Possible role of LTB 4 in the antiviral activity of turbot ( Scophthalmus maximus) leukocyte-derived supernatants against viral hemorrhagic septicemia virus (VHSV)

Abstract

Turbot ( Scophthalmus maximus) blood leukocyte-derived supernatants were tested for antiviral activity against viral hemorrhagic septicemia virus (VHSV). The assays were performed by quantifying the effect of the supernatants on the replication of VHSV in the rainbow trout ( Oncorhynchus mykiss) cell line, RTG-2. Supernatants were obtained by incubating the leukocytes for 17 h at 18°C in L-15 medium supplemented with 5% fetal calf serum (FCS). Testing of leukocyte supernatants indicated that antiviral activity against VHSV resulted in a viral titer reduction of 72.1%. After the supernatants were extracted with calcium ionophore A23187 treatment, the antiviral activity significantly increased, resulting in a viral titer reduction of 99.9%. In order to determine the nature of this antiviral activity, supernatants were produced from leukocytes treated for 17 h with inhibitors of eicosanoid biosynthesis, reactive oxygen intermediates and nitric oxide (NO) production. None of the inhibitors significantly suppressed the supernatant antiviral activity. The presence of oxygen radicals and NO was measured in the case of co-cultures of leukocytes and RTG-2 cells, but no significant differences were found in the VHSV-infected co-cultures compared to non-infected controls. Since previous work demonstrated that leukotriene B 4 (LTB 4) was present in turbot blood leukocyte-derived supernatants, we assessed the effect of the VHSV in vitro infection on turbot leukocyte LTB 4 production by high performance liquid chromatography (HPLC). The levels of LTB 4 were significantly increased in the supernatants after VHSV infection. Furthermore, exogenous LTB 4 significantly inhibited VHSV replication in RTG-2 cells. These findings suggest that LTB 4 may play a significant role in VHSV replication.