Semen-Derived Exosomes Mediate Immune Escape and Transmission of Reticuloendotheliosis Virus.

Reticuloendotheliosis virus (REV) causes immune-suppression disease in poultry, leading to a significant economic burden worldwide. Recent evidence demonstrated that the REV can enter the semen and then induce artificial insemination, but how the virus gets into semen was little known. Accumulating studies indicated that exosomes serve as vehicles for virus transmission, but the role of exosomes in viral shedding through the semen remains unclear. In this study, exosomes purified from the REV-positive semen were shown with reverse transcription-PCR and mass spectrometry to contain viral genomic RNA and viral proteins, which could also establish productive infections both in vivo and in vitro and escape from the REV-specific neutralizing antibodies. More importantly, compared with the infection caused by free virions, the exosome is more efficient for the virus to ensure effective infection and replication, which can also help the REV compromise the efficacy of the host immune response. In summary, this study demonstrated that semen-derived exosomes can medicate the transmission and immune escape of REV, implicating a novel mechanism for REV entering the semen and leading to vertical transmission.

Transcriptional analysis of host responses related to immunity in chicken spleen tissues infected with reticuloendotheliosis virus strain SNV.

In avian species, the Reticuloendotheliosis virus (REV) causes severe immunosuppression and other symptoms, including avian dwarfing syndrome, and chronic tumors in lymphoid and other tissues. The pathogenesis of REV and its interaction with the host have yet to be fully elucidated with transcriptional studies on the changes in host gene expression after REV infection at the body level. In this study, the Spleen Necrosis Virus (SNV) was used to inoculate the one-day-old specific pathogen free (SPF) chicken to simulate congenital infection. We identified 1507 differentially expressed genes (DEGs) at 7, 14 and 21 dpi using Next Generation Sequencing (NGS) technology. Through the Gene Ontology (GO) enrichment and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis of these DEGs, it was found that DEGs were mainly involved in the categories of signal transduction, immune system and signaling molecules and interaction. Among them, Pattern recognition receptors (PRRs), chemokine, T cell receptor, JAK-STAT, TNF, and NF-kappa B signaling pathway, and the Hematopoietic cell lineage play an important role in the tumorigenic and immunosuppressive regulation of REV. In addition, a series of DEGs associated with inflammatory factors (CCL4, TNFRSF18, CDKN2), apoptosis (IRF1, PDCD1, WNT5A), innate immunity (TLR, MAD5, TRIM25), and adaptive immunity (LY6E, CD36, LAG3) were also discovered. We further verified 33 selected immune- relevant DEGs using quantitative RT-PCR (qRT-PCR). These findings provide new insights and research directions for revealing the pathogenesis of REV infection and the interaction between REV and the chicken immune system.

Activation of gga-miR-155 by reticuloendotheliosis virus T strain and its contribution to transformation.

The v-rel oncoprotein encoded by reticuloendotheliosis virus T strain (Rev-T) is a member of the rel/NF-κB family of transcription factors capable of transformation of primary chicken spleen and bone marrow cells. Rapid transformation of avian haematopoietic cells by v-rel occurs through a process of deregulation of multiple protein-encoding genes through its direct effect on their promoters. More recently, upregulation of oncogenic miR-155 and its precursor pre-miR-155 was demonstrated in both Rev-T-infected chicken embryo fibroblast cultures and Rev-T-induced B-cell lymphomas. Through electrophoresis mobility shift assay and reporter analysis on the gga-miR-155 promoter, we showed that the v-rel-induced miR-155 overexpression occurred by the direct binding to one of the putative NF-κB binding sites. Using the v-rel-induced transformation model on chicken embryonic splenocyte cultures, we could demonstrate a dynamic increase in miR-155 levels during the transformation. Transcriptome profiles of lymphoid cells transformed by v-rel showed upregulation of miR-155 accompanied by downregulation of a number of putative miR-155 targets such as Pu.1 and CEBPß. We also showed that v-rel could rescue the suppression of miR-155 expression observed in Marek's disease virus (MDV)-transformed cell lines, where its functional viral homologue MDV-miR-M4 is overexpressed. Demonstration of gene expression changes affecting major molecular pathways, including organismal injury and cancer in avian macrophages transfected with synthetic mature miR-155, underlines its potential direct role in transformation. Our study suggests that v-rel-induced transformation involves a complex set of events mediated by the direct activation of NF-κB targets, together with inhibitory effects on microRNA targets.

Analysis of microRNA expression profile in specific pathogen-free chickens in response to reticuloendotheliosis virus infection.

