The Influence of Temperature on the Antiviral Response of mIgM+ B Lymphocytes Against Hirame Novirhabdovirus in Flounder (Paralichthys olivaceus)

Abstract

Hirame novirhabdovirus (HIRRV) is an ongoing threat to the aquaculture industry. The water temperature for the onset of HIRRV is below 15°C, the peak is about 10°C, but no mortality is observed over 20°C. Previous studies found the positive signal of matrix protein of HIRRV (HIRRV-M) was detected in the peripheral blood leukocytes of viral-infected flounder. Flow cytometry and indirect immunofluorescence assay showed that HIRRV-M was detected in mIgM+ B lymphocytes in viral-infected flounder maintained at 10°C and 20°C, and 22% mIgM+ B lymphocytes are infected at 10°C while 13% are infected at 20°C, indicating that HIRRV could invade into mIgM+ B lymphocytes. Absolute quantitative RT-PCR showed that the viral copies in mIgM+ B lymphocytes were significantly increased at 24 h post infection (hpi) both at 10°C and 20°C, but the viral copies in 10°C infection group were significantly higher than that in 20°C infection group at 72 hpi and 96 hpi. Furthermore, the B lymphocytes were sorted from HIRRV-infected flounder maintained at 10°C and 20°C for RNA-seq. The results showed that the differentially expression genes in mIgM+ B lymphocyte of healthy flounder at 10°C and 20°C were mainly enriched in metabolic pathways. Lipid metabolism and Amino acid metabolism were enhanced at 10°C, while Glucose metabolism was enhanced at 20°C. In contrast, HIRRV infection at 10°C induced the up-regulation of the Complement and coagulation cascades, FcγR-mediated phagocytosis, Platelets activation, Leukocyte transendothelial migration and Natural killer cell mediated cytotoxicity pathways at 72 hpi. HIRRV infection at 20°C induced the up-regulation of the Antigen processing and presentation pathway at 72 hpi. Subsequently, the temporal expression patterns of 16 genes involved in Antigen processing and presentation pathway were investigated by qRT-PCR, and results showed that the pathway was significantly activated by HIRRV infection at 20°C but inhibited at 10°C. In conclusion, HIRRV could invade into mIgM+ B lymphocytes and elicit differential immune response under 10°C and 20°C, which provide a deep insight into the antiviral response in mIgM+ B lymphocytes.