dPRLR causes differences in immune responses between early and late feathering chickens after ALV-J infection.

To understand the differences in immune responses between early feathering (EF) and late feathering (LF) chickens after infection with avian leukosis virus, subgroup J (ALV-J), we monitored the levels of prolactin, growth hormone and the immunoglobulins IgG and IgM in the serum of LF and EF chickens for 8 weeks. Moreover, we analysed the expression of immune-related genes in the spleen and the expression of PRLR, SPEF2 and dPRLR in the immune organs and DF-1 cells by qRT-PCR. The results showed that ALV-J infection affected the expression of prolactin, growth hormone, IgG and IgM in the serum. Regardless of whether LF and EF chickens were infected with ALV-J, the serum levels of the two hormones and two immunoglobulins in EF chickens were higher than those in LF chickens (P < 0.05). However, the expression of immune-related genes in the spleen of positive LF chickens was higher than that in the spleen of positive EF chickens. In the four immune organs, PRLR and SPEF2 expression was also higher in LF chickens than in EF chickens. Furthermore, the dPRLR expression of positive LF chickens was higher than that of negative LF chickens. After infection with ALV-J, the expression of PRLR in DF-1 cells significantly increased. In addition, overexpression of PRLR or dPRLR in DF-1 cells promoted replication of ALV-J. These results suggested that the susceptibility of LF chickens to ALV-J might be induced by dPRLR.

The CCCH-Type Zinc Finger Antiviral Protein Relieves Immunosuppression of T Cells Induced by Avian Leukosis Virus Subgroup J via the NLP-PKC-δ-NFAT Pathway.

The CCCH-type zinc finger antiviral protein (ZAP) can recognize and induce the degradation of mRNAs and proteins of certain viruses, as well as exerting its antiviral activity by activating T cells. However, the mechanism of ZAP that mediates T cell activation during virus infection remains unclear. Here, we found a potential function of ZAP that relieves immunosuppression of T cell induced by avian leukosis virus subgroup J (ALV-J) via a novel signaling pathway that involves norbin-like protein (NLP), protein kinase C delta (PKC-δ), and nuclear factor of activated T cell (NFAT). Specifically, ZAP expression activated T cells by promoting the dephosphorylation and nuclear translocation of NFAT. Furthermore, knockdown of ZAP weakened the reactivity and antiviral response of T cells. Mechanistically, ZAP reduced PKC-δ activity by upregulating and reactivating NLP by competitively binding with viral protein. Knockdown of NLP decreased the dephosphorylation of PKC-δ by ZAP expression. Moreover, we show that knockdown of PKC-δ reduced the phosphorylation levels of NFAT and enhanced its nuclear translocation. Taken together, these data revealed that ZAP relieves immunosuppression caused by ALV-J and mediates T cell activation through the NLP-PKC-δ-NFAT pathway. IMPORTANCE The evolution of the host defense system is driven synchronously in the process of resisting virus invasion. Accordingly, host innate defense factors effectively work to suppress virus replication. However, it remains unclear whether the host innate defense factors are involved in antiviral immune responses against the invasion of immunosuppressive viruses. Here, we found that CCCH-type zinc finger antiviral protein (ZAP) effectively worked in resistance to immunosuppression caused by avian leukosis virus subgroup J (ALV-J), a classic immunosuppressive virus. Evidence showed that ZAP released the phosphatase activity of NLP inhibited by ALV-J and further activated NFAT by inactivating PKC-δ. This novel molecular mechanism, i.e., ZAP regulation of the antiviral immune response by mediating the NLP-PKC-δ-NFAT pathway, has greatly enriched the understanding of the functions of host innate defense factors and provided important scientific ideas and a theoretical basis for research on immunosuppressive viruses and antiviral immunity.

High-frequency and activation of CD4+CD25+ T cells maintain persistent immunotolerance induced by congenital ALV-J infection.

