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

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

Chichen type III interferon produced by silkworm bioreactor induces ISG expression and restricts ALV-J infection in vitro.

Type III interferon (IFN-λ) has recently been shown to exert a significant antiviral impact against viruses in vertebrates. Avian leukosis virus subgroup J (ALV-J), which causes tumor disease and immunosuppression in infected chicken, is a retrovirus that is difficult to prevent and control because of a lack of vaccines and drugs. Here, we obtained chicken IFN-λ (chIFN-λ) using a silkworm bioreactor and demonstrated that chIFN-λ has antiviral activity against ALV-J infection of both chicken embryo fibroblast cell line (DF1) and epithelial cell line (LMH). We found that chIFN-λ triggered higher levels of particular type III interferon-stimulated genes (type III ISGs) including myxovirus resistance protein (Mx), viperin (RSAD2), and interferon-inducible transmembrane protein 3 (IFITM3) in DF1 and LMH cells. Furthermore, over-expression of Mx, viperin, and IFITM3 could inhibit ALV-J infection in DF1 and LMH cells. Therefore, these results suggested that the anti-ALV-J function of chIFN-λ was specifically implemented by induction of expression of type III ISGs. Our data identified chIFN-λ as a critical antiviral agent of ALV-J infection and provides a potentially and attractive platform for the production of commercial chIFN-λ.

Antiviral effect of lithium chloride on replication of avian leukosis virus subgroup J in cell culture.

Lithium chloride (LiCl) has been reported to possess antiviral activity against several viruses. In the current study, we assessed the antiviral activity effect of LiCl on ALV-J infection in CEF cells by using real-time PCR, Western blot analysis, IFA and p27 ELISA analysis. Our results showed that both viral RNA copy number and protein level decreased significantly in a dose and time dependent manner. Time-course analysis revealed that the antiviral effect was more pronounced when CEFs were treated at the post infection stage rather than at early absorption or pre-absorption stages. Further experiments demonstrated that LiCl did not affect virus attachment or entry, but rather affected early virus replication. We also found that inhibition of viral replication after LiCl treatment was associated with reduced mRNA levels of pro-inflammatory cytokines. These results demonstrate that LiCl effectively blocked ALV-J replication in CEF cells and may be used as an antiviral agent against ALV-J.

Gene expression profile and long non-coding RNA analysis, using RNA-Seq, in chicken embryonic fibroblast cells infected by avian leukosis virus J.

Avian leukosis virus J (ALVJ) infection induces hematopoietic malignancy in myeloid leukemia and hemangioma in chickens. However, little is known about the mechanisms underpinning the unique pathogenesis of ALVJ. In this study, we investigated the gene expression profiles of ALVJ-infected chicken cells and performed a comprehensive analysis of the long non-coding RNAs (lncRNAs) in CEF cells using RNA-Seq. As a result, 36 differentially expressed lncRNAs and 91 genes (FC > 2 and q-values < 0.05) were identified. Bioinformatics analysis revealed that these differentially expressed genes are involved in the innate immune response. Target prediction analysis revealed that these lncRNAs may act in cis or trans and affect the expression of genes which are involved in the anti-viral innate immune responses. Toll-like receptor, RIG-I receptor, NOD-like receptor and JAK-STAT signaling pathways were enriched. Notably, the induced expression of innate immunity genes, including B2M, DHX58, IFI27L2, IFIH1, IRF10, ISG12(2), MX, OAS*A, RSAD2, STAT1, TLR3, IL4I1, and IRF1 (FC > 2 and correlation > 0.95), were highly correlated with the upregulation of several lncRNAs, including MG066618, MG066617, MG066601, MG066629, MG066609 and MG066616. These findings identify the expression profile of lncRNAs in chicken CEF cells infected by ALVJ virus and provide new insights into the molecular mechanisms of ALVJ infection.

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

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

Role of gga-miR-221 and gga-miR-222 during Tumour Formation in Chickens Infected by Subgroup J Avian Leukosis Virus.

