Variation in Interferon Sensitivity and Induction
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病菌学杂志 2005年第17期
Center for Biodefense and Emerging Infectious Diseases
Department of Microbiology and Immunology
Department of Pathology, University of Texas Medical Branch, Galveston, Texas 77555-0609
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-, -, and - 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-/ receptors than in wild-type mice, suggesting that IFN-/ is important in controlling replication. In contrast, IFN- 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- 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.
INTRODUCTION
Eastern equine encephalitis virus (EEEV) is an arthropod-borne virus (arbovirus) in the family Togaviridae, genus Alphavirus. The virus possesses a single-stranded, messenger-sense RNA genome of ca. 11.7 kb that encodes four nonstructural proteins (nsP1 to -4) and three main structural proteins (capsid and glycoproteins E1 and E2) (47). The virus is an important veterinary and human pathogen and causes periodic outbreaks in the Americas associated with high morbidity and mortality among humans and equines. In humans, EEEV infection is characterized mainly by fever, lethargy, vomiting, convulsions, and coma. Surviving patients, especially children, are often profoundly affected by neurological and physical sequelae that include mental retardation, hemiparesis, aphasia, emotional instability, convulsions, hemiplegia, strabismus, impaired vision, partial deafness, and speech disorders (45). In equines, pheasants, chickens, sandhill cranes, emus, and pigs, EEEV infection also causes severe and often fatal disease (54).
EEEV was first isolated in the United States from horses during an epizootic in coastal regions of Virginia, Maryland, and Delaware in 1933 (5, 48) and in South America (SA) in 1930 from a horse in Argentina (40). In North America (NA), the enzootic transmission cycle occurs in freshwater swamp foci and involves passerine birds and the ornithophilic mosquito vector, Culiseta melanura. Sporadic epizootic or epidemic transmission involves spillover from the avian swamp cycle and is probably mediated by bridge vectors such as Aedes canadensis, Aedes sollicitans, Aedes vexans, and Coquillettidia perturbans (24, 35, 54). Enzootic transmission in Central and SA is not well understood but probably involves mosquitoes in the Culex (Melanoconion) subgenus and rodent and/or avian reservoir hosts (44, 54).
Antigenic studies have shown that NA strains of EEEV differ from those found in SA and that these two geographic variants also differ in biological and epidemiological characteristics (12-14, 38). Genetic and antigenic studies (9, 38, 55) revealed that NA strains are highly conserved, constituting a unique major lineage, whereas SA strains constitute three major lineages with more antigenic and genetic variation. All strains isolated in NA and most from the Caribbean belong to the NA subtype (lineage I), whereas isolates from Central and SA constitute three SA subtypes (lineages II to IV) (9, 11, 14). Importantly, the NA subtype causes severe disease in both humans and equines, with mortality rates of 30 to 80% in humans and 90 to 95% in equines. In contrast, infection by the SA EEEV subtypes can be fatal in equines, but human infections have rarely been recognized and in most cases were identified only by serosurveys with no evidence of disease. Causey and Theiler (16) demonstrated seroprevalence as high as 25% in children and 31% in adults living in the Amazon basin, and a single human encephalitis case was also reported in Brazil (1). Although EEEV was implicated in equine epizootics in Braganca, Para State, no neurological disease in humans was reported during these outbreaks (15, 50, 53). The EEEV was also isolated repeatedly in Argentina from horses between 1930 and 1958. However, no human neurological disease was reported despite active surveillance and human seroprevalence levels of up to 66% in some locations (42).
Recently, EEEV was isolated from Culex (Melanoconion) pedroi mosquitoes in Puerto Almendras, Iquitos, Peru (M. Turell, USAMRIID [unpublished data]), providing evidence that EEEV circulates throughout the Amazon basin. However, again, no fatal or neurologic human disease associated with EEEV has been recognized in Peru despite active surveillance there for febrile diseases. These epidemiological studies provide strong evidence that, unlike their NA counterparts, SA strains of EEEV generally do not produce severe disease but can infect humans. The reason for this apparent difference in human virulence is unknown.