Congenital avian leukosis virus subgroup J (ALV-J) infection can induce persistent immunotolerance in chicken, however, the underlying mechanism remains unclear. Here, we demonstrate that congenital ALV-J infection induces the production of high-frequency and activated CD4+CD25+ Tregs that maintain persistent immunotolerance. A model of congenital infection by ALV-J was established in fertilized eggs, and hatched chicks showed persistent immunotolerance characterized by persistent viremia, immune organ dysplasia, severe imbalance of the ratio of CD4+/CD8+ T cells in blood and immune organs, and significant decrease in CD3+ T cells and Bu-1+ B cells in the spleen. Concurrently, the mRNA levels of IL-2, IL-10, and IFN-γ showed significant fluctuations in immune organs. Moreover, the frequency of CD4+CD25+ Tregs in blood and immune organs significantly increased, and the frequency of CD4+CD25+ Tregs was positively correlated with changes in ALV-J load in immune organs. Interestingly, CD4+CD25+ Tregs increased in the marginal zone of splenic nodules in ALV-J-infected chickens and dispersed to the germinal center. In addition, the proliferation and activation of B cells in splenic nodules was inhibited, and the number of IgM+ and IgG+ cells in the marginal zone significantly decreased. We further found that the mRNA levels of TGF- ß and CTLA-4 in CD4+CD25+ Tregs of ALV-J-infected chickens significantly increased. Together, high-frequency and activated CD4+CD25+ Tregs inhibited B cells functions by expressing the inhibitory cytokine TGF-ß and inhibitory surface receptor CTLA-4, thereby maintaining persistent immunotolerance in congenital ALV-J-infected chickens.

Prolactin affects the disappearance of ALV-J viremia in vivo and inhibits viral infection.

Based on the RNA-seq data of chicken spleen tissues infected with J subgroup avian leukosis virus (ALV-J), we found that prolactin (PRL) gene was one of differentially expressed gene. We measured ALV-J viremia and PRL levels in the plasma of two groups of ALV-J-infected adult chickens. Furthermore, recombinant chicken PRL (cPRL) was used to assess how cPRL affects ALV-J virus replication both in vivo and in vitro. The results showed that PRL levels in the plasma of adult chickens infected with ALV-J were lower than those of uninfected chickens, and that the difference was more significant in the avian leukemia pathological apparent changes. Notably, the fluctuations in PRL levels might influence the disappearance of ALV-J viremia in chickens. The in vitro results showed that preincubating DF-1 cells with cPRL before ALV-J infection elicited the best antiviral effects. Moreover, these effects were not dose-dependent. in vivo, injection of cPRL into ALV-J-infected chicks could reduce the levels of viremia at the 14 days post infection (dpi). Additionally, the expression of the interferon-stimulated genes oligoadenylate synthetase-like (OSAL) and vasoactive intestinal peptide (VIP) increased, and that of the proinflammatory cytokine-encoding TNTα, IL-1ß, and IL-6 genes decreased in the spleens of ALV-J-infected chicks injected with cPRL, leading to inhibition of viral replication at the 7 dpi. Collectively, our data demonstrated that PRL plays an important antiviral role in the immune response to ALV-J infection. This is the first report of the relationship between ALV-J infection and PRL. It is of great significance for the prevention and control of ALV-J.

Chicken interferon regulatory factor 7 (IRF7) can control ALV-J virus infection by triggering type I interferon production through affecting genes related with innate immune signaling pathway.

