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.

Changes in apoptosis, proliferation and T lymphocyte subtype on thymic cells of SPF chickens infected with reticuloendotheliosis virus.

Reticuloendotheliosis virus (REV), an avian retrovirus is able to infect a variety of birds and can cause immunosuppression. The aim of this study was to investigate the relationship of thymic lymphocytes apoptosis, proliferation and T cell subtype with immunosuppression. In this study, a hundred and twenty one-day old SPF chickens were randomly divided into control groups (group C) and a REV infection groups (group I). The chickens of group I received intraperitoneal injections of REV with 104.62/0.1 ml TCID50. On day 14, 21, 28 and 35 post-inoculation, the chickens of C group and I group were sacrificed by cardiac puncture blood collection, and the thymic lymphocytes was sterile collected. The proliferation ability of lymphocytes was tested by Cell Counting Kit-8. Flow cytometry was performed to detect apoptosis, cell cycle stage and the change in T cell subtype. The RNA genome copy numbers of REV virus were detected using real-time PCR. Real-time PCR and western blotting were performed to analyze the expression of CyclinD1 and Bcl-2. Our results showed that REV genome copy number steadily declined, the proliferation potential of thymic lymphocytes was inhibited, lymphocytes apoptosed, the ratio of CD4+/CD8+ decreased and the expression of CyclinD1 and Bcl-2 were firstly inhibited, then rapidly recovered. Thus, immunosuppression lead by REV is closely related to the change of T cell subtype, apoptosis, and proliferation of thymic lymphocytes.

Assessment on reticuloendotheliosis virus infection in specific-pathogen-free chickens based on detection of yolk antibody.

Reticuloendotheliosis virus (REV) is the most frequent exogenous virus that contaminates attenuated vaccines. Therefore, it is extremely important to select REV-free specific-pathogen-free (SPF) chicken embryos. Generally, REV infection is assessed by detecting REV antibodies in SPF chickens. This present study seeks to evaluate REV infection by replacing serum antibody detection with yolk antibody detection. A cohort of 40 nineteen-week-old SPF chickens were artificially inoculated with REV, with 32 SPF chickens raised in another isolation environment served as a blank control. Eggs and serum from 23-week-old chickens were sampled, and yolks were diluted separately to ratios of 1:150, 1:200, 1:300 and 1:400, which were detected together with serum. We found that the yolk antibody detection findings at a dilution of 1:300 had the highest coincidence rate compared with that based on serum antibody measurements. At a dilution ratio of 1:300 for yolk antibody, 72 chickens were continuously observed for 10 weeks from 25- to 34-weeks-old. Our findings were based on serum antibody or yolk antibody detection, and the evaluation results were completely consistent. Therefore, all serum antibody-positive chickens were yolk antibody-positive, and vice versa. Accordingly, vaccine producers can estimate REV cleanliness in a poultry farm by sampling yolk antibody titers.

Protection induced by a gp90 protein-based vaccine derived from a Reticuloendotheliosis virus strain isolated from a contaminated IBD vaccine.

BACKGROUND: Reticuloendotheliosis is an immunosuppressive disease caused by avian reticuloendotheliosis virus (REV). It is commonly found in poultry farms and has caused a notable economic loss worldwide. Despite this, there is currently no effective vaccine available to protect against REV infection. METHOD: In this study, gp90 protein derived from an REV isolated from a contaminated vaccine was co-administered with cytosine-phosphate-guanine oligodeoxynucleotide (CpG-ODN) adjuvant to hens to determine if it protects their chicks against REV infection. To synthesize the gp90 protein, the gp90 gene was amplified using polymerase chain reaction, expressed in Escherichia coli, and purified. The resulting recombinant protein was injected intramuscularly into breeder hens along with CpG-ODN adjuvant and then serum antibody levels were regularly evaluated. After the fertilized eggs from these vaccinated hens had hatched, the resulting chicks were challenged with a 102.7 50% tissue culture infectious dose (TCID50) of REV at 1 day old and the REV antibody levels in these hatched chickens were evaluated before and after the challenge. Viremia and growth rate were measured weekly and statistically analyzed. RESULTS: The results suggest that the gp90 recombinant protein was successfully prepared and, when used with CpG-ODN adjuvant to immunize breeder hens, induced serological antibody production against REV in both hens and their hatched chicks. In addition, the maternal antibodies induced by the gp90 protein vaccine effectively protected majority of the chicks from REV infection. CONCLUSIONS: Overall, we found the gp90 protein obtained in this study may be a potential vaccine candidate that had good immunogenicity and could be an auxiliary measure to accelerate the eradication of REV.

The experimental transmission of reticuloendotheliosis virus by cock semen.

