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Background: Avian leukosis virus subgroup J (ALV-J) primarily affects poultry, particularly chickens, leading to tumourigenesis and immunosuppression, which results in substantial economic losses. It is important to note that ALV-J is commonly found in indigenous chicken breeds in China, and the virus's vertical transmission characteristics present a significant threat to the preservation of local chicken breeds., Objectives: The study aimed to investigate the characteristics and effects of the recombinant ALV-J strain LY2021J, with a focus on its genetic composition and its potential influence on virulence and pathogenicity., Methods: LY2021J was isolated using DF-1 cells and validated by enzyme-linked immunosorbent assay (ELISA) and IFA. The proviral genome was amplified using segmented PCR and then spliced together using DNASTAR software. Genome-wide genes, including gag, pol, gp85, and long terminal repeat (LTR), were compared. Recombination sites were analysed using RDP5 and SimPlot software. Pathogenicity was evaluated by monitoring symptoms and conducting examinations on SPF chickens., Results: The outbreak of ALV-J in China has caused significant economic losses in the poultry industry. Although largely controlled in white-feather broilers and egg-laying chickens, ALV-J has spread to yellow-feather broilers and local breeds. A strain, LY2021J, isolated from Hetian chickens, showed lower mortality despite severe dysplasia. Genetic analysis revealed high similarity between LY2021J and the Chinese strains JS14NT01 and NX0101, suggesting a shared origin. Recombination with strain ev-1 and specific 3' UTR deletions may explain LY2021J's reduced virulence. Continued monitoring and prevention strategies are essential to mitigate ALV-J's impact., (© 2024 The Author(s). Veterinary Medicine and Science published by John Wiley & Sons Ltd.)

Avian leukemia virus subgroup J (ALV-J) and chicken infectious anemia virus (CIAV) can be vertically transmitted; however, the pathogenicity of vertically transmitted coinfection with these 2 pathogens has not been studied. In this study, we created a model of chick morbidity in which chicks carried either ALV-J, CIAV, or both viruses via embryo inoculation. Thereafter, we analyzed the effects of vertically transmitted coinfection with CIAV and ALV-J on the pathogenicity of ALV-J and performed a purification assay based on hatching, mortality viremia positivity, and detection of fecal ALV-p27 antigen rates, and body weight. The hatching rate of the ALV-J+CIAV group was 68.57%, lower than those of the single infection and control groups. The survival curve showed that the mortality rates of the CIAV and ALV-J coinfection groups were higher than those of the single infection and control groups. Body weight statistics showed that coinfection aggravated the 7-d growth inhibition effect. The results of ALV-p27 antigen detection in cell culture supernatants showed that the positivity rates of the ALV-J and ALV-J+CIAV groups were 100% at all ages and 0% in the control group. The results of ALV-p27 antigen detection by anal swabs showed that the positivity rates of the ALV-J group were 92.86, 90.90, 88.89, and 93.33% at all ages, and that the ALV-J p27 positivity detection rate of anal swabs was lower than that of plasma virus isolation. The immune organ index of the ALV-J+CIAV group was significantly or very significantly lower than those of the single infection and control groups. The immune organ viral load showed that coinfection with CIAV and ALV-J promoted the proliferation of ALV-J and CIAV in immune organs. Coinfection with ALV-J and CIAV reduced chicken embryo hatchability and increased chick mortality and growth inhibition relative to their respective single infections. Additionally, coinfection with ALV-J + CIAV was even more detrimental in inducing immune organ atrophy (e.g., the thymus, spleen, and bursa), and promoted individual virus replication during coinfection., (Copyright © 2024. Published by Elsevier Inc.)

Avian leukosis virus (ALV) can induce various tumors and cause serious production problems. ALVs isolated from chickens were divided into six subgroups (A-J). In 2012, a strain of a putative novel subgroup of ALVs was isolated from Chinese native chickens in Jiangsu Province and named as ALV-K. In this study, three ALV-K strains (JS14LH01, JS13LH14, and JS15SG01) were isolated from chickens with suspected ALV infection in Jiangsu Province. Their complete genomes were amplified, sequenced, and analyzed systematically. The results showed that JS14LH01 and JS13LH14 were ALV-K and ALV-E recombinant strains. Whereas JS15SG01 is an ALV-K, ALV-E, and ALV-J multiple recombinant strain containing the U3 region of ALV-J. The pathogenicity test of JS15SG01 revealed that, compared with previous ALV-K strains, the viremia and viral shedding level of JS15SG01-infected chickens were significantly increased, reaching 100 % and 59 %, respectively. More important, JS15SG01 induced significant proliferation of gliocytes in the cerebral cortex of infected chickens, accompanied by the neurotropic phenomenon. This is the first report about a multiple recombinant ALV-K strain that could invade and injure the brain tissue of chickens in China. Our findings enriched the epidemiologic data of ALV and helped to reveal the evolution of ALV strains prevalent in chicken fields., (Copyright © 2021 Elsevier B.V. All rights reserved.)

The aim of this study was to better understand the sequence characteristics and immune responses in avian leukosis virus subgroup J (ALV-J) infected yellow chicken flocks in South China. We isolated four strains of ALV-J virus from these flocks, which were then identified by several methods, including subtype-specific polymerase chain reaction (PCR), enzyme-linked immunosorbent assay (ELISA), and immunofluorescence assay (IFA). All four viruses were sequenced for their complete genomes and named GD19GZ01, GD19GZ02, GD19GZ03, and GD19GZ04. In comparison with the reference sequence, the homology analysis showed that the gag and pol genes were relatively conserved, whereas env contained much variation. Both GD19GZ01 and GD19GZ02 almost entirely lacked the rTM region and E element, while the latter was retained in GD19GZ03 and GD19GZ04. Moreover, the virus replication levels in GD19GZ03 and GD19GZ04were much higher than those in GD19GZ01 and GD19GZ02. And three virus recombination events in GD19GZ01 and GD19GZ02 were revealed by the results of PDR5 and SimPlot software analysis. Additionally, we found that some interferon-stimulating genes ( CH25H , MX , PKR , OAS , and ZAP ) and inflammatory mediators ( IL-4 , IL-6 , IL-10 , IL-12 , 1L-18 , and TNF-α ) were significantly upregulated in the immune system organs of clinical chickens. Taken together, these findings clarify and reveal the sequence characteristics and trends in the variation of ALV-J infection in yellow chicken flocks of South China., Competing Interests: The authors declare no conflict of interest., (Copyright © 2021 Qihong Zhang et al.)

Avian leukosis virus (ALV) is associated with immune suppression, neoplasia, and reduced performance in chickens. In this study, two strains of ALV were isolated from Luxi gamecocks by DF-1 cell culture and identified by PCR, immunofluorescence assay, and sequencing of the viral genome. These strains were found to be novel recombinant viruses with nucleotide sequence identity of over 93.0% in the LTR and 94.4% in U3 to ALV-J, over 95.0% in the 5'UTR to ALV-C, over 93.4% in gp85 to ALV-B, and over 96.0% in gp37 to ALV-E. These results indicate that these two isolates are recombinants between ALV-J, ALV-C, ALV-E and ALV-B.

