Antiviral effect of nitric oxide during Japanese encephalitis virus infection

Abstract

The ability of Japanese encephalitis virus (JEV) and JEV-induced macrophage derived neutrophil chemotactic factor (MDF) to produce nitric oxide (NO), and the possible antiviral effect of NO during JEV infection, was investigated. Splenic macrophages of JEV infected mice produced maximum NO in vivo at day 7 post infection, and in vitro at 24 h after JEV stimulation. MDF-induced NO production was dose dependent and maximal at 60 min after MDF treatment. The response was sensitive to anti-MDF antibody treatment and the nitric oxide synthase inhibitor NG-monomethyl-L-arginine (L-NMMA). Pretreatment of mice with L-NMMA increased the mortality to 100% in JEV infected mice in vivo and inhibited NO production in vitro, while MDF stimulated macrophages inhibited virus replication with high levels of NO production. MDF treatment increased the survival rate of JEV infected mice. The findings thus demonstrate that MDF induces production of NO during JEV infection, which has an antiviral effect. This may be one of the important mechanisms of natural immunity in controlling the initial stages of JEV infection.

Figure 1

Production of NO by spleen cells of JEV-inoculated mice. Groups of mice were inoculated with JEV intraperitonially (i.p.). Spleens were harvested at different time periods, a single cell suspension was prepared (10 × 10⁶cells/ml) and cultured for 24 h at 37 °C. NO production was assayed in the cell free culture supernatents (▪) as described in Materials and methods. Control (□) mice were inoculated with normal mouse brain suspension. Values are presented as A. M. ± SD from 10 cultures.

Figure 2

Production of NO by JEV stimulated spleen cells in vitro. Normal mouse spleen cell (10 × 10⁶ cells/ml) cultures were inoculated with 10⁴ TCID₅₀ JEV and incubated at 37 °C. At different time periods NO production was assayed (▪) as described in Materials and methods. The control cells (□) were inoculated with normal mouse brain suspension. Each point represents A.M. ± SD from 10 cultures.

Figure 3

MDF induced NO production in vitro, after stimulation of macrophage cultures with 5 μg of MDF (▪). Controls were stimulated with normal mouse splenic macrophage culture supernatent (□). At different time periods NO production was assayed as described in Materials and methods. Values presented as A.M. ± SD from eight cultures.

Figure 4

Inhibition of NO production by treatment with N^G-monomethyl-l-arginine (L-NMMA). Normal mouse macrophage cultures were pretreated with 100 μm L-NMMA for indicated time periods followed by inoculation of 5 μg MDF for 60 min at 37 °C. Control group of cells (*) were treated with MDF only. Cells treated with L-NMMA for different time periods were used for background values. NO production was assayed as described in Materials and methods. Results are presented after deduction of background values as A.M. ± SD from eight cultures.

Figure 5

Effect of L-NMMA treatment on JEV infection. Mice were inoculated with the sublethal dose (10⁵LD₅₀) of JEV (i.c.) with (•) or without (○) treatment of L-NMMA. Mice were treated with L-NMMA (100 μm) daily. As control (□) the mock infected mice were treated with L-NMMA alone. The mortality rate of the mice was monitored daily for three weeks. Each group consisted of 20 mice.

Figure 6

Effect of MDF on JEV induced NO production by macrophages. NO production was assayed in MDF activated (▪) or nonactivated () macrophages after 24 h of in vitro JEV stimulation, as described in Materials and methods. Controls consisted of cells treated with MDF alone () or unstimulated (□). Values are presented as A.M. ± SD of six cultures.

Figure 7

Survival of JEV-infected Swiss albino mice treated with MDF or left untreated. Twenty mice per group were infected with the 10²LD₅₀ of JEV mouse brain suspension(i.c.). Starting from the begining of inoculation, the mice were treated with MDF (5 μg) intravenously daily till the animal died (▪) or left untreated (). As control (□) mock infected mice were also treated with MDF. The survival rate of the mice was monitored daily for three weeks.