Interferons (IFNs) have long been recognized as essential components of the innate immune response to viral infections (3, 4, 30). Specifically, IFN- and - are known to be important in protecting against alphaviral disease (26, 46). Previous studies showed a correlation between alphavirus virulence and resistance to IFN- and -. Some studies with Venezuelan equine encephalitis virus (VEEV) suggested that the IFN- and - resistance or sensitivity phenotype correlated with epizootic potential and equine virulence (31, 46), although others observed little or no difference (2). Although an important role of IFN-/ in the pathogenesis of several alphaviruses, including Sindbis virus (SINV) and Semliki Forest virus (29, 36, 41), has been described, little information is available regarding the role of IFN-, -, and especially of IFN- in the pathogenesis of other alphaviruses, including EEEV. We therefore hypothesized that NA strains of EEEV are more virulent for humans because of a greater resistance to IFN than SA strains and/or because they induce lower levels of IFN. To test these hypotheses, the effects of IFN on EEEV replication were evaluated in Vero cells treated with human IFN-/ and -. Vero cells were chosen because they respond efficiently to human IFN but are unable to produce IFN, eliminating any possibility of autocrine or paracrine effects. For the in vivo studies, the mouse model was used because it allowed us to study the effect of ablating the IFN response on EEEV infection and disease outcome.
MATERIALS AND METHODS
Viruses. Virus isolates (Table 1) were provided by the University of Texas Medical Branch World Reference Center for Emerging Viruses and Arboviruses. All strains were isolated in Vero (African green monkey kidney) cells from sentinel hamsters, monkeys, or mosquitoes and were chosen due to their low-passage histories. Stocks were prepared in mice to avoid selection for attenuated alphavirus mutants that occur after passage in cells expressing glycosaminoglycans (6, 10, 28, 32). One- to three-day-old mice were inoculated intracranially with each virus strain, and a 10% brain suspension was prepared after morbidity or mortality was observed. Virus stock titers were determined by plaque assay in Vero cells. The nucleotide sequence differences among SA strains range from ca. 1 to 2% (e.g., GML903836 versus C-49; all NA strains) to 5% (BeAn5122 versus BeAr300851) for members of the same lineage. All SA strains differed from the NA strains by 18 to 23% at the nucleotide and 5 to 10% at the amino acid level.
Previous studies showed that the amount of IFN induced in the sera and brains of neonatal mice infected with alphaviruses is on the order of 20,000 to 215,000 IU/ml (51, 52). Therefore, to avoid possible confounding effects of IFN in the homogenized brain tissue, virus stocks were diluted ca. 1,000,000-fold in all experiments.
IFN sensitivity assays. Human IFN preparations were kindly provided by the National Institutes of Health, Bethesda, Md. Both human IFN- and IFN- were obtained from human cells by induction with Sendai virus or treatment with polyinosinic polycytidylic acid [poly(I-C)], whereas the human IFN- was a recombinant preparation. Vero cells were seeded in 12-well plates, and 2 days later were pretreated with human IFN-, -, or - and incubated for 24 h. Culture media were then removed, the cells washed three times with phosphate-buffered saline (PBS), and 1,000 PFU of EEEV were added to each well. Negative controls consisted of infected cells not exposed to IFN. At 24, 48, and 72 h postinfection (p.i.), the supernatant fluid was completely removed, the cells were washed twice with PBS, and new media containing the appropriate amount of IFN were added. Virus in the supernatant fluid at selected times was titrated on Vero cells by plaque assay.
Infection of mice. Five- to six-week-old NIH Swiss mice (Harlan Laboratories, Indianapolis, Ind.) were maintained under specific-pathogen-free conditions. The animals were allowed to acclimate to the laboratory for 1 week and then were placed into cohorts of five for subcutaneous infection with 1,000 PFU of EEEV. The animals were monitored daily for clinical signs, including lethargy, paralysis, or death and bled at 24, 48, 72, and 96 h p.i. to determine viremia levels by plaque assay and IFN production.
IFN assays. To quantify levels of IFN-/ produced after EEEV infection of mice, a vesicular stomatitis virus (VSV) plaque reduction assay was used. Serum was exposed to UV light to inactivate EEEV and inactivation was confirmed by a cell culture assay. Serially diluted sera were applied to L929 cells grown in 96-well plates. Controls included the addition of known amounts of mouse IFN-, -, and - (obtained from murine cells induced with Newcastle disease virus) instead of serum samples, and UV-inactivated EEEV was used to determine whether virus particles alone could induce IFN. After a 24-h incubation, cells were washed three times with PBS, and 50 PFU of VSV were added to each well. Negative controls included mock-treated cells infected with VSV. After 1 h of incubation, 1% methylcellulose in minimal essential medium was added. When plaques were visible, the cells were stained with 1% crystal violet and the IFN- and/or IFN- titer was calculated as the highest serum dilution that inhibited 50% of VSV plaques.