Reticuloendotheliosis virus (REV) is an avian retrovirus that causes immunosuppression, growth retardation, and oncogenesis in a variety of birds. REV infection is epidemic in many countries. In this study, we used high-throughput sequencing to identify microRNAs (miRNAs) associated with REV infection. A total of 88 differentially expressed miRNAs were identified in samples collected on days 21 and 28 post-REV infection. Possible target genes of the differentially expressed miRNAs were analyzed. We observed that expression of proapoptotic, proto-oncogene, and carcinogenic cytokine mRNAs was highly upregulated, whereas expression of antiapoptotic cytokine mRNAs was significantly downregulated. Our findings provide a potential link between miRNA expression and the pathogenesis of REV infection.

Genome analysis and pathogenicity of reticuloendotheliosis virus isolated from a contaminated vaccine seed against infectious bursal disease virus: first report in China.

Specific-pathogen-free (SPF) chickens were inoculated with the virus seed of an infectious bursal disease virus (IBDV)-attenuated vaccine, and positive reticuloendotheliosis virus (REV) antibody levels were subsequently detected in the chicken sera, indicating potential REV contamination of the vaccine. After neutralization with IBDV-positive blood serum, the vaccine was inoculated into DF-1 cells for REV isolation and identification. An REV strain, designated IBD-C1605, was identified using an immunofluorescence assay test. Three pairs of primers were employed for the amplification, cloning and sequencing of three overlapping fragments of the IBD-C1605 genome, and the whole-genome sequence of this isolate was obtained after gene assembly. The genome was 8362 base pairs (nt) in length and its homology with the nucleotide sequences of different reference strains varied between 94.2 and 99.2 %. Isolate IBD-C1605 was inoculated into 1-day-old SPF chickens to observe its pathogenicity. Infection with this organism slowed down the weight gain of SPF chickens and caused atrophy of their immune organs, such as the bursa of Fabricius and thymus gland. Furthermore, the chicken antibody levels decreased significantly after Newcastle disease virus and avian influenza virus subtype H9 vaccine immunization. This is the first report on the isolation and identification of REV from attenuated vaccine virus seeds in China, and is also the first study on the pathogenicity of REV from a contaminated vaccine in China. Our findings contribute towards a better understanding of the detrimental effects of vaccine contamination with exogenous viruses such as REV.

The mongoose, the pheasant, the pox, and the retrovirus.

Paleovirology is the study of ancient viruses. The existence of a paleovirus can sometimes be detected by virtue of its accidental insertion into the germline of different animal species, which allows one to date when the virus actually existed. However, the ancient and the modern often connect, as modern viruses have unexpected origins that can be traced to ancient infections. The genomes of two species of mongooses and an egg-laying mammal called an echidna show that a virus currently present in poultry, the reticuloendotheliosis virus (REV), is actually of ancient exotic mammalian origin. REV apparently spread to poultry through a circuitous route involving the isolation of malaria parasites from a pheasant from Borneo housed at the Bronx Zoo that was contaminated with REV. Repeated passage of this virus in poultry adapted the virus to its new host. At some point, the virus got inserted into another virus, called fowlpox virus, which has spread back into the wild. Although REV may still exist somewhere in a mammalian host, its modern form links an 8 million-year-old infection of the ancestor of a mongoose to a virus that now is circulating in wild birds through malaria studies in the mid-20(th) century. These lessons of ancient and modern viruses have implications for modern human pandemics from viral reservoirs and for human interventions that may come with unintended consequences.

Sequence analysis of the whole genome of a recombinant Marek's disease virus strain, GX0101, with a reticuloendotheliosis virus LTR insert.

Marek's disease virus Chinese strain GX0101, isolated in 2001, is the first reported recombinant gallid herpesvirus type 2 (GaHV-2) field strain with one reticuloendotheliosis virus (REV) long terminal repeat (LTR) insert. We constructed an infectious bacterial artificial chromosome (BAC) clone of GX0101, which showed characteristics very similar to those of the parental virus in replication and pathogenicity. Using the GX0101 BAC clone, the complete genome of GX0101 was sequenced and analyzed. The length of the GX0101 genome is 178,101 bp, and it contains only one REV-LTR insert at a site 267 bp upstream of the sorf2 gene.

Pathogenicity of Bordetella avium under immunosuppression induced by Reticuloendotheliosis virus in specific-pathogen-free chickens.