In order to breed new birds with strong disease resistance, it is necessary to first understand the mechanism of avian antiviral response. Interferon regulatory factor 7 (IRF7) is not only a member of type I interferons (IFNs) regulatory factor (IRFs) family, but also a major regulator of the IFN response in mammals. However, whether IRF7 is involved in the host innate immune response remains unclear in poultry, due to the absence of IRF3. Here, we first observed by HE stains that with the increase of the time of ALV-J challenge, the thymus was obviously loose and swollen, the arrangement of liver cell was disordered, and the bursa of fabricius formed vacuolated. Real-time PCR detection showed that the expression level of IRF7 gene and related immune genes in ALV-J group was significantly higher than that in control group (P < 0.05). To further study the role of chicken IRF7 during avian leukosis virus subgroup J (ALV-J) infection, we constructed an induced IRF7 overexpression and interfered chicken embryo fibroblasts (CEFs) cell and performed in vitro infection using low pathogenic ALV-J and virus analog poly(I:C). In ALV-J and poly(I:C) stimulated CEFs cells, the expression level of STAT1, IFN-α, IFN-ß, TLR3 and TLR7 were increased after IRF7 overexpressed, while the results were just the opposite after IRF7 interfered, which indicating that IRF7 may be associated with Toll-like receptor signaling pathway and JAK-STAT signaling pathway. These findings suggest that chicken IRF7 is an important regulator of IFN and is involved in chicken anti-ALV-J innate immunity.

Identification of a novel epitope specific for Gp85 protein of avian leukosis virus subgroup K.

During the past two decades, avian leukosis virus (ALV) caused tremendous economic losses to poultry industry in China. ALV-K as a newly found subgroup in recent years, which made the control and eradication of ALV more difficult as they were originated from the recombination of different subgroups. To date, specific rapid detection methods refer to ALV-K are still missing. Gp85 is the main structural protein of the virus, which mediates the invasion of host cells by the virus and determinates the classification of subgroups. In this study, we prepared a monoclonal antibody (Mab) named Km3 against Gp85 of ALV-K. Immunofluorescence assay showed that Km3 specifically recognized the strains of ALV-K rather than the strains of ALV-A or ALV-J. To explain the subgroups specificity of Km3, the epitope cognized by the Mab was identified by Western blotting using 15 overlapping fragments spanning the Gp85. Finally, the peptide 129AFGPRSIDTLSDWSRPQ145 was identified as the minimal linear epitope recognized by Km3. Alignment of Gp85 from different subgroups showed that the epitope was highly conserved among ALV-K strains, which was quite different from that of the strains from ALV -A, -B and -J. In conclusion, the Mab Km3 may serve as a useful reagent for ALV-K detection and diagnosis in the future.

Screening of immune biomarkers in different breeds of chickens infected with J subgroup of avian leukemia virus by proteomic.

Avian leucosis (AL) is a disease characterized by tumors and is caused by the avian leukosis virus (ALV). Because of the high variability of viruses and complex pathogenic mechanisms, screening and breeding J subgroup of ALV (ALV-J) resistant avian breeds is one of the strategies for prevention and treatment of AL, thus screening of significant immune markers is needed to promote the development of disease-resistant breeds. In this study, data-independent acquisition (DIA) technology was used to detect the DEPs of three breeds of chicken according to different comparison to investigate the potential markers. Results showed special DEPs for spleen development of each breed were detected, such as PCNT, DDB2, and ZNF62. These DEPs were involved in intestinal immune network used in production of IgA signaling pathways and related to immune response which can be used as potential markers for spleen development in different breeds. The DEPs such as RAB44 and TPN involved in viral myocarditis, transcriptional misregulation in cancer, and tuberculosis can be used as potential markers of spleen immune response after ALV-J infection in chickens. Pair-wise analysis was performed for the three breeds after the infection of ALV-J. The proteins such as RFX1, TAF10, and VH1 were differently expressed between three breeds. These DEPs involved in antigen processing and expression, acute myelogenous leukemia, and viral carcinogenesis can be used as potential immune markers after ALV-J infection of different genetic backgrounds. The screening of potential markers at protein level provides a strong theoretical research basis for disease resistance breeding in poultry.

Avian leukosis virus subgroup J induces B cell anergy mediated by Lyn inhibited BCR signal transduction.

