Variation in interferon sensitivity and induction among strains of eastern equine encephalitis virus

Abstract

Eastern equine encephalitis virus (EEEV) causes human encephalitis in North America (NA), but in South America (SA) it has rarely been associated with human disease, suggesting that SA strains are less virulent. To evaluate the hypothesis that this virulence difference is due to a greater ability of NA strains to evade innate immunity, we compared replication of NA and SA strains in Vero cells pretreated with interferon (IFN). Human IFN-alpha, -beta, and -gamma generally exhibited less effect on replication of NA than SA strains, supporting this hypothesis. In the murine model, no consistent difference in IFN induction was observed between NA and SA strains. After infection with most EEEV strains, higher viremia levels and shorter survival times were observed in mice deficient in IFN-alpha/beta receptors than in wild-type mice, suggesting that IFN-alpha/beta is important in controlling replication. In contrast, IFN-gamma receptor-deficient mice infected with NA and SA strains had similar viremia levels and mortality rates to those of wild-type mice, suggesting that IFN-gamma does not play a major role in murine protection. Mice pretreated with poly(I-C), a nonspecific IFN inducer, exhibited dose-dependent protection against fatal eastern equine encephalitis, further evidence that IFN is important in controlling disease. Overall, our in vivo results did not support the hypothesis that NA strains are more virulent in humans due to their greater ability to counteract the IFN response. However, further studies using a better model of human disease are needed to confirm the results of differential human IFN sensitivity obtained in our in vitro experiments.

FIG. 1.

Effect of human IFN-α on EEEV replication. Cells were treated with 50 U of IFN-α and incubated at 37°C for 24 h prior to infection with EEEV (MOI = 0.01). The y axis represents mean levels of suppression of virus replication, calculated by subtracting the mean titers in the treated cells from mean values in the untreated control cells at each time point. Because the peak virus titers in sham-treated control cells occurred at 48 h p.i., the values presented in panel C are based on subtracting the mean value from the treated cells at 72 h from the mean of the sham-treated cells at 48 h p.i.. A significant effect of treatment on virus replication was observed for both NA and SA strains at 24 h p.i. (A) (P < 0.05); however, the suppression in virus replication was only observed with SA strains at 48 h (B) (P < 0.05) and 72 h p.i. (C) (P < 0.05).

FIG. 2.

Effect of human IFN-β on EEEV replication. Cells were treated with 5 U of IFN-β and incubated at 37°C for 24 h prior to infection with EEEV (MOI = 0.01). The y axis represents the mean levels in suppression of virus replication in Vero cells pretreated with IFN-β. The values were calculated by subtracting the mean value obtained in the treated control cells from the sham-treated control cells at each time point. Because the peak virus titers in sham-treated control cells occurred at 48 h p.i., the values presented in panel C are based on subtracting the mean value from the treated cells at 72 h from the mean of the sham-treated cells at 48 h p.i.. Virus replication of both NA and SA strains was inhibited at 24 h p.i. (A); however, it was more evident with most SA strains (GML903836, C-49, and BeAr436087) at 48 (B) and 72 h p.i. (C).

FIG. 3.

Effect of human IFN-γ on EEEV replication. Cells were treated with 25 U of IFN-γ and incubated at 37°C for 24 h prior to infection with EEEV. The y axis represents the mean levels of suppression of virus replication in Vero cells pretreated with IFN-γ. The values were calculated by subtracting the mean value obtained in the treated control cells from the values for sham-treated control cells at the specified time. A significant effect of treatment on virus replication was observed with both NA and SA strains at 24 h p.i. (A). At 48 (B) and 72 h p.i. (C), replication of SA strains was significantly inhibited.

FIG. 4.

IFN-α/β induction and viremia in infected mice. Cohorts of 10 to 15 NIH Swiss mice (5- to 7 weeks old) were infected subcutaneously with 1,000 PFU of virus, and blood samples were collected daily. Significant differences in IFN induction at 48 h p.i. (A) were observed between NA strain 792138 and all other EEEV strains (P < 0.05). IFN levels at 72 h were not significantly different among virus strains (B). Viremia levels confirmed infection in mice and lack of correlation between IFN induction and levels of EEEV replication (C). The detection limit of the plaque assay was 1.7 log₁₀ PFU/ml.

FIG. 5.

Effect of poly(I-C) on mouse survival after EEEV infection. Cohorts of 10 NIH Swiss mice were treated intraperitoneally and 24 h later infected with EEEV. Mortality was recorded daily.

FIG. 6.

Effect of treatment with poly(I-C) and antibodies against IFN treatment on survival after EEEV infection. Cohorts of five mice were treated intraperitoneally with poly(I-C), along with antibodies to IFN or with antibodies alone. Mice treated with high doses of antibodies alone or in combination with 4 μg of poly(I-C) and infected with NA and SA strains succumbed more rapidly to infection than sham-treated mice (P < 0.05). No significant difference in mortality was observed when animals were treated with low doses of antibodies alone, or antibodies plus poly(I-C) (1 μg) and infected with NA and SA strains compared to the sham-treated group (P > 0.05).

FIG. 7.

Viremia and survival in strain 129 SV/EV IFN-α/β receptor-deficient (KO) mice and wild-type, congenic mice. Cohorts of 10 mice (10 to 13 weeks old) were infected subcutaneously, and blood samples were collected at different time points. IFN-α/β receptor KO mice infected with the NA strain 792138 showed increased viremia (A) and more rapid mortality (B) than wild-type mice. In contrast, IFN-α/β receptor KO mice infected with the NA strain FL93-939 showed no significant difference in viremia (C) and only a slight difference in mortality (D) compared to wild-type mice. KO mice infected with SA strains GML903836 and BeAr300851 showed a difference in viremia levels compared to wild-type mice (E and G, respectively) (P < 0.05). (H) Mortality in KO mice was also more rapid with SA strain BeAr300851. The detection limit of the plaque assay was 1.7 log₁₀ PFU/ml.

FIG. 8.

Viremia and survival in strain 129 SV/EV IFN-γ receptor-deficient (KO) and wild-type mice. Cohorts of three IFN-γ receptor-deficient mice were infected subcutaneously with 1,000 PFU. The data from the wild-type mouse group corresponded to a single experiment with 10 animals and are also shown in Fig. 7. IFN-γ receptor KO mice infected with NA strain 792138 showed no significant difference in viremia (A) or mortality (B) compared to wild-type mice (P > 0.05). Similarly, IFN-γ receptor KO mice infected with the SA strain GML903836 showed no difference in viremia (C) or mortality (D) compared to wild-types (P > 0.05). The detection limit of the plaque assay was 1.7 log₁₀ PFU/ml.