The differences among different sub-groups of the avian leukosis virus (ALV) genome are mainly concentrated in the env gene, which binds to cell-specific receptors and determines the characteristics of viral tropism and pathogenicity. In this study, two rescued viruses rGX15MM6-2 (ALV of subgroup J, ALV-J) and rGX14FF03 (ALV of subgroup B, ALV-B) and a recombinant virus rALV-B-Jenv (ALV-B's backbone with ALV-J's env) were generated and tested utilizing both in vitro and in vivo experiments. The results showed that the replication ability of the viruses released in DF-1 cell cultures was listed in order as rGX15MM6-2 > rALV-B-Jenv > rGX14FF03. rGX15MM6-2 caused the most serious suppression of body weight gain, exhibited a significant negative effect on the development of immune organs (P < 0.05) and lower antibody responses to vaccinations with the commercial oil-emulsion vaccines (OEVs) (P<0.05) in the challenged chickens. The viral detection showed that the positive rate in blood from the birds infected with rALV-B-Jenv were respectively higher than those from the birds infected with rGX14FF03 (P < 0.05). At 25 wpi, similar tumors were found in the abdominal cavity of the birds in rGX15MM6-2 and rALV-B-Jenv groups. The results demonstrated that the ALV-J env gene significantly increases the pathogenicity of the recombinant ALV-B. With the increasing incidence of co-infections of different subgroups of ALV in the field, the possibility of viral recombination is increasing and demands further study., (Copyright © 2020 Elsevier B.V. All rights reserved.)

ABSTRACT The prototype fowl glioma-inducing virus (FGVp) causes fowl glioma and cerebellar hypoplasia in chickens. In this study, we investigated whether a strain of avian leukosis virus (ALV), associated with avian osteopetrosis and mesenchymal neoplasms, is able to induce fowl glioma. We encountered avian osteopetrosis and mesenchymal neoplasms, including myxosarcoma and rhabdomyosarcoma, in Japanese native chickens used for both egg-laying and meat production. These birds were also affected by non-suppurative encephalitis and glioma in their brains. Four ALV strains (GifN&#95;001, GifN&#95;002, GifN&#95;004, GifN&#95;005) were isolated, and a phylogenic analysis of env SU showed that these isolates were classified into different clusters from FGVp and the variants previously reported. Whereas the env SU shared a high identity (94.7%) with that of Rous sarcoma virus (strain Schmidt-Ruppin B) (RSV-SRB), the identity between env TM of GifN&#95;001 and that of FGVp was high (94.5%), indicating that GifN&#95;strains may emerge by recombination between FGVp and other exogenous ALVs. Specific-pathogen-free chickens inoculated in ovo with GifN&#95;001 revealed fowl glioma and cerebellar hypoplasia. These results suggest that the newly isolated strains have acquired neuropathogenicity to chickens.

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 β (GADD45β), an important resistance gene involved in the host resistance to ALV-J. Both ALV-J infection and the overexpression of GADD45β inhibited autophagy during the early stages, which prevented the autophagosomes from binding to the lysosomes and resulted in an incomplete autophagic flux. Notably, GADD45β was discovered to interact with MEKK4 in DF-1 cells. The genetic knockdown of GADD45β and MEKK4 using small interfering RNA-affected ALV-J infection, which suggested that ALV-J may promote the binding of GADD45β 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 GADD45β/MEKK4/p38MAPK signaling pathway.

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., (Copyright © 2020 American Society for Microbiology.)

In order to evaluate the influence of the vertical transmission of avian leukosis virus (ALV) from J subgroup (ALV-J) positive parents on the vaccine efficacy of Marek's disease virus (MDV), ALV-J positive male breeders × female breeders of Three-yellow chickens and the ALV negative male breeder × the negative female breeders were used respectively for crossbreeding to produce eggs and the hatching offspring. The commercial CVI988/Rispens vaccine was used to vaccinate the crossbred offspring at 1-day-old. At 7-days-old, the birds were inoculated with the inactivated oil-emulsion vaccines (OEVs) AIV-H5 monovalent and NDV + AIV-H9 bivalent, respectively. Then the birds were challenged with a Chinese very virulent (vv) MDV field strain GXY2 at 14-day-old. The results showed that the viral load of the challenged GXY2 in the offspring from the ALV-J positive breeders was significantly higher than that from the ALV-negative breeders' (P < 0.05), and the mortality and tumor incidence of offspring from the ALV-J positive breeders were higher than those of the ALV-negative breeders. Also the offspring of the ALV-J positive breeders exhibited a significant negative effect on the development of the immune organs (P < 0.05) and lower antibody responses to the vaccinations with the commercial OEVs (P<0.05). The MD vaccine protective index in the offspring from the ALV-J positive breeders was lower than that from the ALV-negative breeders. The results of the study demonstrated that the vertical transmission of ALV from the ALV-J positive parents caused severe immunosuppression and significantly reduced the Marek's disease vaccine efficacy in Three-yellow chickens., Competing Interests: Declaration of Competing Interest The authors declared that they have no competing interests., (Copyright © 2020 Elsevier B.V. All rights reserved.)

The CRISPR/CRISPR-associated protein 9 (Cas9) system is a powerful gene-editing tool originally discovered as an integral mediator of bacterial adaptive immunity. Recently, this technology has been explored for its potential utility in providing new and unique treatments for viral infection. Marek's disease virus (MDV) and avian leukosis virus subgroup J (ALV-J), major immunosuppressive viruses, cause significant economic losses to the chicken industry. Here, we evaluated the efficacy of using MDV as a CRISPR/Cas9-delivery system to directly target and disrupt the reverse-transcribed products of the ALV-J RNA genome during its infection cycle in vitro and in vivo. We first screened multiple potential guide RNA (gRNA) target sites in the ALV-J genome and identified several optimized targets capable of effectively disrupting the latently integrated viral genome and providing efficient defense against new infection by ALV-J in cells. The optimal single-gRNAs and Cas9-expression cassettes were inserted into the genome of an MDV vaccine strain. The results indicated that engineered MDV stably expressing ALV-J-targeting CRISPR/Cas9 efficiently resisted ALV-J challenge in host cells. These findings demonstrated the CRISPR/Cas9 system as an effective treatment strategy against ALV-J infection. Furthermore, the results highlighted the potential of MDV as an effective delivery system for CRISPR/Cas9 in chickens., Competing Interests: Declaration of Competing Interest The authors declare that they have no competing interests., (Copyright © 2020 Elsevier B.V. All rights reserved.)