Immune tolerance induced by avian leukosis virus subgroup J (ALV-J) is a prerequisite for tumorigenesis. Although we had reported that B cell anergy induced by ALV-J was the main reason for immune tolerance, the molecular mechanism still remains unclear. Here, we found SU protein of ALV-J interacted with tyrosine kinase Lyn (a key protein in BCR signaling pathway) by confocal laser scanning microscopy and co-immunoprecipitation test, which suggested that Lyn might play an important role in B cell anergy induced by ALV-J. Correspondingly, the mRNA and protein level of Lyn was significantly up-regulated in B cells after ALV-J infection. Subsequently, the phosphorylated protein levels of Lyn at Tyr507 site were significantly up-regulated in ALV-J-infected B cells after BCR signal activation, but the phosphorylated protein level of Syk (a direct substrate of Lyn) at Tyr525/526 site, Ca2+ flux, and NF-κB p65 protein level were significantly down-regulated. Interestingly, the phosphorylated protein level of Syk at Tyr525/526 site, Ca2+ flux, and NF-κB p65 protein level were both significantly retrieved after the shLyn treatment in B cells infected by ALV-J. In summary, these results indicated that ALV-J activated the negative regulatory effect of phosphorylated Lyn protein at 507 site in BCR signal transduction pathway and then mediated B cell anergy, which will provide a new insight for revealing the pathogenesis of immune tolerance induced by ALV-J.

Systematic Identification of Host Immune Key Factors Influencing Viral Infection in PBL of ALV-J Infected SPF Chicken.

Although research related to avian leukosis virus subgroup J (ALV-J) has lasted for more than a century, the systematic identification of host immune key factors against ALV-J infection has not been reported. In this study, we establish an infection model in which four-week-old SPF chickens are infected with ALV-J strain CHN06, after which the host immune response is detected. We found that the expression of two antiviral interferon-stimulated genes (ISGs) (Mx1 and IFIT5) were increased in ALV-J infected peripheral blood lymphocytes (PBL). A significant CD8+ T cell response induced by ALV-J appeared as early as seven days post-infection (DPI), and humoral immunity starting from 21 DPI differed greatly in the time scale of induction level. Meanwhile, the ALV-J viremia was significantly decreased before antibody production at 14 DPI, and eliminated at 21 DPI under a very low antibody level. The up-regulated CD8+ T cell in the thymus (14DPI) and PBL (7 DPI and 21 DPI) was detected, indicating that the thymus may provide the output of CD8+ T cell to PBL, which was related to virus clearance. Besides, up-regulated chemokine CXCLi1 at 7 DPI in PBL was observed, which may be related to the migration of the CD8+ T cell from the thymus to PBL. More importantly, the CD8 high+ T cell response of the CD8αß phenotype may produce granzyme K, NK lysin, or IFN-γ for clearing viruses. These findings provide novel insights and direction for developing effective ALV-J vaccines.

Development and evaluation of a gp85 protein-based subgroup-specific indirect enzyme-linked immunosorbent assay for the detection of anti-subgroup J avian leukosis virus antibodies.

Avian leukosis virus subgroup J (ALV-J) is an important pathogen for various neoplasms and causes significant economic losses in the poultry industry. Serological detection of specific antibodies against ALV-J infection is important for successful clinical diagnosis. Here, a 293F stable cell line was established to stably express gp85 protein. In this cell line, gp85 protein was expressed at approximately 30 mg/L. A subgroup-specific indirect enzyme-linked immunosorbent assay (iELISA) was developed using ALV-J gp85 protein as coated antigen to detect antibodies against ALV-J. The sensitivity of the iELISA (1:51200 diluted in serum) was 16 times more than that of indirect immunofluorescence assay (IFA; 1:3200 diluted in serum). Moreover, there was no crossreactivity with antibodies against other common avian viruses and other avian leukosis virus subgroups, such as subgroups A and B. The practicality of the iELISA was further evaluated by experimental infection and clinical samples. The results from experimental infection indicated that anti-ALV-J antibodies were readily detected by iELISA as early as 4 weeks after ALV-J infection, and positive antibodies were detected until 20 weeks, with an antibody-positive rate of 11.1% to 33.3%. Moreover, analysis of clinical samples showed that 9.49% of samples were positive for anti-ALV-J antibodies, and the concordance rate of iELISA and IFA was 99.24%. Overall, these results suggested that the subgroup-specific iELISA developed in this study had good sensitivity, specificity, and feasibility. This iELISA will be very useful for epidemiological surveillance, diagnosis, and eradication of ALV-J in poultry farms.