Poly(I-C) treatment. Five- to six-week-old NIH Swiss mice were placed into cohorts of five, and two groups were each pretreated intraperitoneally with 0.5, 1, 2, 4, 10, 65, or 100 μg of poly(I-C) (Amersham Biosciences, NJ), a synthetic, double-stranded RNA that induces IFN by mimicking the genetic material of some viruses or the by-products of some viral infections. Control animals were mock treated with diluent. At 24 h posttreatment, animals were inoculated subcutaneously with 1,000 PFU of EEEV and monitored daily for up to a month for clinical signs of illness and mortality. To confirm that the protection observed with the poly(I-C) treatment was IFN-mediated, mice were treated intraperitoneally with 1 μg of poly(I-C) along with 2,000 U of antibodies to murine IFN-, IFN-, and IFN- (NIH) and with 4 μg of poly(I-C) plus 50,000 U of antibodies. Controls included animals treated with antibodies alone. After 24 h, animals were infected with the NA strain 792138 and the SA strain GML903836, and mortality was recorded daily.
Infection of IFN-/ and IFN- receptor-deficient mice. Ten- to thirteen-week-old strain 129 Sv/Ev (wild-type) mice were purchased from Jackson Laboratories (Bar Harbor, ME), and breeding pairs of the 129 Sv/Ev IFN-/ receptor –/– mice were generously provided by Herbert Virgin (Washington University, St. Louis, MO) and allowed to breed under pathogen-free conditions. Strain 129 Sv/Ev IFN- receptor –/– mice were purchased from Jackson Laboratories. After 1 week of acclimation to the laboratory, mice were subcutaneously inoculated with 1,000 PFU of EEEV and bled 8, 24, 32, 48, 56, 72, and 96 h p.i. for viremia determination. The animals were observed daily for up to a month for clinical signs of illness and mortality, and the median survival time (MST) was determined.
Statistical analysis. Statistical comparisons were performed by using a one-way analysis of variance with Dunn's and Tukey's multiple comparison test to identify significant differences among samples. For survival comparisons, the log-rank test or Mantel-Haenszel test (two-tailed P values) included in the GraphPad Prism program (San Diego, CA) was used. Medians were used to present some data because they are less sensitive to extreme values than are means (37). The MST was calculated as the time at which 50% of the animals succumbed to infection. If the survival curve was horizontal at 50% survival, the MST was calculated as the average of the first and last times at which survival was 50%. If the survival exceeds 50% at the longest time point, then the median survival was not computed. P values of 0.05 were considered significant.
RESULTS
To determine the potential role of IFNs in limiting EEEV infection and to evaluate the hypothesis that NA strains are more virulent for humans because of a greater resistance to IFNs than SA strains, Vero cells were treated with human IFNs. Previous studies have shown that Vero cells respond efficiently to human IFNs but are unable to produce IFN (18, 21), thereby eliminating the confounding effects of autocrine and/or paracrine responses (19).
Sensitivity to human IFN- in vitro. Vero cells were pretreated for 24 h with 50 IU of human IFN-/ml prior to infection with EEEV strains at a multiplicity of infection (MOI) of 0.1. At 24 and 48 h p.i., yields of both NA and SA strains were 10 to 25-fold lower in the IFN-treated cells than in the sham-treated control cells (Fig. 1A) (P < 0.001). At 48 h p.i., the viral yields for the SA strains in the treated cells were again significantly lower (ca. 10- to 25-fold than in control cells that produced in average 7.6 log10 PFU of virus/ml) (P < 0.001). In contrast, viral yields for the NA strains were similar in the treated and sham-treated cells, with viral yields of 8.35 log10 PFU/ml (0-5-fold reduction) (P > 0.05), indicating that IFN- did not have a major effect on the yield of the NA strains at the later time points (Fig. 1B). At 72 h p.i., the yields of SA strains in IFN-treated cells were still lower (10- to 18-fold) than in sham-treated cells (average viral yields 6.5 log10 PFU/ml). In contrast, IFN treatment had no significant effect on viral yields by the NA strains at this time point (Fig. 1C). These results suggested that SA strains were more sensitive to IFN- than the NA strains of EEEV.
Sensitivity to human IFN- in vitro. Differences in sensitivity to 5 U of human IFN- were also detected among EEEV strains. At 24 h p.i., the yield of both NA and SA strains was 8- to 24-fold lower in cells treated with IFN than in sham-treated control cells (Fig. 2A). However, no sustained effect of IFN was observed at 48 h p.i. for the NA strains, which produced similar virus yields in the treated and sham-treated cells (P > 0.05). In contrast, the yield of most of the SA strains remained suppressed at 48 h in IFN-treated cells compared to the control group (P < 0.001), except for strains BeAr300851 and TVP 8512, which exhibited little or no yield reduction like the NA strains (Fig. 2B). Similarly, at 72 h p.i., no difference in virus yield was observed in IFN--treated cells versus sham-treated cells infected with NA strains. In contrast, yields of most of the SA strains (with the exception of strains BeAr300851 and TVP 8512) remained lower in IFN-treated cells than in sham-treated control cells (Fig. 2C).
