Type-I interferon response affects an inoculation dose-independent mortality in mice following Japanese encephalitis virus infection

Abstract

Background: The laboratory mouse model is commonly employed to study the pathogenesis of encephalitic flaviviruses such as Japanese encephalitis virus (JEV). However, it is known that some strains of these viruses do not elicit a typical mortality dose response curve from this organism after peripheral infection and the reason for it has not yet been fully understood. It is suggested that induction of more vigorous Type-I IFN (IFN-I) response might control early virus dissemination following increasing infectious challenge doses of the virus. Thus, the objective of this study was to examine this suggested role of IFN-I in the mortality of mice infected with various doses of JEV.

Methods: Inbred 129 mice and their IFNAR KO (A129) mice were subcutaneously inoculated with 100, 102, 104 or 106 pfu of JaOArS982 strain of JEV. Mice were weighed daily and observed for clinical signs. Virus titers in the brains and spleens of JEV-infected mice were determined by plaque forming assays. The upregulated mRNA levels of genes related to IFN-I response of mice were examined by real-time PCR.

Results: The mortality rates of 129 mice infected with JaOArS982 did not significantly increase despite the increase in inoculation dose and no significant difference of viral loads was observed between their brains. However, there was clear elevation of the mRNA levels of interferon regulatory factor (IRF)3, IRF7, IRF9, MDA5 and RIG-I at 24 hours post-infection depending on the inoculation dose. In A129 mice, length of survival days and the viral loads of spleen and brain were observed to be inoculation dose-dependent.

Conclusions: From these results, it is suggested that early IFN-I response elicited by high inoculation doses of JEV provides an anti-viral effect during the early phase of infection. Accordingly, virus replication is counteracted by IFN-I response at each increasing inoculation dose resulting in the interference of impending severe disease course or fatal outcome; hence, this might explain the inoculation dose-independent mortality in mice caused by Japanese encephalitis virus.

Figure 1

Mortality and viral loads of 129 mice subcutaneously inoculated with the JaOArS982 strain of JEV. Mortality (A) and survival (B) rates of mice inoculated with 10⁰, 10², 10⁴ or 10⁶ pfu of JEV (n=14 per inoculation dose). Mortality rate was recorded 21 days pi. (C) Infectious virus titers in the brains of mice inoculated with 10⁰, 10², 10⁴ or 10⁶ pfu of the JaOArS982 strain of JEV at 5 and 9 days pi (n=3 per inoculation dose and day of sacrifice). Error bars represent the standard errors.

Figure 2

mRNA levels of IFN-I related genes of 129 mice subcutaneously inoculated with the JaOArS982 strain of JEV. mRNA levels were quantified by real-time PCR in the spleens of 129 mice inoculated with 10⁰, 10², 10⁴ or 10⁶ pfu of JEV at 24 (A), 48 (B) 72 (C) hours pi and of uninfected 129 mice (n=3 per group). Uninfected mice show the same data in A, B, and C. Error bars represent the standard errors. P values were calculated by ANOVA. Asterisk indicates the pair that shows significant difference by Tukey’s Multiple Comparison Test.

Figure 3

Mortality and viral loads of A129 mice subcutaneously inoculated with the JaOArS982 strain of JEV. Mortality (A) and survival (B) rates of mice inoculated with 10⁰, 10², 10⁴ or 10⁶ pfu of JEV (n=10 per group). p: Log-lank test. Infectious virus titers in the spleens (C) and brains (D) of mice inoculated with 10⁰, 10², 10⁴ or 10⁶ pfu of the JaOArS982 strain of JEV at 24, 48 and 72 hours pi (n=3 per group). Error bars represent the standard errors. P values were calculated by ANOVA at each time point.