CCCH-type zinc finger antiviral protein mediates antiviral immune response by activating T cells.

The zinc finger antiviral protein (ZAP), as a host restriction factor, inhibits the replication of certain viruses by binding viral mRNA or proteins for degradation. However, little is known about the role of ZAP in the antiviral immune response. We now show that ZAP participates in the antiviral immune response by activating T cells. Overexpression of ZAP significantly inhibited avian leukosis virus subgroup J (ALV-J) replication and reduced the associated inflammatory damage in vivo. In this study, we found that ZAP tended to be expressed in T lymphocytes, especially after ALV-J infection. T lymphocyte proliferation proceeded as usual in response to ALV-J infection in the presence of ZAP, indicating that ZAP endows T lymphocytes with resistance to the immunosuppression caused by ALV-J. Furthermore, ZAP activated cytokine secretion by T lymphocytes by contributing to nuclear translocation of nuclear factors of activated T cells and indirectly promoted anti-ALV-J antibody generation. Together, our findings show that ZAP, acting as an immunomodulatory factor, is involved in the antiviral immune response via T lymphocyte activation.

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

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.

Preparation of a new monoclonal antibody against subgroup A of avian leukosis virus and identifying its antigenic epitope.

This study focuses on preparing the monoclonal antibody (MAb) against subgroup A of avian leukosis virus (ALV-A) and identifying its antigenic epitope. The ALV-A gp85 gene with a size of 1005bp was amplified and expressed into a recombinant protein with a size of 46KD in E.coli. The products expressed after purification were inoculated into BALB/c mice for preparing antibody-secreting splenic lymphocytes and further obtaining hybridoma cells. Finally, one new hybridoma cell (A18GH) secreting MAb against ALV-A was screened, and the MAb was able to detect ALV-A/K strains in an indirect immunofluorescence assay (IFA), but not ALV-B/J strains. A total of 14 overlapping truncated ALV-A gp85 protein segments were expressed and eight peptides containing different antigenic amino acids were artificially synthesized for analyzing the antigenic epitope of the MAb using a western blot or an ELISA, and the results indicate that the antigenic epitope consists of seven amino acids within the 146-ATRFLLR -152 region of the ALV-A gp85 protein. A biological information analysis shows that the antigenic epitope has a high antigenic index and develops a curved linear spatial structure. Further, its 7 amino acids are completely within the 17 representative ALV-A strains, 4 are within the 11 ALV-K strains, and fewer are within the ALV-B/J/E strains. This study will significantly assist in a further understanding of the protein structure and function of ALV-A, and in the establishment of specific ALV-A detection methods.

Oral Immunization of Chickens With Recombinant Lactobacillus plantarum Vaccine Against Early ALV-J Infection.

In this study, a novel oral vaccine of recombinant Lactobacillus plantarum (L. plantarum) containing the gp85 protein was explored, and the effects of this vaccine on the prevention of subgroup J Avian Leukosis Virus (ALV-J) infection were assessed. In the current study, the gp85 protein of ALV-J was expressed on the surface of L. plantarum with the surface-display motif, pgsA, by constructing a shuttle vector pMG36e:pgsA:gp85. Surface localization of the fusion protein was verified by western blotting and flow cytometry. Subsequently, Specific Pathogen Free Hy-Line Brown layer chickens were orally vaccinated with the recombinant L. plantarum and presented with high levels of serum immunoglobulin G (IgG) and secretory immunoglobulin A (sIgA) titers in bile and duodenal-mucosal fluid. After challenged with ALV-J of a 3 × 103 50% tissue culture infective dose (TCID50), serum samples of the chickens were collected and viremia was analyzed. Results showed that, compared to the L. plantarum and PBS control group, the recombinant L. plantarum group showed a significant rise in antibody levels after inoculation, and provide improved protection against ALV-J according to viremia detection. These results indicate that oral immunization with the recombinant L. plantarum provided an effective means for eliciting protective immune response against early ALV-J infection.