Following artificial insemination, the egg-laying rate of a large-scale breeder chicken flock declined by10-15 %. Real-time quantitative polymerase chain reaction (qPCR) analysis detected the presence of reticuloendotheliosis virus (REV) in semen from the breeder cocks used. Six REV strains were successfully isolated from semen randomly extracted from those cocks. Additionally, the whole sequence of SDAUR-S1 was sequenced and analysed. Cock models with continuous production of REV-positive semen were established by intravenous injection with SDAUR-S1. Eggs were then collected from hens after artificial insemination with REV-positive semen, for virus detection. The positive REV antibody rate for egg albumen was 58.3 % and the REV-positive rate for hatched embryos was 8.3 %, which suggested not only that REV can infect cock semen, but can also infect the offspring. In conclusion, the present study is the first to report on the isolation, genome analysis and transmission of REV in cock semen.

Identification of two conserved B-cell epitopes in the gp90 of reticuloendothelial virus using peptide microarray.

Since the gp90 protein of Reticuloendotheliosis virus (REV) plays vital roles in virus neutralization, so detailed analysis of REV-gp90 epitopes is important for the development of epitope based marker vaccines and diagnostic tools for REV infections. In this study, two monoclonal antibodies (mAbs) namely 6C12 and 09980 were used to map the epitopes in REVgp90 using peptide microarray and ELISA. Peptide microarray revealed that mAbs 6C12 and 09980 recognized 216YHPLA220 and 230DPQTSDILEA239 motifs, respectively. This result was confirmed by ELISA using synthetic peptides. Moreover, homology analysis indicated that mAbs defined epitopes are highly conserved among REV strains used in this study. The mAbs and their epitopes identified in this study may have potential applications in development of diagnostic techniques and epitope-based marker vaccines for control of REV infections.

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.

Development of a real-time quantitative RT-PCR to detect REV contamination in live vaccine.

Based on the published Avian reticuloendotheliosis virus (REV) whole genome sequence, primers and TaqMan probes were designed and synthesized, and the TaqMan probe fluorescence real-time quantitative RT-PCR (qRT-PCR) method for detecting the REV pol gene was established by optimizing the reaction conditions. Sensitivity analysis showed that the qRT-PCR method had a sensitivity that was 1,000-fold higher than conventional PCR. Additionally, no amplification signals were obtained when we attempted to detect DNA or cDNA of ALV-A/B/J, MDV, CIAV, IBDV, ARV, NDV, AIV, or other viruses, suggesting a high specificity for our method. Various titers of REV were artificially "spiked" into the FPV and MDV vaccines to simulate REV contamination in attenuated vaccines to validate this qRT-PCR method. Our findings indicated that this qRT-PCR method could detect REV contamination at a dose of 1 TCID50/1,000 feathers, which was 10,000-fold more sensitive than the regular RT-PCR detection (10(4) TCID50/1000 feathers).

[Development of monoclonal antibodies against the gp90 protein of reticuloendotheliosis virus and mapping of their recognition regions].

In order to develop monoclonal antibodies (McAbs) against the gp90 protein of reticuloendotheliosis virus (REV), the His-tagged gp90 protein of REV was used to immunize BALB/c mice. Hybridomas were generated by fusing mouse myeloma cells SP2/0 with the splenocytes from the immunized mice. After screening and 3 rounds of cloning process, 3 hybridomas (3G5-B8, 3G5-A10 and 1G12) that stably secreted McAbs against the REV-gp90 were obtained. The isotypes of the McAbs were determined to be IgG1, IgG1 and IgG2b. The McAbs specifically bound to gp90 in REV-infected DF-1 cells, as demonstrated by Western blotting and indirect immunofluorescence assay. The recognition regions on gp90 that were recognized by 3G5-B8/3G5-A10 and 1G12 were located between amino acids 200 to 245 and 230 to 235, respectively, as demonstrated by Western blotting analysis. These McAbs will be useful in the diagnosis and pathogenesis study of REV.

Transcriptional Profiling of Host Gene Expression in Chicken Embryo Fibroblasts Infected with Reticuloendotheliosis Virus Strain HA1101.

Reticuloendotheliosis virus (REV), a member of the Gammaretrovirus genus in the Retroviridae family, causes an immunosuppressive, oncogenic and runting-stunting syndrome in multiple avian hosts. To better understand the host interactions at the transcriptional level, microarray data analysis was performed in chicken embryo fibroblast cells at 1, 3, 5, and 7 days after infection with REV. This study identified 1,785 differentially expressed genes that were classified into several functional groups including signal transduction, immune response, biological adhesion and endocytosis. Significant differences were mainly observed in the expression of genes involved in the immune response, especially during the later post-infection time points. These results revealed that differentially expressed genes IL6, STAT1, MyD88, TLRs, NF-κB, IRF-7, and ISGs play important roles in the pathogenicity of REV infection. Our study is the first to use microarray analysis to investigate REV, and these findings provide insights into the underlying mechanisms of the host antiviral response and the molecular basis of viral pathogenesis.

