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.

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 novel pH-responsive electrochemiluminescence immunosensor for ALV-J detection based on hollow MnO2 encapsulating Ru(bpy)3Cl2.

A novel pH-responsive sandwich-type electrochemiluminescence immunosensor was successfully developed for the detection of avian leukosis virus subgroup J (ALV-J). The immunosensor consisted of graphene oxide functionalized with tannic acid (rGO-TA) and primary antibodies (Ab1) as the main ALV-J sensing platform. For signal amplification, pH-responsive hollow MnO2 (hMnO2) nanospheres encapsulating Ru(bpy)3Cl2 and carrying secondary antibodies (Ab2) were employed. Under optimal conditions, the immunosensor exhibited a wide ALV-J detection range of 101.80 to 104.30 TCID50/mL (TCID50: 50% tissue culture infective dose) and a low detection limit of 101.71 TCID50/mL (S/N = 3). Importantly, the immunosensor showed high sensitivity, good reproducibility and excellent operational stability. Finally, the immunosensor was evaluated using a real avian serum sample containing ALV-J, with excellent results being achieved.

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

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

Effects of polysaccharide on chicks co-infected with Bordetella avium and Avian leukosis virus.

Chicks' co-infection with immunosuppressive virus and bacteria seriously threaten the development of the poultry industry. In this study, a model was established in which chicks were injected with either subgroup B ALV (ALV-B)+Bordetella avium (B. avium), or ALV-B+B. avium+Taishan Pinus massoniana pollen polysaccharide (TPPPS), or B. avium only, or B. avium+TPPPS. The data showed that the group injected with ALV-B and B. avium exhibited significant inhibition of the immune function and therefore increased pathogenicity compared with the group injected with B. avium-only. Application of TPPPS effectively alleviated immunosuppression, and body weights increased sharply in the TPPPS groups compared with non-TPPPS groups. To some extent, TPPPS may reduce the proliferation of ALV-B. These results suggest that Pinus pollen polysaccharides are beneficial treating co-infections with immunosuppressive virus and bacteria and therefore have potential for development into safe and effective immunoregulator.

Different quasispecies with great mutations hide in the same subgroup J field strain of avian leukosis virus.

Blood samples were collected from a local strain of chickens associated with serious tumor cases in Shandong Province. The samples were inoculated into chicken embryo fibroblast and DF-1 cells for virus isolation and identification, respectively. The inoculated cells were screened for three common chicken tumor viruses. Nine strains of avian leukosis virus subgroup J (ALV-J) were identified, and were designated LY1201-LY1209. The env gene from the LY1201 strain was amplified and cloned. All nine resultant env clones (clones 01-09) were sequenced, and the gp85 and gp37 amino acid regions were subjected to homology analysis. Clones 01 and 03 had 10 amino acid deletions in the gp85 region compared to the other seven clones, suggesting that at least two quasispecies with obvious mutations coexist in the same field strain. Among these nine clones, three had identical gp85 and gp37 sequences, and were recognized as the dominant LY1201 quasispecies. The amino acid sequence homology of gp37 and gp85 among the nine clones was 98.5%-100.0% and 96.6%-100.0% respectively, suggesting that the gp85 region of the env gene can better display the quasispecies diversity of ALV-J than gp37.

Development and validation of an indirect enzyme-linked immunosorbent assay for the detection of Avian leukosis virus antibodies based on a recombinant capsid protein.

Avian leukosis virus (ALV) is associated with tumor development and growth retardation in poultry. Eradication of virus infection at the primary breeder level is the principal method for controlling ALV infection in chickens. An indirect enzyme-linked immunosorbent assay (iELISA) method that utilized the prokaryotically expressed and affinity-purified viral capsid protein antigen p27 was developed for the detection of ALV-specific antibodies in chicken sera. The protocol of iELISA was validated and resulted in a higher agreement value than fluorescent antibody test (FAT) and was shown to be more sensitive and specific compared to the commercial ALV antibody test kit when FAT was used as a reference test. The main advantage of this method is the use of a single immunogenic protein to detect antibodies against all ALV exogenous subgroups. The results show that the developed iELISA is an inexpensive alternative and can potentially be used as a confirmatory test for the presence of anti-ALV antibodies on a large scale.

Subgroup J avian leukosis virus neutralizing antibody escape variants contribute to viral persistence in meat-type chickens.

We have previously demonstrated a high incidence of chickens with persistent viremia even in the presence of neutralizing antibodies (V+A+) against the inoculated parental virus in commercial meat-type chickens inoculated at hatch with subgroup J avian leukosis virus (ALV J) field isolates. In this study, we used an ALV J molecular clone, ADOL pR5-4, to determine the role of neutralizing antibody (NAb) escape mutants in maintaining a high incidence of viral persistence, namely, V+A+ infection profile in commercial meat-type chickens. Chickens were housed as a flock in a pen or housed in isolation in solitary Horsfall-Bauer units for testing for NAb escape variants. The emergence of NAb escape variants was evaluated by sequential autologous virus neutralization (VN) (between virus and antibody from the same sampling period) and heterologous VN (between virus and antibody from preceding and succeeding sampling periods). Sequential virus isolates and corresponding antisera from 18 chickens were examined by VN matrix. In all chickens, autologous virus isolates were not neutralized by corresponding antisera. However, some of these resilient autologous virus isolates were neutralized by antibodies from subsequent sampling intervals. Nucleotide sequence analysis of consecutive isolates from three individually housed chickens with V+A+ infection profile revealed distinct changes within the envelope region, suggesting viral evolution to escape the host immune response. These results demonstrate that the emergence of antibody escape variants in commercial meat-type chickens contributes to ALV J persistence.

Immune-related gene expression in response to H11N9 low pathogenic avian influenza virus infection in chicken and Pekin duck peripheral blood mononuclear cells.

The duck and chicken are important hosts of avian influenza virus (AIV) with distinctive responses to infection. Frequently, AIV infections in ducks are asymptomatic and long-lasting in contrast to the clinically apparent and transient infections observed in chickens. These differences may be due in part to the host response to AIV infection. Using real-time quantitative PCR, we examined the expression of immune-related genes in response to low pathogenic AIV H11N9 infection in peripheral blood mononuclear cells (PBMC) isolated from the blood of chickens and Pekin ducks. While chicken PBMC expressed IL-1beta and IL-6 at high levels similar to mammalian species, duck PBMC expression levels were minimal or unchanged. Similarly, duck IFN-beta expression was nearly unaffected, whereas chicken expression was highly upregulated. Chicken IFN-gamma was expressed to higher levels than duck IFN-gamma, while IFN-alpha was expressed similarly by both species. IL-2 was elevated early in infection in duck PBMC, but returned to baseline levels by the end of the experiment; in contrast, IL-2 was weakly induced in chicken PBMC at late time points. TLR-7 and MHC class I molecule expressions were conserved between species, whereas duck MHC class II expression was downregulated and chicken expression was unchanged. These results show distinct PBMC expression patterns of pro-inflammatory cytokines and IFNs between species. The differences in pro-inflammatory cytokine and IFN expression reflect the asymptomatic and lasting infection observed in ducks and the tendency towards clinical signs and rapid clearance seen in chickens. These results highlight important differences in the host response to AIV of two species thought to be critical in the genesis and maintenance of epidemic strains of AIV.