Subgroup J avian leukosis virus (ALV-J) causes a neoplastic disease in infected chickens. Differential expression patterns of microRNAs (miRNAs) are closely related to the formation and growth of tumors. (1) BACKGROUND: This study was undertaken to understand how miRNAs might be related to tumor growth during ALV-J infection. We chose to characterize the effects of miR-221 and miR-222 on cell proliferation, migration, and apoptosis based on previous microarray data. (2) METHODS: In vivo, the expression levels of miR-221 and miR-222 were significantly increased in the liver of ALV-J infected chickens (p < 0.01). Over-expression of gga-miR-221 and gga-miR-222 promoted the proliferation, migration, and growth of DF-1 cells, and decreased the expression of BCL-2 modifying factor (BMF) making cells more resistant to apoptosis. (3) RESULTS: Our results suggest that gga-miR-221 and gga-miR-222 may be tumour formation relevant gene in chicken that promote proliferation, migration, and growth of cancer cells, and inhibit apoptosis. BMF expression was significantly reduced in vivo 70 days after ALV-J infection. They may also play a pivotal role in tumorigenesis during ALV-J infection.

Detection of avian retroviruses in vaccines by amplification on DF-1 cells with immunostaining and fluorescent product-enhanced reverse transcriptase endpoint methods.

In order to ensure the safety of vaccines produced on avian cells, rigorous testing for the absence of avian retroviruses must be performed. Current methods used to detect avian retroviruses often exhibit a high invalid-test/false-positive rate, rely on hard-to-secure reagents, and/or have readouts that are difficult to standardize. Herein, we describe the development and validation of two consistent and sensitive methods for the detection of avian retroviruses in vaccines: viral amplification on DF-1 cells followed by immunostaining for the detection of avian leukosis virus (ALV) and viral amplification on DF-1 cells followed by fluorescent product-enhanced reverse transcriptase (F-PERT) for the detection of all avian retroviruses. Both assays share an infectivity stage on DF-1 cells followed by a different endpoint readout depending on the retrovirus to be detected. Validation studies demonstrated a limit of detection of one 50% cell culture infectious dose (CCID(50))/ml for retrovirus in a 30-ml test inoculum volume for both methods, which was as sensitive as a classical method used in the vaccine industry, namely, viral amplification on primary chicken embryo fibroblasts followed by the complement fixation test for avian leukosis virus (COFAL). Furthermore, viral amplification on DF-1 cells followed by either immunostaining or F-PERT demonstrated a sensitivity that exceeds the regulatory requirements for detection of ALV strains. A head-to-head comparison of the two endpoint methods showed that viral amplification on DF-1 cells followed by F-PERT is a suitable method to be used as a stand-alone test to ensure that vaccine preparations are free from infectious avian retroviruses.

Proteomics of DF-1 cells infected with avian leukosis virus subgroup J.

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.

Naturally occurring multiple perineuriomas in a chicken (Gallus domesticus).

A 2-year-old, male Japanese native fowl (Gallus gallus domesticus) was presented with an inability to feed and torticollis. At a necropsy, there were cylindrical enlargements and yellow discoloration of multiple peripheral nerves, including nerves of the lumbosacral plexus, brachial plexus, and spinal ganglia. On histologic examination, these lesions consisted of diffuse proliferations of spindle cells with characteristic onion bulb-like structures around residual axons. The spindle cells were immunohistochemically positive for glucose transporter 1 (GLUT1) and negative for S-100 alpha/beta proteins. On the basis of microscopic, histologic, and immunohistochemical findings, the tumors were diagnosed as multiple perineuriomas.

Low pH is required for avian sarcoma and leukosis virus Env-dependent viral penetration into the cytosol and not for viral uncoating.

