d and 21d of age by RNA-seq analysis of the thymuses. Signaling pathways including cytokine-cytokine receptor interactions, peroxisome proliferator-activated receptor (PPAR) signaling pathway, Janus tyrosine kinase/signal transducers and activators of transcription (Jak-STAT) signaling pathway and fatty acid degradation were involved in the interaction between FJ15HT0 and SPF chickens. Interestingly, fold change of ciliary neurotrophic factor receptor α (CNTFRα) in infected donor collected from 2d to 21d showed a significant positive correlation with the corresponding expression of the viral gp85 gene in thymuses (r=0.656, P<0.01) and in livers (r=0.525, P<0.05). It will provide new insights for the molecular pathogenesis of ALV infection.

Sequencing the Biology of Entry: The Retroviral env Gene.

The surface envelope protein of any virus is major determinant of the host cell that is infected and as a result a major determinant of viral pathogenesis. Retroviruses have a single surface protein named Env. It is a trimer of heterodimers and is responsible for binding to the host cell receptor and mediating fusion between the viral and host membranes. In this review we will discuss the history of the discovery of the avian leukosis virus (ALV) and human immunodeficiency virus type 1 (HIV-1) Env proteins and their receptor specificity, comparing the many differences but having some similarities. Much of the progress in these fields has relied on viral genetics and genetic polymorphisms in the host population. A special feature of HIV-1 is that its persistent infection in its human host, to the point of depleting its favorite target cells, allows the virus to evolve new entry phenotypes to expand its host range into several new cell types. This variety of entry phenotypes has led to confusion in the field leading to the major form of entry phenotype of HIV-1 being overlooked until recently. Thus an important part of this story is the description and naming of the most abundant entry form of the virus: R5 T cell-tropic HIV-1.

Avian leukosis virus subgroup J induces VEGF expression via NF-κB/PI3K-dependent IL-6 production.

Avian leukosis virus subgroup J (ALV-J) is an oncogenic virus causing hemangiomas and myeloid tumors in chickens. Interleukin-6 (IL-6) is a multifunctional pro-inflammatory interleukin involved in many types of cancer. We previously demonstrated that IL-6 expression was induced following ALV-J infection in chickens. The aim of this study is to characterize the mechanism by which ALV-J induces IL-6 expression, and the role of IL-6 in tumor development. Our results demonstrate that ALV-J infection increases IL-6 expression in chicken splenocytes, peripheral blood lymphocytes, and vascular endothelial cells. IL-6 production is induced by the ALV-J envelope protein gp85 and capsid protein p27 via PI3K- and NF-κB-mediated signaling. IL-6 in turn induced expression of vascular endothelial growth factor (VEGF)-A and its receptor, VEGFR-2, in vascular endothelial cells and embryonic vascular tissues. Suppression of IL-6 using siRNA inhibited the ALV-J induced VEGF-A and VEGFR-2 expression in vascular endothelial cells, indicating that the ALV-J-induced VEGF-A/VEGFR-2 expression is mediated by IL-6. As VEGF-A and VEGFR-2 are important factors in oncogenesis, our findings suggest that ALV-J hijacks IL-6 to promote tumorigenesis, and indicate that IL-6 could potentially serve as a therapeutic target in ALV-J infections.

Efficient method to optimize antibodies using avian leukosis virus display and eukaryotic cells.

Antibody-based therapeutics have now had success in the clinic. The affinity and specificity of the antibody for the target ligand determines the specificity of therapeutic delivery and off-target side effects. The discovery and optimization of high-affinity antibodies to important therapeutic targets could be significantly improved by the availability of a robust, eukaryotic display technology comparable to phage display that would overcome the protein translation limitations of microorganisms. The use of eukaryotic cells would improve the diversity of the displayed antibodies that can be screened and optimized as well as more seamlessly transition into a large-scale mammalian expression system for clinical production. In this study, we demonstrate that the replication and polypeptide display characteristics of a eukaryotic retrovirus, avian leukosis virus (ALV), offers a robust, eukaryotic version of bacteriophage display. The binding affinity of a model single-chain Fv antibody was optimized by using ALV display, improving affinity >2,000-fold, from micromolar to picomolar levels. We believe ALV display provides an extension to antibody display on microorganisms and offers virus and cell display platforms in a eukaryotic expression system. ALV display should enable an improvement in the diversity of properly processed and functional antibody variants that can be screened and affinity-optimized to improve promising antibody candidates.

Diagnosis and sequence analysis of avian leukosis virus subgroup J isolated from Chinese Partridge Shank chickens.

