ALV-J Strain Research Articles

Diagnosis of Avian Leukosis Virus subgroup J in asymptomatic commercial layers using qPCR

Avian Leukosis Virus (ALV), one of the dangerous retroviruses threatens the poultry industry, is oncogenic and can easily transmit. Over the past three decades, the poultry industry has been significantly affected by subgroup J (ALV-J) in particular, known for its ability to spread both horizontally and vertically. The fact that a commercial vaccine has not yet been developed against ALV increases the risk potential. For these reasons, positive cases should be identified to control ALV infections and minimize infection. A prompt and accurate diagnosis is vital to detecting infected birds. For this purpose, sampling was performed in total 153 blood samples from 14 different asymptomatic commercial layer farm located in Afyonkarahisar, T?rkiye. Herein, the presence, density and prevalence of ALV-J strain and whether it has an oncogenic effect was investigated by qPCR and western blot for the first time in T?rkiye. The average contamination rate of ALV-J was calculated as 70.91% over all samples. However, p27 antigen that is the most abundant polypeptide encoded by the gag gene of ALV could not be detected. High prevalence of ALV-J suggests that ALV strains can be found asymptomatically without showing any symptoms.

3'UTR of ALV-J can affect viral replication through promoting transcription and mRNA nuclear export.

3'UTRs can affect gene transcription and post-transcriptional regulation in multiple ways, further influencing the function of proteins in a unique manner. Recently, ALV-J has been mutating and evolving rapidly, especially the 3'UTR of viral genome. Meanwhile, clinical symptoms caused by ALV-J have changed significantly. In this study, we found that the ALV-J strains containing △-r-TM-type 3'UTR are the most abundant. By constructing ALV-J infectious clones and subgenomic vectors containing different 3'UTRs, we prove that 3'UTRs directly affect viral tissue preference and can promote virus replication as an enhancer. ALV-J strain containing 3'UTR of △-r-TM proliferated fastest in primary cells. All five forms of 3'UTRs can assist intron-containing viral mRNA nuclear export, with similar efficiency. ALV-J mRNA half-life is not influenced by different 3'UTRs. Our results dissect the roles of 3'UTR on regulating viral replication and pathogenicity, providing novel insights into potential anti-viral strategies.

Isolation and characterization of avian leukosis virus subgroup J associated with hemangioma and myelocytoma in layer chickens in China.

A strain of avian leukosis virus (ALV) belonging to a new envelope subgroup J (ALV-J) emerged in 1988 as a new subgroup of ALV and spread rapidly throughout the world. Due to the infection and spread of ALV-J, the global poultry industry experienced a significant loss. Although the disease had been prevented and controlled effectively by culling domestic chickens in the infected zone, a few field cases of ALV-J infection were reported in China in recent years. This study was conducted to characterize the genome and analyze the lesions and histopathology of the ALV-J strain named HB2020, which was isolated from layer chickens in Hubei Province, China. The full-length proviral genome sequence analysis of ALV-J HB2020 revealed that it was a recombinant strain of ev-1 and HPRS-103 in the gag gene in comparison to ALV-J prototype HPRS-103. In the 3′-untranslated region (3'UTR) of the nucleotide sequence, there were found 205-base pairs (bp) deletion, of which 175 were detected in the redundant transmembrane (rTM) region. Besides, the surface glycoprotein gene gp85 had five mutations in a conservative site, whereas the transmembrane protein gene gp37 was relatively conserved. The animal experiments conducted later on this strain have shown that HB2020 can cause various neoplastic lesions in chickens, including enlarged livers with hemangiomas and spleens with white nodules. Additionally, as the exposure time increased, the number of tumor cells that resembled myelocytes in the blood smears of infected chickens gradually increased. These results indicated that HB2020 on recombination with ALV subgroup E (ALV-E) and ALV-J could induce severe hemangiomas and myelocytomas. This inference might provide a molecular basis for further research about the pathogenicity of ALV and emphasize the need for control and prevention of avian leukosis.