To determine whether geese are susceptible to infection by avian leukosis virus (ALV), 702 serum samples from domestic and foreign goose breeds were screened for p27 antigen as well as being inoculated into DF-1 cell cultures to isolate ALV. Although 5.7% of samples were positive for p27 antigen, reactivity appeared to be non-specific because no ALV was detected in the corresponding DF-1 cultures. To further determine whether geese are susceptible to ALV-J isolated from chickens, ALV-J strain JS09GY7 was artificially inoculated into 10-day-old goose embryos, with one-day-old hatched goslings then screened for p27 antigen and the presence of ALV. In all cases, the results of both tests were negative. Liver tissues from the 1-day-old goslings were screened using a polymerase chain reaction-based assay, which failed to amplify ALV-J gene fragments from any of the samples. Further, no histopathological damage was observed in the liver tissues. ALV-J was further inoculated intraperitoneally into one-day-old goslings, with cloacal swabs samples and plasma samples then collected every 5 days for 30 days. All samples were again negative for the presence of p27 antigen and ALV, and liver tissues from the challenged geese showed no histopathological damage and were negative for the presence of ALV-J gene fragments. Furthermore, p27 antigen detection, PCR-based screening, and indirect immunofluorescence assays were all negative following the infection of goose embryo fibroblasts with ALV-J. Together, these results confirm that virulent chicken-derived ALV-J strains cannot infect geese, and that p27 antigen detection in goose serum is susceptible to non-specific interference.

One natural recombinant avian leukosis virus (ALV) strain GX14DJ3-18 was isolated from a native gamecock by DF-1 cell culture and identified with Polymerase Chain Reaction (PCR), immunofluorescence assay and the viral genome's nucleotide sequencing. This strain was revealed as a novel recombinant virus with nucleotide sequence similarities of 95.4% Long Terminal Repeated (LTR), 95.8% 5', UTR, 97.9% gag, and 92.9% 3'untranslated regions (UTR) in ALV-J. Also we found sequence similarities of 99.3% pol and 99.0% gp37 in ALV-E, and 89.9% gp85 in ALV-A. The simulated congenital infection with GX14DJ3-18 in Three-Yellow chickens exhibited a significant negative effect on the development of immune organs (P < 0.05). Also, lower antibody responses were found to vaccinations with the commercial vaccines of Newcastle disease virus and with subtypes H5 and H9 of avian influenza virus (P < 0.05). The incidence of tumor or tumor-like lesions in the challenged birds was 14.28% (5/35), while none were observed in the un-challenged control group (0/35). These results suggested that GX14DJ3-18 is a novel recombinant ALV that can induce pathogenicity in the commercial Three-Yellow chickens. We speculated that cross-provincial sales of gamecocks in which ALVs have not been eradicated thoroughly might be a potential route for the transmission of ALVs to commercial chickens., (© The Author(s) 2019. Published by Oxford University Press on behalf of Poultry Science Association.)

Avian leukosis virus subgroup K (ALV-K) is composed of newly emerging isolates, which, in sequence analyses, cluster separately from the well-characterized subgroups A, B, C, D, E, and J. However, it remains unclear whether ALV-K represents an independent ALV subgroup with regard to receptor usage, host range, and superinfection interference. In the present study, we examined the host range of the Chinese infectious isolate JS11C1, an ALV-K prototype, and we found substantial overlap of species that were either resistant or susceptible to ALV-A and JS11C1. Ectopic expression of the chicken tva gene in mammalian cells conferred susceptibility to JS11C1, while genetic ablation of the tva gene rendered chicken DF-1 cells resistant to infection by JS11C1. Thus, tva expression is both sufficient and necessary for JS11C1 entry. Receptor sharing was also manifested in superinfection interference, with preinfection of cells with ALV-A, but not ALV-B or ALV-J, blocking subsequent JS11C1 infection. Finally, direct binding of JS11C1 and Tva was demonstrated by preincubation of the virus with soluble Tva, which substantially decreased viral infectivity in susceptible chicken cells. Collectively, these findings indicate that JS11C1 represents a new and bona fide ALV subgroup that utilizes Tva for cell entry and binds to a site other than that for ALV-A. IMPORTANCE ALV consists of several subgroups that are particularly characterized by their receptor usage, which subsequently dictates the host range and tropism of the virus. A few newly emerging and highly pathogenic Chinese ALV strains have recently been suggested to be an independent subgroup, ALV-K, based solely on their genomic sequences. Here, we performed a series of experiments with the ALV-K strain JS11C1, which showed its dependence on the Tva cell surface receptor. Due to the sharing of this receptor with ALV-A, both subgroups were able to interfere with superinfection. Because ALV-K could become an important pathogen and a significant threat to the poultry industry in Asia, the identification of a specific receptor could help in the breeding of resistant chicken lines with receptor variants with decreased susceptibility to the virus., (Copyright © 2019 American Society for Microbiology.)

Avian leukosis virus subgroup J has been found to infect many types of chickens with various genetic backgrounds. The ALV-J strain NX0101, which was isolated from broiler breeders in 2001, mainly induces the formation of myeloid cell tumors. However, strain HN10PY01, which was recently isolated from laying hens, mainly induces the formation of myeloid cell tumors and hemangioma. In order to determine the difference in pathogenicity of the 2 strains in broiler chickens, 2 groups of chicken embryos were infected with NA0101 and HN10PY01 separately. A comparison was made of the mortality, oncogenicity, body weights, indexes for immune organs, levels of ALV group-specific antigen p27, and mRNA expression levels of the tumor-related gene, p53, in ALV-J-infected birds and immune organs of theses chickens in response to Newcastle Disease Virus (NDV) and avian influenza virus subtype H9 (AIV-H9) vaccination. The results indicated that strain NX0101 was highly pathogenic in broiler chickens and led to a 30% mortality rate and 45% oncogenicity, compared with the HN10PY01-infected birds. Weight of chickens was also significantly lower after 15 wk (P < 0.05). In addition, the mRNA expression levels of tumor-related p53 in medulla, liver, and lung in broilers infected with strain NX0101 were significantly higher than those infected with strain HN10PY01 (P < 0.05). These results indicated that strain NX0101 had a higher replication ability in broiler chickens. The findings of this study will contribute to further elucidating the mechanisms underlying host susceptibility and tumor classification in ALV-J-infected chickens., (© 2019 Poultry Science Association Inc.)

In this study, a recombinant ALV with ALV-K env and ALV-J backbone was generated (designated ALV-K-env-J) and tested in vitro and in vivo. The growth curve in DF1 cells showed that the recombinant virus replicated more efficiently in comparison with the ALV-J and ALV-K. Although all the infected chickens showed growth retardation compared with the non-infected chickens, the viral and serological detection showed that the positive rate and virus load detected in blood and cloaca, and the positive rate and titer of antibody against p27 from the chickens infected with ALV-K-env-J were higher than those from the chickens infected with the ALV-K, but less than those from the chickens infected with the ALV-J. All these data clearly demonstrated that the recombination event in this study increased the pathogenesis of ALV-K, and the potential recombination between different ALV subgroups should be worried when the clinical co-infections occur., (Copyright © 2019 Elsevier B.V. All rights reserved.)