The diagnosis of avian leukosis virus subgroup J (ALV-J) infection in Chinese Partridge Shank chickens was confirmed by necropsy, histopathological examinations, antibody tests, viral isolation, immunofluorescence assays, and sequence analysis. Myelocytoma, myeloma, and fibrosarcoma were simultaneously found in Partridge Shank flock with ALV-J infection. Sequence analysis of the env genes of ALV-J demonstrated that both gp85 and gp37 were highly homologous among the three strains from local chickens of those among ALV-J strains isolated from white meat-type chickens. The phylogenetic trees indicated that the three strains isolated in this study were closely related to reference strains isolated in so-called Chinese yellow chickens and some strains isolated from white meat-type chickens, both from the USA and China. The observed ALV-J infection was the first report on Partridge Shank chickens, and myelocytoma, myeloma, and fibrosarcoma were found at the same time in this batch of local chickens.

gga-miR-375 plays a key role in tumorigenesis post subgroup J avian leukosis virus infection.

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.

A rapid profiling assay for avian leukosis virus subgroup E proviruses in chickens.

Endogenous retroviral elements (ERVs) are prolific components of the genomes of complex species, typically occupying more sequence space than do essential, protein-encoding genes. Much of what we know today about the structure and function, as well as the evolution and pathogenic potential, of ERVs was fleshed out over several decades during the last century using the avian leukosis virus subgroup E-related (ALVE) family of endogenous retroviruses of chickens as a model system. A critical enabling factor in the elucidation of ALVE structure and function is the ability to detect and unambiguously identify specific ALVE proviral elements and to develop accurate element profiles for individual chickens under study. Currently, the most common approach for ALVE locus detection involves element-specific PCR assays carried out using primers that target host DNA near the insertion site of the provirus (i.e., the upstream and downstream flanks of the unoccupied site). Here we describe a new approach for proviral detection that exploits restriction enzyme sites in flanking DNA to develop ALVE element profiles more rapidly than with assays currently in use. Moreover, unlike element-specific PCR tests, the "profiling" assay detects novel ALVEs for which insertion sites have not yet been identified as well as previously characterized elements.

Genetic diversity and phylogenetic analysis of glycoprotein gp85 of avian leukosis virus subgroup J wild-bird isolates from Northeast China.

Avian leukosis virus subgroup J (ALV-J), first isolated in 1989, preferentially infects meat-type birds. Chinese layer flocks have experienced outbreaks of this virus since 2008. To analyze the status of ALV-J infection in wild birds in China, 585 wild birds collected from three provinces of Northeast China from 2010 to 2012 were tested, and six ALV-J strains were isolated for the first time. Furthermore, the gp85 genes of the six strains were amplified, cloned, and sequenced. The results indicated that two different ALV-J strains coexisted in Chinese wild birds from 2010 to 2012. These results not only expand the epidemiological data available for ALV-J and provide necessary information for the further understanding of the evolution of ALV-J, but they also highlight the potential role of wild-bird migration in the spread of ALV-J.

A 19-nucleotide insertion in the leader sequence of avian leukosis virus subgroup J contributes to its replication in vitro but is not related to its pathogenicity in vivo.

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.

Gene detection, virus isolation, and sequence analysis of avian leukosis viruses in Taiwan country chickens.

Avian leukosis virus (ALV) infection in Taiwan Country chickens (TCCs) was investigated by using gene detection, virus isolation, and sequence analysis. The blood samples of 61 TCC flocks at market ages from a slaughter house were screened for exogenous ALVs using polymerase chain reaction to investigate the ALV infection status. The buffy coats from three breeder and four commercial chicken flocks were cocultured with DF-1 cells to isolate the virus. The full proviral DNA genomes of two ALV isolates were sequenced, analyzed, and compared with reference ALV strains. The gene detection results showed that 60 and 43 of the 61 flocks were infected with subgroup A of ALV (ALV-A) and subgroup J of ALV (ALV-J), respectively. Virus isolation results showed that five ALV-As and two ALV-Js were isolated from those seven TCC flocks. The full sequences of the isolates showed that isolate TW-3577 possessed a myeloblastosis-associated virus 1 gp85 coding region and an ALV-J 3'-untranslated region (3'UTR) and was similar to ordinary ALV-A. However, TW-3593 was unique. The 3'UTR of this isolate displayed high identity to endogenous counterpart sequence and its gp85 was different from all subgroups. This unique ALV is common in Taiwan.