A novel entry mechanism has been proposed for the avian sarcoma and leukosis virus (ASLV), whereby interaction with specific cell surface receptors activates or primes the viral envelope glycoprotein (Env), rendering it sensitive to subsequent low-pH-dependent fusion triggering in acidic intracellular organelles. However, ASLV fusion seems to proceed to a lipid mixing stage at neutral pH, leading to the suggestion that low pH might instead be required for a later stage of viral entry such as uncoating (L. J. Earp, S. E. Delos, R. C. Netter, P. Bates, and J. M. White. J. Virol. 77:3058-3066, 2003). To address this possibility, hybrid virus particles were generated with the core of human immunodeficiency virus type 1 (HIV-1), a known pH-independent virus, and with subgroups A or B ASLV Env proteins. Infection of cells by these pseudotyped virions was blocked by lysosomotropic agents, as judged by inhibition of HIV-1 DNA synthesis. Furthermore, by using HIV-1 cores that contain a Vpr-beta-lactamase fusion protein (Vpr-BlaM) to monitor viral penetration into the cytosol, we demonstrated that virions bearing ASLV Env, but not HIV-1 Env, enter the cytosol in a low-pH-dependent manner. This effect was independent of the presence of the cytoplasmic tail of ASLV Env. These studies provide strong support for the model, indicating that low pH is required for ASLV Env-dependent viral penetration into the cytosol and not for viral uncoating.

Tissue tropism and bursal transformation ability of subgroup J avian leukosis virus in White Leghorn chickens.

In Experiment 1, a monoclonal antibody against the envelope glycoprotein (gp85) of subgroup J avian leukosis virus (ALV-J) was used to study the distribution of ALV-J in various tissues of White Leghorn chickens inoculated as embryos with the strain ADOL-Hcl of ALV-J. At 2 and 6 wk of age, various tissues from infected and control uninfected chickens were tested for the presence of ALV-J gp85 by immunohistochemistry. In Experiment 2, using the methyl green-pyronine (MGP) stain, sections of bursa of Fabricius (BF) from chickens of line 15I5 x 7(1), inoculated with ALV-J or Rous-associated virus-1 (RAV-1), a subgroup A ALV, at hatch were examined for transformation of bursal follicles at 4 and 10 wk of age. In Experiment 1, specific staining indicative of the presence of ALV-J gp85 was noted at both 2 and 6 wk of age in the adrenal gland, bursa, gonads, heart, kidney, liver, bone marrow, nerve, pancreas, proventriculus, spleen, and thymus. In Experiment 2, by 10 wk of age, transformed bursal follicles were detected in MGP-stained sections of BF in only one of five (20%) chickens inoculated with ALV-J at hatch, compared with five of five (100%) chickens inoculated with RAV-1. The data demonstrate distribution of ALV-J gp85 in various tissues of White Leghorn chickens experimentally inoculated as embryos with the virus. The data also confirm our previous observation that ALV-J is capable of inducing transformation of bursal follicles, albeit the incidence is less frequent than that induced by subgroup A ALV.

Retroviral entry mediated by receptor priming and low pH triggering of an envelope glycoprotein.

Avian leukosis virus (ALV) has been used as a model system to understand the mechanism of pH-independent viral entry involving receptor-induced conformational changes in the viral envelope (Env) glycoprotein that lead to membrane fusion. Here, we report the unexpected finding that ALV entry depends on a critical low pH step that was overlooked when this virus was directly compared to the classical pH-dependent influenza A virus. In contrast to influenza A virus, receptor interaction plays an essential role in priming ALV Env for subsequent low pH triggering. Our results reveal a novel principle in viral entry, namely that receptor interaction can convert a pH-insensitive viral glycoprotein to a form that is responsive to low pH.

Isolation and some characteristics of a subgroup J-like avian leukosis virus associated with myeloid leukosis in meat-type chickens in the United States.