A sensitive and specific photoelectrochemical (PEC) immunosensor was fabricated for subgroup J avian leukosis viruses (ALV-J) analysis based on a dual signal-on strategy. Gold nanoparticles (AuNPs) decorated graphitic carbon nitride (AuNPs/g-C 3 N 4 ) as photoelectrochemical species and primary antibody (Ab 1 ) against ALV-J were immobilized onto ITO electrode in turn. An ALP-CdTe-Ab 2 bio-conjugant was fabricated by assembling second antibody (Ab 2 ) and alkaline phosphatase (ALP) to CdTe quantum dots (QDs) surface. The PEC immunosensor was fabricated by successively anchoring the target ALV-J and ALP-CdTe-Ab 2 bio-conjugants onto electrode surface via the immune recognition. By virtue of the matched energy levels between CdTe QDs and AuNPs/g-C 3 N 4 , ALP-CdTe-Ab 2 bio-conjugants could serve as the PEC active probes for photocurrent enhancement. Moreover, the photocurrent response could be further enhanced attributed to the ALP catalytic chemistry to in situ produce ascorbic acid for electron donating, achieving an effective dual signal-on mode for PEC assay. On the basis of the ALV-J titers-dependent photocurrent increment, the fabricated PEC immunosensor showed high sensitivity, specificity and stability for ALV-J assay in a wide linear range with a low detection limit of 85 TCID 50 /mL. This PEC immunosensor with the dual signal-on strategy may open up a promising platform for more target analytes in novel immune analysis and clinical diagnostics., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Background: The pathogenesis of immunological tolerance caused by avian leukosis virus subgroup J (ALV-J), an oncogenic retrovirus, is largely unknown., Results: In this study, the development, differentiation, and immunological capability of B cells and their progenitors infected with ALV-J were studied both morphologically and functionally by using a model of ALV-J congenital infection. Compared with posthatch infection, congenital infection of ALV-J resulted in severe immunological tolerance, which was identified as the absence of detectable specific antivirus antibodies. In congenitally infected chickens, immune organs, particularly the bursa of Fabricius, were poorly developed. Moreover, IgM-and IgG-positive cells and total immunoglobulin levels were significantly decreased in these chickens. Large numbers of bursa follicles with no differentiation into cortex and medulla indicated that B cell development was arrested at the early stage. Flow cytometry analysis further confirmed that ALV-J blocked the differentiation of CD117 + chB6 + B cell progenitors in the bursa of Fabricius. Furthermore, both the humoral immunity and the immunological capability of B cells and their progenitors were significantly suppressed, as assessed by (a) the antibody titres against sheep red blood cells and the Marek's disease virus attenuated serotype 1 vaccine; (b) the proliferative response of B cells against thymus-independent antigen lipopolysaccharide (LPS) in the spleen germinal centres; and (c) the capacities for proliferation, differentiation and immunoglobulin gene class-switch recombination of B cell progenitors in response to LPS and interleukin-4(IL-4) in vitro., Conclusions: These findings suggested that the anergy of B cells in congenitally infected chickens is caused by the developmental arrest and dysfunction of B cell progenitors, which is an important factor for the immunological tolerance induced by ALV-J.

Avian leukosis virus (ALV) is an avian oncogenic retrovirus that induces leukemia-like proliferative diseases in chickens. ALV infection can result in the development of immunological tolerance and persistent viremia. Since effective vaccines against ALV are not yet available, its current prevention primarily depends on detection and eradication to establish exogenous ALV-free poultry flocks. In this study, a rapid and simple colloidal gold test strip method, specific for the group-specific antigen, p27 protein, was developed and systematically evaluated for the detection of ALV from different samples. The detection limit of this assay was as low as 6.25 ng/ml for p27 protein and 80 TCID 50 /ml for different subgroups of ALV. Besides, the test strip showed high specificity in the detection of different subgroups of ALV, including ALV-A, ALV-B, ALV-J, and ALV-K, with no cross-reaction with other avian pathogens. Furthermore, we artificially infected specific pathogen-free (SPF) chickens with ALV-J, collected cloacal swabs, and examined viral shedding using both test strips and ELISA. Results from the test strip were highly consistent with that from ELISA. In addition, 1104 virus isolates from anti-coagulant blood samples, 645 albumen samples, and 4312 meconium samples were tested, and the test strip results agreed with those of ELISA kit up to 97.1%. All the results indicated that the colloidal gold test strip could serve as a simple, rapid, sensitive, and specific diagnostic method for eradication of ALV in poultry farms.

Aberrant expression of microRNAs (miRNAs) is known to be involved in cancer progression caused by subgroup J avian leukosis virus (ALV-J) in liver tissues. To advance our understanding of the related pathological mechanisms and virus-host interactions, seven previously reported miRNAs were selected for a comparative analysis of miRNA expression between infected and uninfected DF-1 cells, including six miRNAs related to tumorigenesis (let-7b/7i, miR-221/222, miR-125b, miR-375 and miR-2127. The results showed that six of the seven miRNAs except gga-miR-375 were upregulated in cells infected with NX0101 (caused myeloma (ML)) and GD1109 (caused hemangioma (HE)) at 1 h post infection. On day 2 post-infection, all seven miRNAs were upregulated in infected DF-1 cells. On day 6 post-infection, gga-let-7b, gga-miR-125b, and gga-miR-375 were downregulated whereas gga-miR-221 and gga-miR-222 were upregulated in DF-1 cells infected with the two ALV-J strains of different phenotypes. However, expression of gga-let-7i was reduced in DF-1 cells infected with NX0101 and was increased in those infected with GD1109; gga-miR-2127 expression showed no significant difference between infected and uninfected cells. This study is the first to report the changes in the miRNA expression levels in DF-1 cells during the course of ALV-J infection, and suggests a relationship between its pathological mechanisms and miRNAs., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

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., Competing Interests: The authors have declared that no competing interests exist.

The function of the chicken's major histocompatibility complex (MHC or B complex) class I major (BF2) and minor (BF1) glycoproteins is compared for their expression, ability to present viral antigens to cytotoxic T lymphocytes (CTLs), and interaction with natural killer (NK) cells. MHC-restricted CTLs recognized virus antigen in the context of the BF2*21 major glycoprotein but not the BF1*21 minor glycoprotein. Marek's disease virus (MDV), a large DNA virus known to reduce the cell surface expression of class I glycoprotein, reduced the expression of BF2 glycoprotein while BF1glycoprotein expressions are remained as no change or slight increase. In addition, the expression of BF1*21 class I glycoprotein protected target cells from NK cell lysis while the expression of the BF2*21 class I glycoprotein enhanced NK cell lysis of target cells. Therefore, BF1 and BF2 provide two different cellular immune functions; BF1 negatively regulates the NK cell killing activity and BF2 restricts the antigen specific CTL immune response.