Cytokine inducible SH2-containing protein potentiate J subgroup avian leukosis virus replication and suppress antiviral responses in DF-1 chicken fibroblast cells

Cytokine-inducible Srchomology2 (SH2)-containing protein (CIS) belongs to the suppressors of cytokine signaling (SOCS) protein family function as a negative feedback loop inhibiting cytokine signal transduction. J subgroup avian leukosis virus (ALV-J), a commonly-seen avian virus with a feature of immunosuppression, poses an unmeasurable threat to the poultry industry across the world. However, commercial medicines or vaccines are still no available for this virus. This study aims to evaluate the potential effect of chicken CIS in antiviral response and its role on ALV-J replication. The results showed that ALV-J strain SCAU-HN06 infection induced CIS expression in DF-1 cells, which was derived from chicken embryo free of endogenous avian sarcoma-leukosis virus (ASLV) like sequences. By overexpressing CIS, the expression of chicken type I interferon (IFN-I) and interferon-stimulated genes (ISGs; PKR, ZAP, CH25H, CCL4, IFIT5, and ISG12) were both suppressed. Meanwhile, data showed that CIS overexpression also increased viral yield. Interestingly, knockdown of CIS enhanced induction of IFN-I and ISGs and inhibited viral replication. Collectively, we proved that modulation of CIS expression not only affected SCAU-HN06 replication in vitro but also altered the expression of IFN-I and ISGs that act as an essential part of antiviral innate immune system. Our data provide a potential target for developing antiviral agents for ALV-J.

Isolation and Identification of Subgroup J Avian Leukosis Virus Inducing Multiple Systemic Tumors in Parental Meat-Type Chickens.

Avian leukosis virus (ALV) continues evolving to obtain new genomic characters to enhance its pathogenicity. In the present study, an ALV-J strain LH20180301 was isolated from broiler breeder chickens that reached the speak of paralyzation before 20-week-old. The necropsy chickens showed subcutaneous and muscular hemorrhage, and developed tumors in multiple organs including bone, liver, spleen, and kidney. The complete provirus was then cloned and sequenced to investigate the molecular characteristics and oncogenicity etiology of this virus associated with the outbreak of disease. The genomic structure of the reported ALV-J strain LH20180301 was highly conservative with other ALVs. Recombination events between the virus with endogenous virus were identified in the viral genome. Compared with the ALV-J original HPRS-103 strain, the major recombination sites of the viral genome with ev-1 were located in 5′ UTR-gag and 3′ UTR regions. Phylogenetic analysis of group specific antigen gp85 encoding protein showed that the LH20180301 branched with ALV-J prevalent in “yellow chickens” of local breeds in South China. Nine amino acids (N58, D60, K70, A71, K108, N112, N113, N121, R272) in the gp85 were highly conserved among ALV-J isolates before 2012, but various mutations were found in the late isolates including LH20180301. In addition, the LH20180301 strain also had the same deletion pattern of 3′ UTR with them. Therefore, LH20180301 might derive from the same ancestor with those viruses and may be the trend of ALV-J evolution in China. The defined new genomic characters in the gp85 and 3′ UTR region of ALV-J might provide the molecular basis for its enhanced oncogenicity.

Complete genome sequence of a novel recombinant avian leukosis virus isolated from a three-yellow chicken.

In this study, an avian leukosis virus (ALV) strain (GX-2020-01) was isolated from a three-yellow chicken, and its complete genome was 7570bp long with the typical organization "5'LTR-gag-pol-env-3'LTR." Phylogenetic analysis and sequence comparison revealed that it belongs to the ALV-J subgroup. However, the LTR region of GX-2020-01 is highly similar to that of reference strains of ALV-K/E (96.61%-97.10%), demonstrating that this novel isolate is a natural recombinant. The replication efficiency of GX-2020-01 was significantly lower than the previously isolated ALV-J strain (NX0101), indicating that the recombination event might have resulted in slower virus replication, making it harder for it to be detected through routine testing.