Several subgroup J-like avian leukosis viruses (ALV-Js) were isolated from broiler breeder (BB) and commercial broiler flocks experiencing myeloid leukosis (ML) at 4 wk of age or older. In all cases, diagnosis of ML was based on the presence of typical gross and microscopic lesions in affected tissues. The isolates were classified as ALV-J by 1) their ability to propagate in chicken embryo fibroblasts (CEF) that are resistant to avian leukosis virus (ALV) subgroups A and E (C/AE) and 2) positive reaction in a polymerase chain reaction with primers specific for ALV-J. The prototype strain of these isolates, an isolate termed ADOL-Hc1, was obtained from an adult BB flock that had a history of ML. The ADOL-Hc1 was isolated and propagated on C/AE CEF and was distinct antigenically from ALV of subgroups A, B, C, D, and E, as determined by virus neutralization tests. Antibody to ADOL-Hc1 neutralized strain HPRS-103, the prototype of ALV-J isolated from meat-type chickens in the United Kingdom, but antibody to HPRS-103 did not neutralize strain ADOL-Hc1. On the basis of both viremia and antibody, prevalence of ALV-J infection in affected flocks was as high as 87%. Viremia in day-old chicks of three different hatches from a BB flock naturally infected with ALV-J varied from 4% to 25%; in two of the three hatches, 100% of chicks that tested negative for virus at hatch had evidence of viremia by 8 wk of age. The data document the isolation of ALV-J from meat-type chickens experiencing ML as young as 4 wk of age. The data also suggest that strain ADOL-Hc1 is antigenically related, but not identical, to strain HPRS-103 and that contact transmission of ALV-J is efficient and can lead to tolerant infection.

Production of avian leukosis virus particles in mammalian cells can be mediated by the interaction of the human immunodeficiency virus protein Rev and the Rev-responsive element.

In human immunodeficiency virus type 1-infected cells, the efficient expression of viral proteins from unspliced and singly spliced RNAs is dependent on two factors: the presence in the cell of the viral protein Rev and the presence in the viral RNA of the Rev-responsive element (RRE). We show here that the HIV-1 Rev/RRE system can increase the expression of avian leukosis virus (ALV) structural proteins in mammalian cells (D-17 canine osteosarcoma) and promote the release of mature ALV virions from these cells. In this system, the Rev/RRE interaction appears to facilitate the export of full-length unspliced ALV RNA from the nucleus to the cytoplasm, allowing increased production of the ALV structural proteins. Gag protein is produced in the cytoplasm of the ALV-transfected cells even in the absence of a Rev/RRE interaction. However, a functional Rev/RRE interaction increases the amount of Gag present intracellularly and, more strikingly, results in the release of mature ALV particles into the supernatant. RCAS virus containing an RRE is replication-competent in chicken embryo fibroblasts; however, we have been unable to determine whether the particles produced in D-17 cells are as infectious as the particles produced in chicken embryo fibroblasts.

A system for tissue-specific gene targeting: transgenic mice susceptible to subgroup A avian leukosis virus-based retroviral vectors.

Avian leukosis viruses (ALVs) have been used extensively as genetic vectors in avian systems, but their utility in mammals or mammalian cell lines is compromised by inefficient viral entry. We have overcome this limitation by generating transgenic mice that express the receptor for the subgroup A ALV under the control of the chicken alpha sk-actin promoter. The skeletal muscles of these transgenic animals are susceptible to efficient infection by subgroup A ALV. Because infection is restricted to cell lineages that express the transgene, the method has utility for studies of development and oncogenesis and will provide models for tissue-specific gene therapy.

Retroviral capture of c-erbB proto-oncogene sequences: rapid evolution of distinct viral genomes carrying mutant v-erbB genes with different transforming capacities.