Superinfection of Marek's disease virus (MDV) and avian leukosis virus subgroup J (ALV-J) causes lethal neoplasia and death in chickens. However, whether there is synergism between the two viruses in viral replication and pathogenicity has remained elusive. In this study, we found that the superinfection of MDV and ALV-J increased the viral replication of the two viruses in RNA and protein level, and synergistically promoted the expression of IL-10, IL-6, and TGF-β in chicken embryo fibroblasts (CEF). Moreover, MDV and ALV-J protein expression in dual-infected cells detected by confocal laser scanning microscope appeared earlier in the cytoplasm and the nucleus, and caused more severe cytopathy than single infection, suggesting that synergistically increased MDV and ALV-J viral-protein biosynthesis is responsible for the severe cytopathy. In vivo, compared to the single virus infected chickens, the mortality and tumor formation rates increased significantly in MDV and ALV-J dual-infected chickens. Viral loads of MDV and ALV-J in tissues of dual-infected chickens were significantly higher than those of single-infected chickens. Histopathology observation showed that more severe inflammation and tumor cells metastases were present in dual-infected chickens. In the present study, we concluded that synergistic viral replication of MDV and ALV-J is responsible for the enhanced pathogenicity in superinfection of chickens.

In recent years, cases of avian leukosis virus (ALV) infection have become more frequent in China. We isolated 6 ALV strains from yellow feather broiler breeders in south China from 2014 to 2016. Their full genomes were sequenced, compared, and analyzed with other reference strains of ALV. The complete genomic nucleotide sequences of GD150509, GD160403, GD160607, GDFX0601, and GDFX0602 were 7482 bp in length, whereas GDFX0603 was 7480 bp. They shared 99.7% to 99.8% identity with each other. Homology analysis showed that the gag , pol , long terminal repeats (LTRs), and the transmembrane region (gp37) of the env genes of the 6 viruses were well conserved to endogenous counterpart sequences (>97.8%). However, the gp85 genes displayed high variability with any known chicken ALV strains. Growth kinetics of DF-1 cells infected with the isolated ALV showed viral titers that were lower than those infected with the GD13 (ALV-A), CD08 (ALV-B), and CHN06 (ALV-J) on day 7 post-infection. The infected Specific-pathogen-free (SPF) chickens could produce continuous viremia, atrophy of immune organs, growth retardation and no tumors were observed. These subgroup ALVs are unique and may be common in south China. The results suggested that updating the control and eradication program of exogenous ALV for yellow feather broiler breeders in south China needs to be considered because of the emergence of the new subgroup viruses., Competing Interests: The authors declare no conflict of interest.

Avian leukemia subgroup J (ALV-J) is one of the most detrimental neoplastic diseases in poultry production. However, the differences between somatic cells and immune cells post-infection remain poorly understood. The aim of our study was to detect the different responses in chicken to infection with ALV-J in different cell lines. In this study, we detected transcriptome expression changes during infection with ALV-J in chicken embryo fibroblast (CEF) and HD11 cell lines. RNA-Seq was used to determine the expression levels of mRNA transcripts from the two cell types after infection with ALV-J at 1, 4, and 7 dpi, and gene ontology analyses were used to cluster differentially expressed genes into pathways. Quantitative real-time PCR confirmed the expression of 336 and 269 differentially expressed genes in CEF and HD11 lines, respectively, involved in innate immunity (OASL, CCL4), adaptive immunity (LYZ, CD72), apoptosis and autophagy (WISP2, COMP), inflammation (JSC, IL8), and tumorgenesis (PCNA, GPX3). The notable signal transduction pathways included the PPARs signaling pathway and ECM-receptor interactions in CEF, and the Toll-like receptor, NOD-like receptor, and RIG-I-like receptor signaling pathways in HD11. To our knowledge, this is the first study to use high-throughput sequencing methods to investigate viral infection in different cell types. The results of the present study form a foundation for developing potential biological markers for viral infection.

J subgroup avian leukosis virus (ALV-J) is an exogenous retrovirus of avian. A key feature of ALV-J infection is leading to severe immunosuppressive characteristic of diseases. Viral components of retrovirus were reported closely associated with immunosuppression, and several similarities between exosomes and retrovirus preparations have lead to the hypotheses of retrovirus hijacker exosomes pathway. In this study, we purified exosomes from DF-1 cells infected and uninfected by ALV-J. Electron microscopy and mass spectrometry (MS) analysis showed that ALV-J not only increased the production of exosomes from ALV-J infected DF-1 cells (Exo-J) but also stimulated some proteins expression, especially ALV-J components secreted in exosomes. Immunosuppressive domain peptide (ISD) of envelope subunit transmembrane (TM) and gag of ALV-J were secreted in Exo-J. It has been reported that HIV gag was budded from endosome-like domains of the T cell plasma membrane. But env protein was first detected in exosomes from retrovirus infected cells. We found that Exo-J caused negative effects on splenocytes in a dose-dependant manner by flow cytometric analysis. And low dose of Exo-J activated immune activity of splenocytes, while high dose possessed immunosuppressive properties. Interestingly, Exo-J has no significant effects on the immunosuppression induced by ALV-J, and the immunosuppressive effects induced by Exo-J lower than that by ALV-J. Taken together, our data indicated that Exo-J supplied a microenvironment for the replication and transformation of ALV-J., (Copyright © 2017. Published by Elsevier Ltd.)

Avian leukosis virus subgroup (ALV-J) is an oncogenic neoplasm-inducing retrovirus that causes significant economic losses in the poultry industry. Recent studies have demonstrated circular RNAs (circRNAs) are implicated in pathogenic processes; however, no research has indicated circRNAs are involved in resistance to disease. In this study, over 1800 circRNAs were detected by circRNA sequencing of liver tissues from ALV-J-resistant (n = 3) and ALV-J-susceptible chickens (n = 3). 32 differentially expressed circRNAs were selected for analyzing including 12 upregulated in ALV-J-resistant chickens and 20 upregulated in ALV-J-susceptible chickens, besides, the top five microRNAs (miRNAs) for 12 upregulated circRNAs in ALV-J-resistant chickens were analyzed. Gene ontology and KEGG pathway analyses were performed for miRNA target genes, the predicted genes were mainly involved in immune pathways. This study provides the first evidence that circRNA alterations are involved in resistance to ALV-J-induced tumor formation. We propose circRNAs may help to mediate tumor induction and development in chickens.

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.

In our previous study, six subgroup J strains of avian leukosis virus (ALV-J)-associated acutely transforming viruses carrying different lengths of the v-fps oncogene, designated as Fu-J and Fu-J1-5, were isolated and characterized from fibrosarcomas in ALV-J-infected chickens. In the present study, the oncogenic potential of Fu-J and Fu-J1-5 was investigated using a reverse genetics technique. Six replication-defective viruses, named rFu-J and rFu-J1-5, were rescued with the replication-competent rescued ALV-J strain rSDAU1005 as a helper virus by co-transfection of chicken embryo fibroblast monolayers with infectious clone plasmids. Experimental bird studies were performed, demonstrating that only the rescued rFu-J virus carrying the complete v-fps oncogene with rSDAU1005 as the helper virus could induce acute fibrosarcoma after inoculation in specific-pathogen-free (SPF) chickens. These results provide direct evidence that the replication-defective acutely transforming Fu-J virus, with the complete v-fps oncogene, was associated with acute fibrosarcoma in chickens infected with ALV-J in the field, as reported previously.

