Introduction
The growth of the aquaculture industry is being hindered by increasing mortality caused by infectious diseases and environmental stressors. In particular, viral infections cause significant damage in aquaculture fish due to the lack of effective treatments. Therefore, understanding and studying the antiviral immune system of fish is essential to deal with viral diseases.
NLRC5 (NOD-like receptor family CARD domain containing 5) belongs to the NOD-like receptor family and is known to be involved in immune responses by regulating histocompatibility complex (MHC) I gene expression and type 1 interferon in mammals, but its role in teleost remain unclear. In this study, we established an Nlrc5 knockout (KO) zebrafish model to investigate the role of NLRC5 in the antiviral immune response of teleost, and demonstrated its importance through viral hemorrhagic septicemia virus (VHSV) infection experiments.
Materials and methods
Nlrc5 KO zebrafish were generated using the CRISPR/Cas9 system. VHSV infection was induced in both wild-type (WT) and Nlrc5 KO zebrafish via intraperitoneal (IP) injection (1×106 and 5×106 TCID50/fish), and mortality rates, viral replication, and downstream gene (cytokines, class-I MHC pathway components, neutrophil markers) expression were subsequently compared.
Results and discussion
CRISPR/Cas9-mediated Nlrc5 knockout zebrafish were identified to have a 10 bp deletion sequence at the sgRNA target site, resulting in a frameshift mutation that produced a truncated 206-amino acid peptide. The mutated protein contained only the CARD domain, while other functional domains, including the NACHT domain, were completely absent.
In the VHSV infection experiment using WT and Nlrc5 KO zebrafish, the Nlrc5-deficient fish exhibited higher mortality, increased virus replication, and severe pathological symptoms (including hemorrhage, and eye extrusion). Gene expression analysis following VHSV infection revealed that ifnφ1 was significantly upregulated, while expression of cytokines involved in neutrophil migration and MHC-I genes was significantly downregulated in Nlrc5 KO zebrafish compared to WT zebrafish. In addition, neutrophil migration analysis confirmed reduced neutrophil recruitment to the caudal fin injury site in Nlrc5 knockout zebrafish. These results demonstrate that Nlrc5 is essential for the antiviral immune response to VHSV infection in zebrafish, primarily through regulation of the MHC class I pathway.
Collectively, this study provides evidence for the role of Nlrc5 in antiviral immunity in teleost, enhancing our understanding of innate immune regulation and highlighting its significance in antiviral immune system.