Key role of TLR3 in type I IFN expression and apoptosis induction in IBDV-infected chicken fibroblast cells

Abstract

Infectious Bursal Disease Virus (IBDV) (Avibirnavirus genus, Birnaviridae family) is a non-enveloped virus with a double-stranded RNA (dsRNA) genome. IBDV causes a highly contagious and immunosuppressive disease in domestic chickens (Gallus gallus), representing a major threat to the global poultry industry. Apoptotic cell death and exacerbated innate immune responses have been implicated in IBDV pathogenesis. Previous studies from our laboratory demonstrated the crucial role of type I interferon (IFN) in triggering apoptosis in IBDV-infected cell cultures. Genomic IBDV dsRNA is recognized by the cytoplasmic pattern recognition receptor (PRR) melanoma differentiation-associated gene 5 (MDA5) in chicken cells, triggering type I IFN responses. However, the contribution of the endosomal PRR Toll-like receptor 3 (TLR3) dsRNA sensor on type I IFN production upon IBDV infection has not been studied, despite several studies have demonstrated that its expression is significantly upregulated upon IBDV infection. Here, we demonstrate that ablation of TLR3 gene expression in DF-1 chicken fibroblasts results in a complete blockade of IBDV-induced apoptosis, a marked reduction in IFN production, and a significant enhancement of virus progeny yields. Notably, this effect appears to be specific to IBDV, as similar outcomes were not observed with other RNA viruses. Our findings also suggest that TLR3 may also play a role in viral release into the extracellular space. Additionally, receptor interacting protein kinase 1 (RIPK1), a protein that interacts with TLR3 through the adaptor Toll/IL-1 receptor (TIR) domain-containing adaptor-inducing interferon-{beta} (TRIF), was shown to contribute to both IFN production and apoptosis in response to IBDV infection or dsRNA stimulation in DF-1 cells. Overall, this study provides new insights into the innate immune recognition of IBDV, highlighting the central role of TLR3 in mediating antiviral responses in chicken cells. Author summaryInfectious Bursal Disease Virus (IBDV) is the etiological agent of a devastating syndrome known as IBD or Gumboro disease, which affects domestic chickens (Gallus gallus), leading to significant economic losses in the poultry industry worldwide. The virus primarily targets immature B lymphocytes causing their killing by apoptosis, resulting in severe immunosuppression. Diverse studies have suggested that exacerbation of the innate immune response is related to IBDV pathogenesis. Here, we have investigated the role of the cellular sensor of double-stranded RNA (dsRNA) Toll-like receptor 3 (TLR3) on the fate of IBDV-infected cells. Deletion of the TLR3 gene completely blocks apoptosis induced in IBDV-infected DF-1 cells, drastically reduces interferon (IFN) production and improves viral replication efficiency. We also demonstrated the participation of receptor interacting protein kinase 1 (RIPK1), a known downstream mediator of TLR3, in both, IFN production and apoptosis induction in response to IBDV infection. These findings provide new molecular insights into the mechanisms underlying the robust type I IFN response observed during IBDV infection and contribute to a deeper understanding of IBDV pathogenesis.