Subgroup J avian leukosis virus (ALV-J) is an oncogenic retrovirus known to induce tumor formation and immunosuppression in infected chickens. One of the organs susceptible to ALV-J is the bone marrow, from which specialized antigen-presenting cells named dendritic cells (BM-DCs) are derived. Notably, these cells possess the unique ability to induce primary immune responses. In the present study, a method of cultivating and purifying DCs from chicken bone marrow in vitro was established to investigate the effects of ALV-J infection on BM-DC differentiation or generation. The results indicated that ALV-J not only infects the chicken bone marrow mononuclear cells but also appears to inhibit the differentiation and maturation of BM-DCs and to trigger apoptosis. Moreover, substantial reductions in the mRNA expression of TLR1, TLR2, TLR3, MHCI, and MHCII and in cytokine production were detected in the surviving BM-DCs following ALV-J infection. These findings indicate that ALV-J infection disrupts the process of bone marrow mononuclear cell differentiation into BM-DCs likely via altered antigen presentation, resulting in a downstream immune response in affected chickens., (Copyright © 2016 Elsevier B.V. All rights reserved.)

To investigate the effects of co-infections of subgroup J avian leukosis virus (ALV-J) and Eimeria tenella on the pathogenesis in specific-pathogen-free (SPF) white leghorn chickens, groups of chickens were infected with ALV-J strain NX0101 at one day of age or with E. tenella at 14 days of age or both. The control group was left uninfected and was mock-inoculated with phosphate buffer saline (PBS). Mortality rates, body weights, cecal lesions, and viremia of infected chickens in each group were evaluated. Immune status was evaluated by measuring several parameters: immune organ weight/body weight index, specific humoral responses to inactivated NDV vaccine and to inoculated E. tenella, proportions of blood CD3+CD4+ and CD3+CD8α+ lymphocytes and transcriptional levels of cytokines in blood and cecal tonsils. The results show that co-infections of ALV-J and E. tenella induced a higher mortality rate and a lower body weight in SPF chickens compared to single-pathogen infection. In co-infected chickens, ALV-J accelerated the disease symptoms induced by E. tenella, and the E. tenella extended the ALV-J viremia. Thymus atrophy, decrease in the humoral response levels to pathogens and the NDV vaccine, modifications in the blood lymphocyte sub-populations and transcriptional cytokine disorders were found in co-infected chickens compared to chickens infected with one pathogen alone and to controls. We underline a synergy between ALV-J and E. tenella that results in increasing pathogenesis in SPF chickens., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Transduction of oncogenes by ALVs and generation of acute transforming viruses is common in natural viral infections. In order to understand the molecular basis for the rapid oncogenicity of Fu-J, an acutely transforming avian leukosis virus isolated from fibrosarcomas in crossbreed broilers infected with subgroup J avian leukosis virus (ALV-J) in China, complete genomic structure of Fu-J virus was determined by PCR amplification and compared with those of Fu-J1, Fu-J2, Fu-J3, Fu-J4, and Fu-J5 reported previously. The results showed that the genome of Fu-J was defective, with parts of gag gene replaced by the complete v-fps oncogene and encoded a 137 kDa Gag-fps fusion protein. Sequence analysis revealed that Fu-J and Fu-J1 to Fu-J5 were related quasi-species variants carrying different lengths of v-fps oncogenes generated from recombination between helper virus and c-fps gene. Comparison of virus carrying v-fps oncogene also gave us a glimpse of the molecular characterization and evolution process of the acutely transforming ALV.

Endogenous retroviruses (ERVs) are important retroelements that reside in host genomes. However, ERV expression patterns and regulatory mechanisms are poorly understood. In this study, chicken embryo fibroblasts (CEFs) and MSB1 cells infected with Marek's disease virus (MDV) exhibited significantly increased expression of env from the endogenous retrovirus ALVE. In contrast, env expression was significantly lower in CEF and MSB1 cells infected with exogenous avian leukosis virus J (ALVJ) at the early infection stage. Furthermore, env was found to be ubiquitously expressed in various chicken tissues, with high expression in certain tissues at 2 days of age and low levels in most tissues, including immune organs (thymus, spleen and bursa) as well as the brain and heart, at 35 days of age. Sequence analysis revealed miR-155 target sites in env transcripts, which was verified using a firefly luciferase reporter assay, and treatment with miR-155 agomir significantly decreased levels of env transcripts in MSB1 and CEF cells. Together, these findings suggest that the env gene from the endogenous retrovirus ALVE is regulated by miR-155.

Many pathogens trigger caspase-1-mediated innate immune responses. Avian leukosis virus subgroup J (ALV-J) causes serious immunosuppression and diverse tumors in chicks. The caspase-1 inflammasome mechanism of response to ALV-J invading remains unclear. Here we investigated the expression of caspase-1, the inflammasome adaptor NLRP3, IL-1β and IL-18 in response to ALV-J infection in the liver of chick. We found caspase-1 mRNA expression was elevated at 5 dpi and peaked at 7 dpi in ALV-J infected animals. Corresponding to this, the expressions of NLRP3 and proinflammatory cytokines IL-1β and IL-18 were significantly increased at 5 or 7 dpi. In addition, caspase-1 protein expression and inflammatory cell infiltration were induced after virus infection. These results indicated that ALV-J infection could trigger the caspase-1- mediated inflammatory response in chicks. Thus, an understanding of the inflammatory responses can provide a better insight into the pathogenicity of ALV-J and a possible anti-virus target for ALV-J infection., (Copyright © 2016 Elsevier B.V. All rights reserved.)

To investigate the molecular mechanisms of the oncogenic effects of avian leukosis virus subgroup J (ALV-J), we examined mutations in and the expression of p53 in the myelocytomas distributed in the liver, spleen, trachea, and bone marrow, as well as in fibrosarcomas in the abdominal cavity and hemangiomas in skin from chickens that were naturally or experimentally infected with ALV-J. Two types of mutations in the p53 gene were detected in myelocytomas of both the experimentally infected and the naturally infected chickens and included point mutations and deletions. Two of the point mutations have not been reported previously. Partial complementary DNA clones with a 122-bp deletion in the p53 gene ORF and a 15-bp deletion in the C-terminus were identified in the myelocytomas. In addition, moderate expression of the mutant p53 protein was detected in the myelocytomas that were distributed in the liver, trachea, spleen, and bone marrow. Mutant p53 protein was not detected in the subcutaneous hemangiomas or in the abdominal fibrosarcomas associated with natural and experimental ALV-J infection, respectively. These results identify mutations associated with abnormal expression of p53 in ALV-J-associated myelocytomas, suggesting a role in tumorigenesis., (© The Author(s) 2014.)