Geese not susceptible to virulent subgroup J avian leukosis virus isolated from chickens

ABSTRACT 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.

Difference in pathogenicity of 2 strains of avian leukosis virus subgroup J in broiler chicken

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.

Preparation of the Secretory Recombinant ALV-J gp85 Protein Using Pichia pastoris and Its Immunoprotection as Vaccine Antigen Combining with CpG-ODN Adjuvant.

This study focuses on preparing the secretory recombinant J subgroup of avian leukosis virus (ALV-J) gp85 protein using Pichia pastoris and evaluating its immunoprotection as vaccine antigen combining with CpG-ODN adjuvant. The secretory recombinant plasmid pPIC9-gp85 containing ALV-J gp85 gene was designed and was transfected into the genome of P. pastoris (GS115) cells. The recombinant plasmid was expressed under the induction of methanol. The expressed products in the medium of the cells were purified and identified with endoglycosidase digestion assay and western blot mediated with monoclonal antibody (MAb) JE9. The purified product combining with CpG-ODN adjuvant was inoculated intramuscularly into 7-day-old chickens and three booster inoculations were performed on 21 days post first inoculation (dpfi), 42, and 56 dpfi. The antibody responses and cellular immune responses were detected, and the protective effects were analyzed after challenge with ALV-J. The results showed that the secretory pPIC9-gp85 plasmid was successfully constructed and could be stably expressed in GS115 cells. The expressed products were N-acetylglucosylated and could specifically combine with MAb (JE9). The secreted gp85 protein combining with CpG-ODN adjuvant could induce higher antibody response and spleen lymphocyte proliferation response and IFN-γ-inducing response, and could protect all the inoculated chickens against the viremia and the immunosuppressive lesions caused by ALV-J challenge. The results of neutralizing test in vitro suggested that the antisera with some ALV-J antibody titers could neutralize ALV-J strain and inhibit the growth of virus in vitro. The result of IFA showed that IgG antibody in the antisera could specifically combine with ALV-J strain in cells. It can be concluded that the secretory recombinant gp85 protein, as a new acetylglucosylated gp85 protein, was successfully prepared and combining with CpG-ODN adjuvant could protect the inoculated chickens against ALV-J infection. This study first reported the methods on preparing the secretory recombinant ALV-J gp85 protein using P. pastoris and evaluated its immunoprotection.

Effect of Dual Infection with Eimeria tenella and Subgroup J Avian Leukosis Virus on the Cecal Microbiome in Specific-Pathogen-Free Chicks.

Understanding gut microflora alterations associated with gut parasites and other pathogens that drive these alterations may help to promote the understanding of intestinal flora’s role in multiple-infected individuals. This study examined the effects of dual infection with Eimeria tenella and subgroup J avian leukosis virus (ALV-J) on the chick cecal microbiome. Specific-pathogen-free (SPF) chicks were infected with either ALV-J strain NX0101 at 1 day of age or E. tenella at 14 days of age, another group was infected with both pathogens. Cecal contents from chicks were extracted at the 21 days of age and examined using 16S rRNA genes illumina sequencing. A genus-level opportunistic pathogen enrichment and a decrease in possible resident probiotics were observed in response to all infection groups. Of note, E. tenella mainly induced a sharp decrease in the richness and diversity of cecal microflora from infected chicks because of the serious E. tenella-induced damage to intestinal tissues. ALV-J infection led to structural changes and increased the richness and diversity of the cecal microflora. As for E. tenella and ALV-J dual infected chicks, a marked enrichment of opportunistic pathogens in addition to some other bacteria that may play a role involving cecal microbiota carbohydrate transport and metabolic functions were also found compared to single pathogen-infected chicks. Overall, this study provides valuable insights into the SPF chick cecal microbial community, the modulations of this community in response to different pathogenic infections of single or dual infections, and the interactions between different pathogens and hosts from the perspective of intestinal microflora.