Multiple infections of Bordetella avium (B. avium) with virus, especially immunosuppressive virus, have become more and more severe in chickens in China. The increasing morbidity and mortality of its complications have amplified concerns about the impact of B. avium on animal health. To evaluate the pathogenicity of B. avium under immunosuppression status, we developed four types of Reticuloendotheliosis virus (REV) infection models. After a comparison of body weight, relative immune organ index, Newcastle disease virus antibody titers and lymphocyte ratio, we chose the early age with low dose infection as our immunosuppressive model. To investigate the pathogenicity of B. avium under this model, a study was completed in which chickens were inoculated with REV-only, B. avium-only, both agents (REV -B. avium) or first inoculated with REV and 5 d later with B. avium (REV/B. avium). Results revealed that antibody titers to B. avium, concentrations of IFN-γ and SIgA were decreased in coinfected chickens when compared to the B. avium-only chickens, but the changing trend was similar between REV/B. avium and B. avium-only groups. Overall, REV did enhance the pathogenicity of B. avium. However, B. avium-only did not cause severe immune dysfunction unless chicks were coinfected with REV. REV preceding infection with B. avium showed mild impairment, which needs further exploration.

New pathogenetic characters of reticuloendotheliosis virus isolated from Chinese partridge in specific-pathogen-free chickens.

Avian reticuloendotheliosis virus (REV) infection can induce a runting syndrome, immunosuppression, acute reticulum cell neoplasia and lymphomas in a variety of domestic and wild birds. To evaluate the pathogenicity and oncogenicity of REV-JX0927 that isolated from Chinese partridge, experimental inoculated day-old specific-pathogen-free (SPF) White Leghorn chickens were examined at regular intervals. The examination procedures included hematology, serology and histopathology; also including immunohistochemistry and apoptosis assay. Body weight, relative immune organs weight and apoptosis assay results revealed that the immunosuppression of infected birds is associated with apoptosis of lymphocytes in lymphoid tissues, especially in thymus induced by REV-JX0927. Hematology and apoptosis assay results showed that the 7th week of post-infection is a critical time point for lymphocytes to be transformed into tumor cells. Histopathology evidences demonstrated that REV-JX0927 induced reticuloendotheliosis at early stage (1 week), and lymphosarcomas at middle stage (after 7 weeks). In addition, squamous-cell carcinoma, adenocarcinoma and aneurysm were found in infected birds. Arteritis was associated with concentration of serum protein and fat. REV antigen expression was observed in infected birds through the experimental period. REV has high tropism for proventriculus, kidney, liver, lymphoid tissues, pancreas, lymphosarcoma cells and blood vessels. Data from this study showed that several new pathogenitic characters caused by REV-JX0927 were observed. It indicated that REV-JX0927 is a multipotential oncogenic retrovirus.

Construction and characterization of the infectious clone of Reticuloendotheliosis virus carrying a genetic marker.

Reticuloendotheliosis virus (REV) causes an immunosuppressive and oncogenic disease, which posed substantial threats to the commercial poultry industry. However, researches on the molecular epidemiology and the pathogenic mechanism of REV are still scant. In this study, a full-length genome cDNA of Reticuloendotheliosis virus (REV) strain HLJR0901, which was isolated from layer in China, was cloned, sequenced, and analyzed. The genome of HLJR0901 was 8284 bp long, and the GenBank accession number is GQ415646. Sequence analysis showed that HLJR0901 was more similar to some REV strains that are common in the USA and Taiwan than to the early Chinese REV isolate. Next, the first efficient and genetically stable infectious clone with the genetic markers of the layer strain of REV was developed. The rescued and parental viruses had similar biological characteristics in vitro and in vivo. This study not only contributes to novel molecular epidemiology data, but also facilitates researching the gene functions and evolution of REV.

Reticuloendotheliosis virus strain T induces miR-155, which targets JARID2 and promotes cell survival.

The oncogenic microRNA miR-155 is upregulated by several oncogenic viruses. The precursor of miR-155, termed bic, was first observed to cooperate with myc in chicken B-cell lymphomas induced by avian leukosis proviral integrations. We identified another oncogenic retrovirus, reticuloendotheliosis virus strain T (REV-T), that upregulates miR-155 in chicken embryo fibroblasts. We also observed very high levels of miR-155 in REV-T-induced B-cell lymphomas. To study the role of miR-155 in these tumors, we identified JARID2/Jumonji, a cell cycle regulator and part of a histone methyltransferase complex, as a target of miR-155. The overexpression of miR-155 decreased levels of endogenous JARID2 mRNA. We confirmed that miR-155 directly targets both human and chicken JARID2 by assaying the repression of reporters containing the JARID2 3'-untranslated regions. Further, the overexpression of a sponge complementary to miR-155 in a tumor cell line increased endogenous JARID2 mRNA levels. The overexpression of JARID2 in chicken fibroblasts led to decreased cell numbers and an increase in apoptotic cells. The overexpression of miR-155 rescued cells undergoing cytopathic effect caused by infection with subgroup B avian retroviruses. Therefore, we propose that miR-155 has a prosurvival function that is mediated through the downregulation of targets including JARID2.