To study interactions between avian leukosis virus subgroup J (ALV-J) and reticuloendotheliosis virus (REV) and the effects of co-infection on pathogenicity of these viruses, 1-day-old broiler chicks were infected with ALV-J, REV or both ALV-J and REV. The results indicated that co-infection of ALV-J and REV induced more growth retardation and higher mortality rate than ALV-J or REV single infection (P < 0.05). Chickens co-infected with ALV-J and REV also showed more severe immunosuppression than those with a single infection. This was manifested by significantly lower bursa of Fabricius and thymus to body weight ratios and lower antibody responses to Newcastle disease virus and H9-avian influenza virus (P < 0.05). Perihepatitis and pericarditis related to severe infection with Escherichia coli were found in many of the dead birds. E. coli was isolated from each case of perihepatitis and pericarditis. The mortality associated with E. coli infection in the co-infection groups was significantly higher than in the other groups (P < 0.05). Among 516 tested E. coli isolates from 58 dead birds, 12 serotypes of the O-antigen were identified in two experiments. Different serotypes of E. coli strains were even isolated from the same organ of the same bird. Diversification of O-serotypes suggested that perihepatitis and pericarditis associated with E. coli infection was the most frequent secondary infection following the immunosuppression induced by ALV-J and REV co-infection. These results suggested that the co-infection of ALV-J and REV caused more serious synergistic pathogenic effects, growth retardation, immunosuppression, and secondary E. coli infection in broiler chickens.

Epidemiological studies suggest that retroviruses, including human immunodeficiency virus type 1, are associated with cardiomyopathy and myocarditis, but a causal relationship remains to be established. We encountered unusual cardiomyocyte hypertrophy and mitosis in Japanese native fowls infected with subgroup A of the avian leukosis viruses (ALVs-A), which belong to the genus Alpharetrovirus of the family Retroviridae and mainly induce lymphoid neoplasm in chickens. The affected hearts were evaluated by histopathology and immunohistochemistry, viral isolation, viral genome sequencing and experimental infection. There was non-suppurative myocarditis in eighteen fowls and seven of them had abnormal cardiomyocytes, which were distributed predominantly in the left ventricular wall and showed hypertrophic cytoplasm and atypical large nuclei. Nuclear chains and mitosis were frequently noted in these cardiomyocytes and immunohistochemistry for proliferating cell nuclear antigen supported the enhancement of mitotic activity. ALVs were isolated from all affected cases and phylogenic analysis of envSU genes showed that the isolates were mainly classified into two different clusters, suggesting viral genome diversity. In ovo experimental infection with two of the isolates was demonstrated to cause myocarditis and cardiomyocyte hypertrophy similar to those in the naturally occurring lesions and cardiac hamartoma (rhabdomyoma) in a shorter period of time (at 70 days of age) than expected. These results indicate that ALVs cause myocarditis as well as cardiomyocyte abnormality in chickens, implying a pathogenetic mechanism different from insertional mutagenesis and the existence of retrovirus-induced heart disorder.

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.

Background: Subgroup J avian leukosis virus (ALV-J) infection can induce tumor-related diseases in chickens. Previous studies by our laboratory demonstrated that ALV-J infection of DF-1 cells resulted in altered activity and phosphorylation of AKT. However, little is known about the subsequent activation of host DF-1 cells., Results: In the current study, autophagy inhibition was observed for ALV-J infected DF-1 cells. Our data showed that the autophagosome protein, microtubule-associated protein 1 light chain 3-II (LC3-II), was reduced considerably in DF-1 cells infected with active ALV-J, while no change was observed for cells infected with inactivated ALV-J. Autophagy inhibition was also confirmed by fluorescence microscopy and transmission electron microscopy. Interestingly, when autophagy was promoted by rapamycin, the titers of ALV-J replication were decreased, and the replication level of ALV-J was significantly enhanced when atg5 (autophagy-related gene 5) was knocked out., Conclusions: These results suggested that ALV-J infection could down-regulate autophagy in DF-1 cells during viral replication. This study is the first to report on the relationship between ALV-J infection and autophagy in DF-1 cells.

Background: Clinical cases of hemangioma associated with subgroup J avian leukosis virus (ALV-J) have been reported in commercial chicken layer flocks since 2006. We attempted to reproduce hemangioma through experimental infection with ALV-J to evaluate viral pathogenicity in layer birds and their progenies., Results: Body weight and indexes for immune organs of chickens infected with ALV-J strain SCDY1 were lower than those in controls. Proliferation of lymphocytes was observed in many tissues, and viral integration was detected in the genome of lymphocytes at 14 days post-infection, along with virus shedding. ALV-J was also efficiently transmitted from eggs to progenies. Embryo hatchability and progeny mortality were lower than those for controls. The efficiencies of virus shedding and virus integration in the lymphocytes of progenies were higher than those in parents., Conclusions: ALV-J is able to inhibit the growth of infected chickens, and causes damage to immune organs. Vertical transmission of ALV-J appears to be more deleterious than horizontal transmission.

Poly(ADP-ribose) polymerase 1 (PARP-1) is a cellular enzyme with a fundamental role in DNA repair and the regulation of chromatin structure, processes involved in the cellular response to retroviral DNA integration. However, the function of PARP-1 in retroviral DNA integration is controversial, probably due to the functional redundancy of the PARP family in mammalian cells. We evaluated the function of PARP-1 in retroviral infection using the chicken B lymphoblastoid cell line DT40. These cells lack significant PARP-1 functional redundancy and efficiently support the postentry early events of the mammalian-retrovirus replication cycle. We observed that DT40 PARP-1(-/-) cells were 9- and 6-fold more susceptible to infection by human immunodeficiency virus type 1 (HIV-1)- and murine leukemia virus (MLV)-derived viral vectors, respectively, than cells expressing PARP-1. Production of avian Rous-associated virus type 1 was also impaired by PARP-1. However, the susceptibilities of these cell lines to infection by the nonretrovirus vesicular stomatitis virus were indistinguishable. Real-time PCR analysis of the HIV-1 life cycle demonstrated that PARP-1 did not impair reverse transcription, nuclear import of the preintegration complex, or viral DNA integration, suggesting that PARP-1 regulates a postintegration step. In support of this hypothesis, pharmacological inhibition of the epigenetic mechanism of transcriptional silencing increased retroviral expression in PARP-1-expressing cells, suppressing the differences observed. Further analysis of the implicated molecular mechanism indicated that PARP-1-mediated retroviral silencing requires the C-terminal region, but not the enzymatic activity, of the protein. In sum, our data indicate a novel role of PARP-1 in the transcriptional repression of integrated retroviruses.

In the past 5 years, an atypical clinical outbreak of avian leukosis virus subgroup J (ALV-J), which contains a unique 205-nucleotide deletion in its 3' untranslated region (3'UTR), has become epidemic in chickens in China. To determine the role of the 205-nucleotide deletion in the pathogenicity of ALV-J, a pair of viruses were constructed and rescued. The first virus was an ALV-J Chinese isolate (designated HLJ09SH01) containing the 205-nucleotide deletion in its 3'UTR. The second virus was a chimeric clone in which the 3'UTR contains a 205-nucleotide sequence corresponding to a region of the ALV-J prototype virus. The replication and pathogenicity of the rescued viruses (rHLJ09SH01 and rHLJ09SH01A205) were investigated. Compared to rHLJ09SH01A205, rHLJ09SH01 showed a moderate growth advantage in vitro and in vivo, in addition to exhibiting a higher oncogenicity rate and lethality rate in layers and broilers. Increased vascular endothelial growth factor A (VEGF-A) and vascular endothelial growth receptor subtype 2 (VEGFR-2) expression was induced by rHLJ09SH01 more so than by rHLJ09SH01A205 during early embryonic vascular development, but this increased expression disappeared when the expression levels were normalized to the viral levels. This finding suggests that the expression of VEGF-A and VEGFR-2 is associated with viral replication and may also represent a novel molecular mechanism underlying the oncogenic potential of ALV-J. Overall, our findings not only indicate that the unique 205-nucleotide deletion in the ALV-J genome occurred naturally in China and contributes to increased pathogenicity but also point to the possible mechanism of ALV-J-induced oncogenicity.

