BackgroundOutbreaks of Marek’s disease (MD), caused by Marek’s disease virus (MDV), primarily occur in 10–12-week-old hens.Case presentation We report a case of MD in a breeding flock of 24–30-week-old vaccinated broilers in China. The clinical signs in the affected chickens appeared at 24 weeks, and the incidence of tumours peaked at 30 weeks. The morbidity and mortality of the hens were 5 % and 80 %, respectively. Hematoxylin–eosin staining of the tissues showed the typical characteristics of MD. MDV infection was confirmed in the hens with an agar gel diffusion precipitation assay for the MD antigen in the feather follicle epithelium. An MDV strain, designated AH1410, was isolated from the blood lymphocytes. Sequence analyses of the pp38, meq, and gB genes revealed that strain AH1410 had molecular features consistent with a virulent, previously identified MDV.ConclusionOur data provide evidence that not only is MDV becoming more virulent, but that the period of its onset in chickens is expanding. These findings provide the basis the molecular surveillance and further study of virulent MDV mutants and control strategies for MD in China.

MicroRNA-155 (miR-155) has been as an important controller of TLR3 signalling. However, the interactions between miR-155 and TLR3 are poorly understood. Here, we focused on the regulation of the relationship between miR-155 and TLR3. Sequence analyses and firefly luciferase reporter assay revealed that miR-155 target were present in the coding sequences (CDS) of TLR3. And the expression of the TLR3 protein could be inhibited by a miR-155 mimic or by a virally encoded orthologue in chick embryo fibroblast cells. Notably, endogenous miR-155 induction emerged a negative regulation on TLR3 expression in TLR2, 4 and 7 ligands stimulated HD11 cells, an avian macrophage cell line. Moreover, treatment with the miR-155 antagomir increased TLR3 levels while significantly decreased the abundance of TLR3 with miR-155 agomir. In addition, our data showed that miR-155 could inhibit IFN-β production possibly though TLR3 signal pathway. All these findings might reveal a new mechanism by which miR-155 can regulate the TLR3 immune response.

Cell-penetrating peptide (CPP) is a promising cargo for delivering bioactive molecules. In this study, the N terminus of VP1 from chicken anemia virus, designated as CVP1, was found to carry enriched arginine residues with α-helix. By confocal imaging, flow cytometry and MTT assay, we identified CVP1 as a novel, safe and efficient CPP. CVP1-FITC peptide could entry different types of cells tested with dose dependence, but without cytotoxic effects. Compared with TAT-FITC peptide, the CVP1-FITC peptide showed much higher cell-penetrating activity. Moreover, CVP1 could successfully deliver β-glycosidase, poly (I:C) and plasmid into HCT116 cells. Inhibitors and temperature sensitivity analysis further indicated that the cell-penetrating activity of CVP1 was based on ATP-dependent and caveolae-mediated endocytosis. All these data demonstrate that CVP1 has efficient cell-penetrating activity and great potential for developing a novel delivery vector.

Toll-like receptor 3 (TLR3) is a critical component of the innate immune system against viral infection and controls the activation of adaptive immunity. The role of TLR3 in Marek's disease virus (MDV) infection is not clear. In this study, we found that the abundance of TLR3 mRNA was significantly higher in chicken embryo fibroblast cells (CEF) infected with MDV than in a control group. Activated TLR3 signaling via TLR3 ligand stimulation inhibited replication of the RB1B strain of MDV in CEF cells. In contrast, CEF cells transfected with TLR3 siRNA promoted RB1B infection and replication. However, treatment with other TLR ligands, whether stimulatory (LPS, imiquimod and CpG) or inhibitory (TLR2/4 inhibitor and/or MyD88 inhibitor), had little effect on RB1B infection and replication. In addition, we found that the expression trend of TLR3 mRNA in RB1B-infected CEF cells was similar to that of mdv1-mir-M4-5p (a functional ortholog of oncogenic miR-155 encoded by MDV). Inconsistent with this, the TLR3 protein level was sharply reduced in RB1B-infected CEF cells at 96 hpi, while there was an at least 200-fold increase in miR-M4-5p at the same time point. Additionally, CEF cells transfected with an mdv1-mir-M4-5p mimic promoted RB1B infection and replication, while an mdv1-mir-M4-5p inhibitor inhibited RB1B infection and replication. Similar results were observed in CEF cells transfected with a gga-miR-155 mimic or inhibitor. These findings suggest that TLR3 and MDV-encoded miRNAs might be involved in MDV infection.

Marek’s disease virus (MDV) is an α-herpesvirus that causes immune suppression and T lymphoma in chickens. Toll-like receptor 3 (TLR3) is critical for the host immune response against MDV infection. Previously, our team demonstrated that pre-treatment of TLR3 agonist poly (I:C) inhibited Marek’s disease virus infection in chicken embryo fibroblasts (CEFs). However, whether TLR3 inhibits the aggravation of MDV infection is unknown. In the current study, we found that TLR3 activation in MDV-infected CEFs effectively inhibited virus spread. Using pharmacological approaches, we revealed that pro-inflammatory cytokines and interferon-β induced by TLR3 could restrict Marek’s disease virus infection. This study contributes to elucidating the function and mechanism of the TLR3 pathway in host immune responses against MDV infection.