Avian tumor viruses: persistent and evolving pathogens.

Most neoplasias of lymphoid and other hematopoietic cells in commercial poultry are caused by viruses which belong to one of four distinct groups. Marek's disease virus (MDV) is an oncogenic herpesvirus. Avian leukosis virus (ALV), reticuloendotheliosis virus (REV) and lymphoproliferative disease virus (LPDV) are oncogenic retroviruses. Each group is distinguished by nucleic acid type, molecular structure, antigenicity, epidemiology, host range and other characteristics. However, most of these viruses have in common a unique ability to persist, both in the host and in the ecosystem. In addition, both the viruses and the virus-host relationships for several members of the group have demonstrated a propensity to evolve with time, creating new dilemmas for diagnosis and control. A focus on the persistence and evolution of avian tumor viruses will be used to address a number of current issues with individual viruses of economic importance. Issues of primary concern include (1) the evolution of MDV towards greater virulence with concomitant reduction of vaccine efficacy and expansion of host range, (2) the emergence of subgroup J ALV as a major pathogen in meat-type breeder stocks, and (3) the increasing prevalence of REV and its evolving role as a pathogen in chickens and turkeys.

Oncogenic retroviruses of cattle, chickens and turkeys: potential infectivity and oncogenicity for humans.

Reticuloendotheliosis virus naturally infects and cause lymphoid leukosis in chickens and turkeys. We investigated whether it can infect/transfect a variety of human cells in vitro. Successful infection and transfection were achieved with limited replication of virus. These findings, together with the knowledge that other oncogenic retroviruses of chickens and cattle, viz. The avian leukosis/sarcoma viruses and bovine leukemia virus show similar infectivity/oncogencity in vitro for humans, and the recent demonstration of antibodies to avian leukosis/sarcoma virus and reticuloendotheliosis virus in human sera, suggest that these viruses may have a role in the etiology of certain cancers in humans. There is now urgent need to investigate this, particularly because recent epidemiological studies consistently demonstrate excess of cancers in groups occupationally exposed to these viruses.

The v-Rel oncoprotein complexes with new Rel- and RelA-related proteins in transformed cells.

The v-Rel oncoprotein of the Rev-T retrovirus interacts with a number of cellular proteins in transformed chicken spleen cells including p40/I kappa B alpha, p68c-Rel, hsc70, and the p124 and p115 precursors for the p50 and p52 subunits of NF kappa B. Here we report that v-Rel associates with at least three other cellular proteins of 75-85 kDa in these cells, as well as with a protein related to human RelA. Western blot analysis of v-Rel immune complexes showed cross-reactivity between all of these factors and antibodies raised against Rel sequences, but none appeared to represent isoforms of c-Rel. Synchronization experiments revealed that the expression and/or association of these proteins with v-Rel varied throughout the cell cycle. Combined with the previously described interaction of v-Rel with known members of the Rel family, these studies strengthen the hypothesis that the interaction of v-Rel with multiple Rel-related proteins may be important for the transformation of lymphoid cells.

Structural genes, not the LTRs, are the primary determinants of reticuloendotheliosis virus A-induced runting and bursal atrophy.

Reticuloendotheliosis virus strain A (REV-A) and chicken syncytial virus (CSV), two replication competent avian retroviruses, differ in the extent to which they induce a runting syndrome that includes anemia, lymphoid organ atrophy, and reduced body size. We have isolated an infectious clone of CSV, the less pathogenic of the two viruses, and compared it to REV-A. Partial DNA sequence analysis suggests that it differs from REV-A by no more than 1 to 2% at the nucleotide level. Analysis of viral interference indicates that these two viruses use the same cell receptor for infection of both fibroblasts and hematopoietic cells, DNA sequence of the CSV and REV-A long terminal repeats (LTRs) reveals that these structures differ principally by two small insertions (5 and 19 bp) present in the U3 region of REV-A. The larger of these may encode enhancer sequences that have been reported to influence transcription rates in vitro. Measurement of steady-state levels of viral RNA in infected cells, however, as well as circulating virus in infected chicks indicates that the different pathogenic responses elicited by these two viruses are not due to large differences in viral transcription or replication. Chimeric viruses were constructed in which the LTRs from one virus were used to express the structural genes of the second virus. Infection of 1-day-old chicks by parental virus as well as the reciprocal chimeric constructs demonstrated that the ability to induce both runting and bursal atrophy segregated with the structural genes of REV-A. Infection of birds with additional chimeric viruses in which the env genes of REV-A and CSV were exchanged indicated that the pathogenic response resulting from REV-A infection was due to at least two regions of the viral genome encoding structural genes.