Subgroup J avian leukosis virus (ALV-J) isolate GDKP1202 was isolated from a 50-day-old local yellow commercial broiler in the Guangdong province of China in 2012. Here we report the complete genomic sequence of the GDKP1202 isolate, which caused high mortality, serious growth suppression, thymic atrophy, and liver enlargement in commercial broilers. A novel potential binding site (5'-GGCACCTCC-3') for c-myb was identified in the GDKP1202 genome. These findings will provide additional insights into the molecular characteristics in the genomes and pathogenicity of ALV-J.

Subgroup J avian leukosis virus (ALV-J) is an oncogenic retrovirus primarily causing myeloid leukosis (ML) in broilers. Although ALV is well under control in a few countries including the USA, poultry industry in many parts of the world continues suffering from serious economic loss due to sporadic or widespread ALV infection, especially ALV-J infection. ALV-J infection of chickens is reportedly mediated by a cellular receptor. So far, however, no genetic variant of the receptor gene that confers resistance to ALV-J has been identified. To advance our understanding on epigenetic factors that are involved in the event of ALV-J infection, we examined the expression of miRNAs in livers of 10-week-old chickens uninfected or infected with ALV-J by miRNA microarray analysis. Our data showed there were 12 miRNAs differentially expressed in liver between the uninfected and infected groups (P<0.01). Of which, the expressions of seven miRNAs (gga-mir-221, gga-mir-222, gga-mir-1456, gga-mir-1704, gga-mir-1777, gga-mir-1790, and gga-mir-2127,) were upregulated by ALV-J infection and may be involved in oncogenicity. The other five miRNAs (gga-let-7b, gga-let-7i, gga-mir-125b, gga-mir-375, and gga-mir-458) were significantly downregulated. The downregulated miRNAs may play important roles in tumor suppression. This finding paves the way for further exploration of epigenetic influence on tumorigenicity upon ALV-J infection., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Avian leukosis virus subgroup J (ALV-J) is an avian oncogenic retrovirus that has led to severe economic losses in the poultry industry in China in recent years. The pathogenesis of virus infection and virus-host interactions are still not well elucidated. In this paper, we investigated the expression changes for cellular proteins in DF-1 cells infected with ALV-J. Comparative analyses revealed that the majority of the altered proteins in DF-1 cells appeared at 6-12h after ALV-J infection. Mass spectrometry identified 74 altered cellular proteins, including 30 up-regulated proteins and 44 down-regulated proteins. Some of these proteins are involved in cell cytoskeleton, metabolic processes, response to stimulus and immune responses. Other proteins, such as DJ-1, UCHL1, VDAC1 and HMGB1, have some relationship to apoptosis or oncogenesis. The changes in the transcriptional profile of DJ-1, UCHL1, VDAC1 and HMGB1 in infected as compared to uninfected DF-1 cells were confirmed by real-time RT-PCR. Our work gives some information about differential protein expression in cells infected with ALV-J, which will help us to understand viral pathogenicity., (Copyright © 2012 Elsevier B.V. All rights reserved.)

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.

To study the correlation between 50% tissue-culture infective dose (TCID50) value and p27 antigen S/P value of Avian leukosis virus subgroup J and discuss their significance, chicken embryo fibroblast (CEF) cells were inoculated with Avian leukosis virus subgroup J strain NX0101 and samples were tested continuously for ten days after changing maintenance media. The correlation between TCID50 and p27 antigen S/P value of ten days were then analysized. Simultaneously, DF-1 cells were inoculated with NX0101 and passaged to 20 generations. Samples taken from 1st generation, 5th generation, 10th generation, 15th generation and 20th generation were tested for the TCID50 titer and the p27 antigen S/P value separately. A significant Pearson correlation was found between them in CEF cells (r = 0.85277; P < 0.0001) and in DF-1 cells (r = 0.93000; P = 0.0220). This study provided an important parameter for predicting TCID50 by detecting the p27 antigen S/P value.

Fowl glioma-inducing virus (FGV), which belongs to avian leukosis virus subgroup A, causes the so-called fowl glioma and cerebellar hypoplasia in chickens. In the present study, the complete nucleotide sequences of four isolates (Tym-43, U-1, Sp-40 and Sp-53) related to the FGV prototype were determined and their pathogenicity was investigated. Phylogenetic analysis showed that the 3'-long terminal repeat of all isolates grouped together in a cluster, while sequences of the surface (SU) proteins encoded by the env gene of these viruses had 85 to 96% identity with the corresponding region of FGV. The SU regions of Tym-43, U-1 and FGV grouped together in a cluster, but those of Sp-40 and Sp-53 formed a completely separate cluster. Next, C/O specific-pathogen-free chickens were inoculated in ovo with these isolates as well as the chimeric virus RCAS(A)-(FGVenvSU), constructed by substituting the SU region of FGV into the retroviral vector RCAS(A). The four variants induced fowl glioma and cerebellar hypoplasia and the birds inoculated with Sp-53 had the most severe lesions. In contrast, RCAS(A)-(FGVenvSU) provoked only mild non-suppurative inflammation. These results suggest that the ability to induce brain lesions similar to those of the FGV prototype is still preserved in these FGV variants.

In the 3 years leading up to November 2009, 6 different types of naturally occurring neoplasms associated with avian leukosis virus subgroup J (ALV-J) were diagnosed by histopathology, polymerase chain reaction (PCR) and immunohistochemistry (IHC) in 140 layer hens out of approximately 100,000. The most prevalent tumor type was hemangioma (50%) in commercial layer flocks; the second most prevalent neoplasm type was myelocytoma (38.6%); a small number of ALV-J positive lymphomas (4.3%) that were not associated with Marek's disease (MD) or lymphoid leukosis (LL) was observed. Histiocytic sarcomas (2.1%) were found mainly in the spleen, liver and kidney. Fibrosarcomas (2.8%) presented as metastatic thigh, liver, lung and kidney neoplasms. Three cases of intestinal adenocarcinoma (2.1%) were found associated with ALV-J. Chickens with multiple tumors were a common phenomenon. Usually, hemangiomas plus myelocytomas (8.6%), myelocytomas plus histiocytic sarcomas (2.1%), hemangioma plus myelocytoma and lymphoma (3.6%) were found in various viscera organs. The present report describes the occurrence of multiple neoplasms associated with ALV-J in field layer hens.