Immunomodulatory effects of Taishan Pinus massoniana pollen polysaccharide and propolis on immunosuppressed chickens.

Co-infection of reticuloendotheliosis virus (REV) and avian leukosis virus subgroup J (ALV-J), which can cause suppressed immunity and vaccination failure, frequently occurs in chicken flocks in China. Taishan Pinus massoniana pollen polysaccharide (TPPPS) and propolis (PP) have been proven to possess immune modulatory effects and improve the immune effects of vaccines. This study aimed to investigate the immune modulatory ability of TPPPS and PP on chickens co-infected with immunosuppressive viruses. Prior to the study, chickens were artificially established as REV and ALV-J co-infection models. Four randomly assigned groups of these immunosuppressed chickens were successively administered with TPPPS, PP, mixture of TPPPS and PP (TPPPS-PP), or phosphate-buffered saline (PBS) for three days. At nine days old, the four immunosuppressed groups, as well as one normal group, were inoculated with the attenuated Newcastle disease (ND) vaccine. During the monitoring period, the indices of immune organ weight, lymphocyte transformation rates, CD4(+) and CD8(+) T-lymphocyte counts in peripheral blood, IL-2 and IFN-γ secretions, serum antibody titers of ND vaccine, and viral loads in spleens were determined. The results showed that chickens administered with TPPPS, PP, or TPPPS-PP could significantly enhance the levels of the above immune parameters compared to chickens in the PBS group. We observed the strongest immunity in the TPPPS-PP group, which indicates that the combination of TPPPS and PP versus TPPPS or PP alone, could generate better effects on improving the immune system effectiveness of immunosuppressed chickens.

[Immunogenicity of co-expressed p30-gp90 against reticuloendotheliosis virus].

OBJECTIVE: To study the immunogenicity of co-expression of p30, one of a group specific antigens, and glycoprotein gp90 genes of Reticuloendotheliosis virus (REV). METHODS: p30 and gp90 genes were amplified with the template of plasmid pPB101 containing the whole sequence of spleen necrosis virus (SNV), and cloned into pET-28a(+) vector. The positive clone pET-p30-gp90 was identified by enzyme analysis and sequencing. The co-expressed protein p30-gp90 was confirmed by SDS-PAGE and Western blot analysis. After quantitation, the purified protein p30-gp90 was immunized to Balb/c mice three times to prepare the p30-gp90 specific anti-serum. Afterwards, we detected the REV infected Chick Embryo Fibroblasts (CEF) by Immunofluorescence assay (IFA) with the prepared anti-serum. RESULTS: The p30-gp90 was co-expressed efficiently by SDS-PAGE and Western blot analysis. The anti-serum of p30-gp90 reacts with REV infected CEF in IFA. CONCLUSION: The expressed protein of p30-gp90 keeps good immunogenicity.

Protection of chickens against reticuloendotheliosis virus infection by DNA vaccination.

The present study was undertaken to evaluate the ability of DNA vaccination to protect chickens against reticuloendotheliosis virus (REV) infection and to determine whether codon optimization and the woodchuck hepatitis virus posttranscriptional regulatory element (WPRE) could improve the immunogenicity of the DNA vaccines. The wild-type and codon-optimized gp90 genes of REV were cloned into pCAGGS vector, and designated as pCAGgp90 and pCAGoptigp90, respectively. Plasmids pCAGWgp90 and pCAGWoptigp90 containing WPRE were also constructed. To evaluate vaccine efficacy, 3-week-old specific pathogen free chickens were injected with the constructed plasmids twice at 3-week intervals and challenged with REV 3 weeks post boost. Plasmids pCAGoptigp90 and pCAGWgp90 elicited significantly higher humoral and cellular immune responses than pCAGgp90, while chickens immunized with pCAGWoptigp90 showed the highest immune responses among the groups. Chickens immunized with pCAGgp90, pCAGoptigp90, pCAGWgp90 or pCAGWoptigp90 had 53%, 67%, 73% or 87% protection, respectively, as evidenced by the absence of REV viremia, while the empty vector pCAGGS only conferred 13% protection against viremia. These results highlight the potential value of DNA vaccination in the prevention of REV infection and suggest that codon optimization and WPRE could increase the efficacy of DNA vaccines.

Detection of anti-reticuloendotheliosis virus antibody by blocking enzyme-linked immunosorbent assay with expression envelope protein.