The antiviral property of Sargassum fusiforme polysaccharide for avian leukosis virus subgroup J in vitro and in vivo.

Avian Leukosis Virus Subgroup J (ALV-J) is an oncogenic retrovirus, mainly spread by vertical and horizontal transmission, which have caused severe losses in world poultry industry. Sargassum fusiforme polysaccharide (SFP), a marine algae sulfated polysaccharide, has attracted more attention due to its variously biological activities. In this study, the anti-ALV-J property of SFP was assessed in vivo and in vitro. The results demonstrated that different Mw of SFPs showed virustatic activity to ALV-J in vitro by combining with the virus when ALV-J adsorbed onto the host cells. When treated with SFPs, the ALV-J gene and protein expression reduced clearly and SFP-3 (Molecular weight 9 kDa) had the best antiviral effect. Results in vivo showed that the immunosuppression of the ALV-J infected chickens were relieved by SFP-3. Moreover, SFP-3 obviously inhibit the viral shedding and alleviated the organs damage caused by ALV-J. This study offered a new method for ALV-J treatment and enriched the potential application of SFP.

Novel carbon quantum dots can serve as an excellent adjuvant for the gp85 protein vaccine against avian leukosis virus subgroup J in chickens.

To evaluate whether carbon quantum dots (CQDs) can serve as an excellent adjuvant for the gp85 protein vaccine, this study produced recombinant gp85 protein against the avian leukosis virus subgroup J (ALV-J) in chickens. Functionalized CQDs were prepared and then linked to the recombinant gp85 protein. A total of 36 chickens were divided into 3 groups, namely, 2 experimental groups and 1 control group. Chickens from the experimental groups were inoculated twice intramuscularly with purified recombinant gp85 protein with CQDs as adjuvant or Freund's adjuvant emulsion at 14 and 21 D, whereas those from the control group were inoculated with an equivalent volume of PBS. At 35 D, the chickens were challenged with a 102.4 50% tissue culture infective dose of ALV-J. Blood samples were collected from each chicken at weekly intervals for serum antibody and viremia analyses. Results indicated that immunization with gp85-CQDs or gp85-Freund's adjuvant induced the inoculated chickens to produce positive serum antibodies (sample-to-positive ratio >0.6) at the 3rd week and persisted over 9 wk. Antibody levels in the gp85-CQDs group were higher than those in the gp85-Freund's adjuvant group. Differences were significant at 21 D (P < 0.05) and extremely significant from 28 D to 70 D (P < 0.01). Additionally, results of viremia showed higher protection in the gp85-CQDs group than in the Freund's adjuvant group. These findings highlighted the potential of CQDs as excellent candidate nanovehicles for vaccine delivery.

Long non-coding RNA and MicroRNA profiling provides comprehensive insight into non-coding RNA involved host immune responses in ALV-J-infected chicken primary macrophage.

Avian leukosis virus subgroup J (ALV-J) infection can cause tumors and immunosuppression in infected chickens. Macrophages play a crucial role in host defense against invading pathogens. In the present study, whole transcriptome analysis was performed to analyze the host factors including genes, microRNA (miRNA), long non-coding RNA (lncRNA) and their regulatory network in chicken primary monocyte-derived macrophages (MDMs). In total, 128 differentially expressed (DE) lncRNAs and 15 DE miRNAs were identified in MDMs at 3 h post infection (hpi), and 30 DE lncRNAs and 8 DE miRNAs were identified in MDMs at 36 hpi during ALV-J infection. We further constructed the DE lncRNAs-mRNAs, miRNA-mRNAs and lncRNAs-miRNA-mRNAs interaction networks. The results suggested that DE lncRNAs and miRNAs are involved in the regulation of CCND3 and SOCS5 in Jak-STAT signaling pathway via ceRNA network in ALV-J-infected MDMs at 3 hpi. In addition, lncRNAs including XLOC_672329, ALDBGALG0000001429, XLOC_016500 and ALDBGALG0000000253 cis-regulating CH25H, CISH, IL-1ß and CD80 respectively in MDMs at 3 hpi participated in host antiviral responses. Our findings give a comprehensive view of the connection between non-coding RNA and ALV-J in chicken primary macrophages, and provide an excellent resource for further studies of epigenetic effects on ALV-J disease resistance breeding as well as immune system and genomic researches.