Poultry oncogenic retroviruses and humans.

Viruses of the avian leukosis/sarcoma group (ALSV) and reticuloendotheliosis viruses (REV) are highly prevalent in chickens and turkeys and naturally cause tumors in them. Commercial chickens are positive for antibodies, and a proportion actually carry infectious virus. Virus may be present in chicken products and in eggs, thus human exposure is virtually universal. The viruses show little potential for producing infectious viral particles in mammalian cells; nevertheless, they have the capacity to infect and transform mammalian cells (including human cells) in vitro, and to induce tumors in a variety of mammals, including primates. Most, but not all, of the serological studies in humans have been negative. Given the known behavior of these viruses in mammals, this was not unexpected. Moreover, there were methodological problems with most of the studies. There is some epidemiological evidence associating putative poultry exposure with cancer in humans. However, this has not been rigorously investigated. This paper is a comprehensive review of the extent of the carcinogenic potential these viruses show for humans. It is concluded, virological evidence indicates, that these viruses could conceivably have a carcinogenic potential for humans, but if so, at a level much less than in chickens. Whether this is insignificant, or translates to a real risk, is not known at the moment. Therefore, there is a need for definitive studies to completely rule out this possibility.

A nonimmunosuppressive helper virus allows high efficiency induction of B cell lymphomas by reticuloendotheliosis virus strain T.

We have documented the effect of two nondefective helper viruses, reticuloendotheliosis virus A (REV-A) and chick syncytial virus (CSV) infection on bursal tissue. REV-A infection results in bursal atrophy, destroying both its structural and functional integrity. In contrast, the bursae in CSV-infected chicks, while reduced slightly in size, appear both structurally and functionally normal. REV-A-induced bursal atrophy is not a result of viral replication in the B-lymphocyte as (a) both viruses are capable of inducing, with equal efficiency, the formation of preneoplastic lesions containing proliferating B lymphocytes and (b) it appears that equivalent amounts of viral antigen are expressed in the bursae of chicks infected with either virus. We have examined the phenotype of tumors induced by the replication-defective virus REV-T when replicated by the two different helper viruses, REV-A and CSV. In REV-T(REV-A)-infected chicks, the majority of tumors that develop are negative for IgM expression. In contrast, the majority of tumors induced by REV-T(CSV) infection are IgM+. This finding is confirmed by recovery of IgM- cell lines from REV-T(REV-A)-infected chicks and IgM+ cell lines from REV-T(CSV)-infected chicks. In addition, repopulation studies show that bursal-derived cells that are IgM+ serve as target cells for REV-T(CSV)-induced lymphomas. This study demonstrates, therefore, that REV-T can induce IgM+, B cell lymphomas with high efficiency. We conclude that infections by the helper viruses, REV-A and CSV, differ dramatically in their effects on the composition of the population of cells that serve as targets for REV-T-induced neoplasia.

Studies of reticuloendotheliosis virus-induced lymphomagenesis in chickens.

Seventy-three percent of chickens inoculated with the chick syncytial strain of reticuloendotheliosis virus (REV) at hatching developed lymphomas by 39 weeks of age. Neonatal treatment with cyclophosphamide or surgical bursectomy at 2, 4, 8, or 12 weeks of age significantly (P less than 0.01) reduced lymphoma development. In a further experiment, surgical bursectomy of REV-infected chickens followed by intravenous inoculation of the chickens with a single cell suspension of their own bursa cells at 2, 4, 9, or 13 weeks of age resulted in lymphoid tumors in chickens treated at 9 or 13 weeks but not in chickens treated at 2 or 4 weeks of age. Furthermore, this treatment did not shorten the incubation period for lymphoma development. These findings argue very strongly that transforming target cells are primarily in the bursa of Fabricius. The data also suggest that a minimum residency of 4 weeks in the bursa is required for infected bursa cells to become transformed. Therefore, lymphomagenesis induced by REV in chickens appears similar to that induced by the avian leukosis virus group.