Viral proliferation and expression of tumor-related gene in different chicken embryo fibroblasts infected with different tumorigenic phenotypes of avian leukosis virus subgroup J

Subgroup J avian leukosis virus (ALV-J) causes a neoplastic disease in infected chickens. The ALV-J strain NX0101, which was isolated from broiler breeders in 2001, mainly induced formation of myeloid cell tumors. However, strain HN10PY01, which was recently isolated from laying hens, mainly induces formation of myeloid cell tumors and hemangioma. To identify the molecular pathological mechanism underlying changes in host susceptibility and tumor classification induced by these two types of ALV-J strains, chicken embryo fibroblasts derived from chickens with different genetic backgrounds (broiler breeders and laying hens) and an immortalized chicken embryo fibroblasts (DF-1) were prepared and infected with strain NX0101 or HN10PY01, respectively. The 50% tissue culture infective dose (TCID50) and levels of ALV group-specific antigen p27 and heat shock protein 70 in the supernatant collected from the ALV-J infected cells were detected. Moreover, mRNA expression levels of tumor-related genes p53, c-myc, and Bcl-2 in ALV-J-infected cells were quantified. The results indicated that the infection of ALV-J could significantly increase mRNA expression levels of p53, c-myc, and Bcl-2. Strain HN10PY01 exhibited a greater influence on the three tumor-related genes in each of the three types of cells when compared with strain NX0101, and the TCID50 and p27 levels in the supernatant collected from HN10PY01-infected cells were higher than those collected from NX0101-infected cells. These results indicate that the infection of the two ALV-J strains influenced the gene expression levels in the infected cells, while the newly isolated strain HN10PY01 showed higher replication ability in cells and induced higher expression levels of tumor-related genes in infected cells. Furthermore, virus titers and expression levels of tumor-related genes and cellular stress responses of cells with different genetic backgrounds when infected with each of the two ALV-J strain were different, indicating that genetic backgrounds influenced the capabilities of the virus to infect and proliferate. The findings of this study provide useful data to further elucidate the mechanism underlying host susceptibility and tumor classification in ALV-J-infected chickens and cells.

Rescue of avian leukosis subgroup-J-associated acutely transforming viruses carrying different lengths of the v-fps oncogene and analysis of their tumorigenicity.

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.

Synergy of subgroup J avian leukosis virus and Eimeria tenella to increase pathogenesis in specific-pathogen-free chickens

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.

Transcription analysis of the response of chicken bursa of Fabricius to avian leukosis virus subgroup J strain JS09GY3

BackgroundAvian leukosis virus subgroup J (ALV-J) causes tumours and immunosuppression in chickens. The host-ALV interactions at the transcriptional level are unknown. In this study, gene expression profiling was performed to analyse the bursa response induced by ALV-J strain JS09GY3 in chickens. ResultsA total of 594 gene transcripts displaying differential expression during ALV-J infection were identified. These differentially expressed genes are involved in binding, biological regulation, metabolic processes (MYF6 and FABP3), response to stimulus (F13A1 and CNGA3) and immune system processes (LY86, CATHL2, CCL4, and OASL), and several differentially expressed genes (e.g., ETV7, MMP9, and NOV) are involved in tumourigenesis. Eight differentially expressed genes were confirmed by quantitative reverse transcription-PCR (qRT-PCR). Based on pathway analysis, the notable signalling pathways mainly included cytokine–cytokine receptor interaction, the JAK-STAT signalling pathway and the RIG-1-like receptor signalling pathway. ConclusionsThe gene expression profile obtained in this study may aid a better understanding of the molecular pathogenesis of ALV-J infection in chickens.