The evolution of oncogene-transducing retroviruses was followed by studying the genomes of five new, erbB carrying retroviruses. These viruses, isolated from cells of one chicken infected with Rous Associated virus 1 (RAV-1), had captured c-erbB sequences as a consequence of RAV-1 integration into the host genome. Their genome structures were distinct; however, their v-erbB genes had sustained identical 5' and 3' deletions and the v-erbB-env junctions were identical at the nucleotide level. The results therefore strongly suggest that all five viruses originate from the same capture event. Sequence analyses of the v-erbB genes from three of these viruses revealed that one of them had undergone no further mutation and lacked detectable capacity to transform cells, therefore probably representing an 'early' form of transducing virus. The two other v-erbB genes contained distinct mutations and differed in their potential to induce fibroblast- and erythroblast transformation; they therefore probably represent later derivatives of the virus that captured the erbB oncogene. The data suggest that the initial retrovirus rapidly underwent many alterations after capture of c-erbB sequences, already in the RAV-1 infected bird as well as during subsequent in vitro isolation procedures. The changes involve both major rearrangements of the genome as well as point mutations that activated the erbB oncogene.

Use of helper cells with two host ranges to generate high-titer retroviral vectors.

We have recently described Avian Leukosis Virus (ALV)-based packaging cell lines that can produce helper-free ALV-based retrovirus vectors with A, B, C, and E envelope host ranges. Here, we report that lacZ retroviral vectors of subgroup C or E can infect helper cells of subgroup A (Isolde) which are then able to produce high titers of lacZ recombinant viruses of subgroup A. Superinfection of helper cells by lacZ recombinant virus was performed by cocultivating packaging cells with two subgroup specificities (A and E), but this did not result in increased recombinant virus titers. This "ping-pong" process caused the emergence of replication-competent (RC) viruses which are shown to result from recombination between the viral sequences of the helper cell lines and the cis-acting sequences of the lacZ recombinant virus.

Avian retroviral RNA encapsidation: reexamination of functional 5' RNA sequences and the role of nucleocapsid Cys-His motifs.

RNA packaging signals (psi) from the 5' ends of murine and avian retroviral genomes have previously been shown to direct encapsidation of heterologous mRNA into the retroviral virion. The avian 5' packaging region has now been further characterized, and we have defined a 270-nucleotide sequence, A psi, which is sufficient to direct packaging of heterologous RNA. Identification of the A psi sequence suggests that several retroviral cis-acting sequences contained in psi+ (the primer binding site, the putative dimer linkage sequence, and the splice donor site) are dispensable for specific RNA encapsidation. Subgenomic env mRNA is not efficiently encapsidated into particles, even though the A psi sequence is present in this RNA. In contrast, spliced heterologous psi-containing RNA is packaged into virions as efficiently as unspliced species; thus splicing per se is not responsible for the failure of env mRNA to be encapsidated. We also found that an avian retroviral mutant deleted for both nucleocapsid Cys-His boxes retains the capacity to encapsidate RNA containing psi sequences, although this RNA is unstable and is thus difficult to detect in mature particles. Electron microscopy reveals that virions produced by this mutant lack a condensed core, which may allow the RNA to be accessible to nucleases.

Packaging cells for avian leukosis virus-based vectors with various host ranges.

Using our previously described Haydée semipackaging cell line (F. L. Cosset, C. Legras, Y. Chebloune, P. Savatier, P. Thoraval, J. L. Thomas, J. Samarut, V. M. Nigon, and G. Verdier, J. Virol. 64:1070-1078, 1990) which produces avian leukosis virus gag and pol proteins, we have constructed packaging cells with subgroups B, C, and E envelope specificities. This allows us to produce helper-free avian leukosis virus particles carrying the lacZ reporter gene and the A, B, C, or E subgroup specificities. Titers of the recombinant lacZ virus are shown to be dependent upon the type of the env subgroup and the target avian cell.

Type A oncornavirus-like particles in turkey bone marrow cells transformed by avian leukemia virus.

Type A oncornavirus-like particles (TAOLP) were found in the cytoplasm of turkey bone marrow cells transformed by strain Mc-31 avian leukemia virus. They resembled morphologically the cores of budding virions and the cores of immature extracellular Mc-31 virus particles. TAOLP were observed more often in T3Mc31 cells where cytopathological changes existed. TAOLP were not found in the materials from in vivo experiments. A relationship is likely to exist between TAOLP and Mc-31 virus and TAOLP could represent a side product of Mc-31 virus replication.