TLR3 and MDA5 knockout DF-1 cells enhance replication of avian orthoavulavirus 1

Authors: Lee, Chang; MAHESH, K - The Ohio State University; NGUNJIRI, JOHN - The Ohio State University; GHORBANI, AMIR - The Ohio State University; LEE, KICHOON - The Ohio State University

Submitted to: Avian Diseases
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 12/22/2023
Publication Date: 3/7/2023
Citation: Lee, C.W., Mahesh, K.C., Ngunjiri, J.M., Ghorbani, A., Lee, K. 2023. TLR3 and MDA5 knockout DF-1 cells enhance replication of avian orthoavulavirus 1. Avian Diseases. 67(1):94-101. https://doi.org/10.1637/aviandiseases-D-22-00065.
DOI: https://doi.org/10.1637/aviandiseases-D-22-00065

Interpretive Summary: With the recent advances in genetic modification techniques called the “CRISPR/Cas9” system, it is possible to produce cells that lack function of certain genes (gene knockout) more efficiently. Using the technique, we developed chicken cells that lost the function of two genes (TLR3 and MDA5) known to recognize pathogens and mediate antiviral response. We confirmed that the cells do not respond to chemical that specifically bind to those two receptors (TLR3 and MDA5). In addition, the newly developed chicken knockout cells supported the replication of Newcastle disease (economically important disease in poultry) vaccine virus better which may due to the reduced ability of the knockout cells to induce defensive immune response against the virus. In addition to its practical applicability in growing viruses, the knockout cells will be useful in studying the roles and relevance of those genes in viral replication and pathogenesis in avian species.

Technical Abstract: Despite the essential role of innate immunity in defining the outcome of viral infections, the roles played by different components of the avian innate immune system are poorly delineated. Here, we investigated the potential implication of avian toll-like receptor 3 (TLR3) and melanoma differentiation-associated gene 5 (MDA5) receptors of double-stranded RNA (dsRNA) in induction of the interferon pathway and avian orthoavulavirus 1 (AOAV-1) replication in chicken-origin DF-1 fibroblast cells. TLR3 and MDA5 knockout DF-1 cells were generated using our avian-specific CRISPR/Cas9 system and stimulated with a synthetic dsRNA ligand polyinosinic:polycytidylic acid [poly(I:C)] or infected with AOAV-1 (previously known as Newcastle disease virus). Poly(I:C) treatment in cell culture media resulted in significant upregulation of IFNa, IFNß, and Mx1 gene expression in wild type (WT) DF-1 cells but not in TLR3-MDA5 double knockout cells. Interestingly, poly(I:C) treatment induced rapid cell degeneration in WT and MDA5 knockout cells, but not in TLR3 knockout or TRL3-MDA5 double knockout cells, directly linking poly(I:C)-induced cell degeneration to TLR3-mediated host response. The double knockout cells supported significantly higher replication of AOAV-1 virus than the WT cells. However, no correlation between the level of virus replication and type I IFN response was observed. Our study suggests that innate immune response is host and pathogen specific and further investigation is needed to understand the relevance of dsRNA receptor-mediated immune responses in viral replication and pathogenesis in avian species.