Reproduction of hemangioma by infection with subgroup J avian leukosis virus: the vertical transmission is more hazardous than the horizontal way

BackgroundClinical 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.ResultsBody 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.ConclusionsALV-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.

The Expression and Characterization of Gp85 Gene of Subgroup J Avian Leukosis Virus Associated with Hemangioma

The subtype of avian leukosis virus (ALV) was mainly determined by the gp85 glycoprotein. A subtype J ALV strain SCDY1 associated with hemangioma was isolated from grandparent breeding chicken and the highly antigenic region of its gp85 gene was amplified and expressed in Rosetta Escherichia coli using the pET-32a(+)vector. The fusion protein, which was expressed at a high level, was similar antigenically to the native gp85 protein as determined by Western blot assay using polyclonal antibodies against ALV-J strain. The fusion protein was also purified. This research lays a foundation for using this recombinant protein for development of indirect enzyme-linked immunosorbent assay (ELISA) for serum antibody detection or for production of monoclonal antibodies against prevalent ALV-J.

An ALV-J isolate is responsible for spontaneous haemangiomas in layer chickens in China

Subgroup J avian leukosis virus (ALV-J), first isolated in 1989, mainly induces tumours of myeloid leukosis (ML) in meat-type chickens. In 2006, ALV-J strain SCAU-HN06 was isolated from commercial layer hens with spontaneous haemangiomas in China. To confirm its role in the induction of haemangioma, we constructed a full-length copy of the proviral genome from SCAU-HN06, recovered virus from DF-1 cells detected by enzyme-linked immunosorbent assay, characterized its growth property and investigated its pathogenicity. The recovered virus appeared to be identical to SCAU-HN06 analysed by both blast gene sequences and indirect immunofluorescence assay. It also showed similarities in growth to the parental wild-type virus in vitro. The pathogenicity of the rescued and parental virus in specific-pathogen-free White Leghorn chickens was investigated. Both SCAU-HN06 and rSCAU-HN06 could induce haemangioma, with incidence of 52% and 42.8% respectively. Overall, our findings indicated that the ALV-J strain SCAU-HN06 was the causal agent inducing haemangiomas rather than ML in certain layer chickens.

The PI3K/Akt pathway is involved in early infection of some exogenous avian leukosis viruses

Avian leukosis virus (ALV) is an enveloped and oncogenic retrovirus. Avian leukosis caused by the members of ALV subgroups A, B and J has become one of the major problems challenging the poultry industry in China. However, the cellular factors such as signal transduction pathways involved in ALV infection are not well defined. In this study, our data demonstrated that ALV-J strain NX0101 infection in primary chicken embryo fibroblasts or DF-1 cells was correlated with the activity and phosphorylation of Akt. Akt activation was initiated at a very early stage of infection independently of NX0101 replication. The specific phosphatidylinositol 3-kinase (PI3K) inhibitors LY294002 or wortmannin could suppress Akt phosphorylation, indicating that NX0101-induced Akt phosphorylation is PI3K-dependent. ALV-A strain GD08 or ALV-B strain CD08 infection also demonstrated a similar profile of PI3K/Akt activation. Treatment of DF-1 cells with the drug 5-(N, N-hexamethylene) amiloride that inhibits the activity of chicken Na(+)/H(+) exchanger type 1 significantly reduced Akt activation induced by NX0101, but not by GD08 and CD08. Akt activation triggered by GD08 or CD08 was abolished by clathrin-mediated endocytosis inhibitor chlorpromazine. Receptor-mediated endocytosis inhibitor dansylcadaverine had a negligible effect on all ALV-induced Akt phosphorylation. Moreover, viral replication of ALV was suppressed by LY294002 in a dose-dependent manner, which was due to the inhibition of virus infection by LY294002. These data suggest that the activation of the PI3K/Akt signalling pathway by exogenous ALV infection plays an important role in viral entry, yet the precise mechanism remains under further investigation.