A balanced game: chicken macrophage response to ALV-J infection.

Avian leukosis virus subgroup J (ALV-J) infection can cause tumors and immunosuppression in infected chickens. Macrophages play a central role in host defense against invading pathogens. In this study, we discovered an interesting phenomenon: ALV-J replication is weakened from 3 hours post-infection (hpi) to 36 hpi, which was verified using Western blotting and RT-PCR. To further investigate the interaction between ALV-J and macrophages, transcriptome analysis was performed to analyze the host genes' function in chicken primary monocyte-derived macrophages (MDM). Compared to the uninfected control, 624 up-regulated differentially expressed genes (DEG) and 341 down-regulated DEG at 3 hpi, and 174 up-regulated DEG and 87 down-regulated DEG at 36 hpi were identified in chicken MDM, respectively. ALV-J infection induced strong innate immune responses in chicken MDM at 3 hpi, instead of 36 hpi, according to the analysis results of Gene Ontology and KEGG pathway. Importantly, the host factors, such as up-regulated MIP-3α, IL-1ß, iNOS, K60, IRG1, CH25H, NFKBIZ, lysozyme and OASL were involved in the host defense response during the course of ALV-J infection. On the contrary, up-regulated EX-FABP, IL4I1, COX-2, NFKBIA, TNFAIP3 and the Jak STAT pathway inhibitors including CISH, SOCS1 and SOCS3 are beneficial to ALV-J survival in chicken macrophages. We speculated that ALV-J tropism for macrophages helps to establish a latent infection in chicken MDM from 6 to 36 hpi. The present study provides a comprehensive view of the interactions between macrophages and ALV-J. It suggests the mechanisms of defense of chicken macrophages against ALV-J invasion and how ALV-J escape the host innate immune responses.

Comparison of Viremia, Cloacal Virus Shedding, Antibody Responses and Pathological Lesions in Adult Chickens, Quails, and Pigeons Infected with ALV-A.

Subgroup A of the avian leukosis virus (ALV-A) can cause severe pathological lesions and death in infected chickens, and its reported hosts have increased recently. To assess the susceptibility of adult chickens, quails, and pigeons to ALV-A, three sets of 250-day-old birds were intraperitoneally inoculated with ALV-A. Viremia and cloacal virus shedding were dynamically detected using an immunofluorescence assay (IFA), ALV-P27 antigen ELISA or RT-PCR; pathological lesions were assessed using tissue sections; ALV-A in tissues was detected by IFA; and ALV-A antibody responses were detected using antibody ELISA kits and an immune diffusion test. The results indicated that persistent viremia occurred in 80% (8/10) of infected chickens, and transient viremia occurred in 17% (2/12) of infected quails, but no viremia occurred in infected pigeons. Cloacal virus shedding occurred intermittently in 80% (8/10) of infected chickens and in 8% (1/12) of infected quails but did not occur in infected pigeons. Severe inflammatory pathological lesions occurred in the visceral tissues of most infected chickens, and mild lesions occurred in a few of the infected quails, but no pathological lesions occurred in the infected pigeons. The ALV-A virus was detected in the visceral tissues of most infected chickens but not in the infected quails and pigeons. Obviously different ALV-A antibody responses occurred in the infected chickens, quails and pigeons. It can be concluded that adult chickens, quails and pigeons have dramatically different susceptibilities to ALV-A. This is the first report on artificial infection by ALV-A in different birds.