Assessment of TLR3-TRIF-NF-κB Innate Immune Signaling Pathway Induced by Newcastle Disease Virus and Construction of TLR3-Deficient BHK-21 Cells

The innate immune system plays a significant role in inhibiting viral infection and proliferation. To explore the signaling pathway of the innate immune response induced by Newcastle disease virus (NDV) in BHK-21 cells, the mRNA expression levels of pattern recognition receptors such as TLR3, TLR7, TLR8, and NOD2, as well as those of cytokines including TNF-α, IL-6, IL-1β, IL-10, IL-17, TRIF, TRAF3, and NF-κB were determined by transcriptome sequencing, qRT-PCR, and ELISA. All these results indicated that the NDV LaSota strain primarily induces the TLR3-TRIF-NF-κB signaling pathway in BHK-21 cells. Subsequently, an NF-κB inhibitor, a TLR3 inhibitor, and a TRAF3 inhibitor were separately used, and the viral titer was measured during the NDV LaSota strain infection of BHK-21 cells. The results displayed that the inhibition of TLR3 or NF-κB effectively increased the viral titer of NDV. Hence, the CRISPR/Cas9 system was used to create a BHK-TLR3—cell clone in which TLR3 was knocked out, and the cells were assessed for morphology, cell density, cell viability, and genetic stability. Finally, the cells were infected with the NDV LaSota strain. The results showed that the BHK-TLR3—cell line is a stable cell line with markedly enhanced NDV LaSota strain production. Furthermore, the knockout of the TLR3 gene significantly inhibited the expression of IFN-α, IFN-β, IL-1β, and TNF-α, especially that of IL-1β and TNF-α. In summary, the present research revealed the main signaling pathway of the innate immune response induced by the NDV LaSota strain and provides a promising basis for NDV production.