Isolation and analysis of a novel grass carp toll-like receptor 4 (tlr4) gene cluster involved in the response to grass carp reovirus

The mammalian response to lipopolysaccharide (LPS) is mainly mediated by Toll-Like Receptor 4 (TLR4). Fish and mammalian TLR4 vary; fish TLR4 ligands are unknown. Isolation of fish tlr4 genes is difficult due to their complex genomic structure. Three bacterial artificial chromosome (BAC) clones containing grass carp tlr4 were obtained. Four tlr4 genes, with a varied genomic structure and different protein domains were subsequently isolated by constructing a subcloned library and rapid amplification of cDNA ends (RACE). The four tlr4 genes were expressed during development from 12 h post-fertilization, in all healthy adult fish tissues tested, and significantly increased in grass carp reovirus (GCRV)-infected liver and muscle, suggesting the tlr4 genes play a role in GCRV infection. This study effectively separated each gene in the tlr4 gene cluster, implies that grass carp TLR4 proteins have different ligand recognition specificities to mammalian TLRs, and provides information on the functional evolution of TLRs.

► Fish TLR4s are poorly characterized and difficult to identify.
► We identified and cloned four genes in a novel grass carp tlr4 gene cluster.
► The four tlr4 genes have varied genomic sequences and protein domains.
► Grass carp TLR4 proteins may have different ligand specificities to mammalian TLRs.
► Grass carp TLR4s may play a role in GCRV infection.