The current reticuloendotheliosis virus (REV) antibody detection kit that uses enzyme-linked immunosorbent assay (ELISA) needs concentrated virus, which is difficult to obtain due to its poor propagation in cells. In addition, this kit detects only chicken antibody but not other species. To overcome these disadvantages, we cloned and expressed REV env gene to develop monoclonal antibodies (mAbs), which we used for antibody detection in ELISA. Three mAbs were prepared from mice. These three mAbs could recognize REVs from ducks and geese by immunodot assay. In addition, the epitopes that the three mAbs recognized were determined by using three different env protein fragments by western blotting. One mAb was used to develop a blocking ELISA (bELISA) coated with expressed env protein to detect anti-REV antibody in chicken serum. This assay had a 98.8% (79/80) agreement with a commercial ELISA kit. Another 146 chicken sera with known neutralization antibodies were used as positive controls to evaluate this bELISA. The sensitivity and specificity this bELISA were 88.9% (40/45) and 94.8% (91/96), respectively. Thus, this bELISA could be used for anti-REV antibody detection in birds.

Enhancement of humoral and cellular immunity in chickens against reticuloendotheliosis virus by DNA prime-protein boost vaccination.

Reticuloendotheliosis virus (REV) causes an oncogenic, immunosuppressive and runting syndrome in multiple avian hosts worldwide. In this study, an optimal vaccination strategy was developed to enhance the immune responses against REV infection. Chickens were vaccinated twice intramuscularly with plasmid pCAGgp90 encoding gp90 protein of REV, or with recombinant gp90 protein, or vaccinated with plasmid pCAGgp90 and then boosted with recombinant gp90 protein. The humoral immune responses were monitored by ELISA and virus neutralizing test. In addition, lymphocyte proliferation response, cytokine production and protection effectiveness against REV infection were also evaluated. Although all vaccinated groups developed immune responses, chickens primed with pCAGgp90 plasmid and boosted with rgp90 protein developed higher levels of antibodies compared with those immunized with pCAGgp90 plasmid or rgp90 protein alone. Furthermore, enhanced cellular immune responses were induced following priming with the pCAGgp90 plasmid and boosting with the rgp90 protein. In addition, the DNA prime-protein boost vaccine yielded 100% protection of chickens from REV viremia caused by challenge infection. These findings demonstrated that a DNA prime-protein boost vaccination strategy could enhance both humoral and cellular immune responses in chickens, highlighting the potential value of such an approach in the prevention of REV infection.

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.

Identification of a conserved B-cell epitope on reticuloendotheliosis virus envelope protein by screening a phage-displayed random peptide library.

BACKGROUND: The gp90 protein of avian reticuloendotheliosis-associated virus (REV-A) is an important envelope glycoprotein, which is responsible for inducing protective antibody immune responses in animals. B-cell epitopes on the gp90 protein of REV have not been well studied and reported. METHODS AND RESULTS: This study describes the identification of a linear B-cell epitope on the gp90 protein by screening a phage-displayed 12-mer random peptide library with the neutralizing monoclonal antibody (mAb) A9E8 directed against the gp90. The mAb A9E8 recognized phages displaying peptides with the consensus motif SVQYHPL. Amino acid sequence of the motif exactly matched (213)SVQYHPL(219) of the gp90. Further identification of the displayed B cell epitope was conducted using a set of truncated peptides expressed as GST fusion proteins and the Western blot results indicated that (213)SVQYHPL(219) was the minimal determinant of the linear B cell epitope recognized by the mAb A9E8. Moreover, an eight amino acid peptide SVQYHPLA was proven to be the minimal unit of the epitope with the maximal binding activity to mAb A9E8. The REV-A-positive chicken serum reacted with the minimal linear epitopes in Western blot, revealing the importance of the eight amino acids of the epitope in antibody-epitope binding activity. Furthermore, we found that the epitope is a common motif shared among REV-A and other members of REV group. CONCLUSIONS AND SIGNIFICANCE: We identified (213)SVQYHPL(219) as a gp90-specific linear B-cell epitope recognized by the neutralizing mAb A9E8. The results in this study may have potential applications in development of diagnostic techniques and epitope-based marker vaccines against REV-A and other viruses of the REV group.

Probable congenital transmission of reticuloendotheliosis virus caused by vaccination with contaminated vaccines.

Contaminated vaccine is one unexpected and potential origin of virus infection. In order to investigate the most likely cause of disease in a broiler breeder company of Shandong Province, all 17 batches of live-virus vaccines used in the affected flocks and 478 tissue samples were tested by dot-blot hybridization, nested PCR, and IFA. The results suggested the outbreak of disease was most probably due to the vaccination of REV-contaminated MD-CVI988/Rispens vaccines and ND-LaSota+IB-H120 vaccines. Furthermore, the REV was probably transmitted to the commercial chickens through congenital transmission.

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.