These results demonstrated that NA strains were generally more resistant than most SA strains to the antiviral effects of IFN-.
Sensitivity to human IFN- in vitro. When Vero cells were treated with 25 U of IFN-, similar results to those obtained with IFN- were observed (Fig. 3). At 24 h p.i., both NA and SA strain viral yields were suppressed 10 to 26-fold by IFN treatment (Fig. 3A). At 48 h p.i. (Fig. 3B), viral yields were ca. 10- to 11-fold lower in IFN-treated cells infected with NA strains compared to the sham-treated control cells (P < 0.05), whereas yields of the SA strains were reduced even more (18- to 38-fold) (P < 0.001). By 72 h p.i., reductions in virus yield in IFN-treated cells were only two- to eightfold compared to sham-treated cells infected with NA strains. In contrast, the antiviral effect lasted longer in the SA strains infections, with yields still significantly lower (10- to 30-fold) in IFN-treated cells (Fig. 3C). These results demonstrated that NA strains were generally more resistant than SA strains to the antiviral effects of IFN-.
Induction of IFN-/ in vivo. To test the hypothesis that SA EEEV strains induce higher levels of IFN-/ than NA strains, mice were used because they are the only inexpensive animal model with reagents available for manipulation of the innate immune response. Five- to six-week-old mice were infected subcutaneously with 1,000 PFU, and blood samples were taken daily to measure viremia and IFN-/ induction. IFN titers were determined by using the plaque reduction biological assay with VSV because of its reliability, economy, efficiency, and reproducibility (21). Neither NA nor SA strains induced detectable levels of IFN-/ by 24 h p.i., nor did UV-inactivated EEEV used as a control. However, at 48 h p.i. (Fig. 4A), some differences in the IFN-/ induction were observed among EEEV strains. Animals infected with SA strains induced variable amounts of IFN-/, ranging from 70 to 110 U/ml, whereas animals infected with NA strains induced 45 to 160 U/ml. A significant difference in IFN induction was observed between the NA strain 792138, which induced the lowest IFN levels, and the other EEEV strains (P < 0.05); however, no significant difference was observed when the other EEEV strains were compared. No significant differences in IFN induction were observed when the IFN levels were averaged and compared between NA and SA strains (mean of 87.2 for NA strains versus 88.9 for SA strains). At 72 h p.i., the levels of IFN-/ rapidly declined in all animals (Fig. 4B), with no significant differences among EEEV strains. The relative inability of NA strain 792138 to induce IFN-/ was not due to poor viral replication in the mice because all animals became viremic after infection with titers in the range of those observed for the other EEEV strains (Fig. 4C). In summary, these results indicated that IFN-/ induction in the mouse model does not appear to correlate with EEEV strain human virulence.
IFN sensitivity in vivo. To determine whether SA strains of EEEV are more sensitive to IFN than NA strains in vivo, adult mice were pretreated with several doses of poly(I-C), a potent IFN inducer and immune-modulator known to protect against viral infections (33, 39, 58). Treatment with poly(I-C) is also simpler to standardize than direct IFN treatment and less expensive, permitting larger sample sizes and greater statistical power. At 24 h posttreatment with 0.5 to 100 μg, animals were infected with SA and NA EEEV strains, and clinical signs of infection and mortality were monitored daily. All animals treated with 10 μg of poly(I-C) survived EEEV infection regardless of the strain used. When animals were pretreated with 0.5 μg of poly(I-C), some differences in survival were observed after infection with different EEEV strains (Fig. 5). No significant difference in survival was observed between mice infected with the NA strain 792138 and the SA strain GML903836 compared to sham-treated controls. However, treated mice infected with the NA strain FL93-939 and the SA strain BeAr300851 had significant increases in survival compared to control groups (P < 0.05). Interestingly, these strains induced the highest IFN levels in the animals (160 and 110 U/ml, respectively), suggesting an additive interaction between transcription factors activated by poly(I-C) and by virus-induced IFN.
When poly(I-C) treatment was increased to 1 to 4 μg, a dose-dependent protective effect was observed in most experiments. Mice treated with 1 μg of poly(I-C) and infected with the NA strain 792138 did not show any significant difference in survival compared to the control mice. In contrast, animals treated with the same poly(I-C) dose and infected with other EEEV strains showed a delay in the disease progression and a reduced mortality compared to the control groups (P < 0.05).
To determine more specifically the role of IFN in protection against EEEV infection, mice were treated with 1 μg of poly(I-C), along with simultaneous intraperitoneal administration of 2,000 U of antibodies to murine IFN-, IFN-, and IFN-. Controls included animals treated with antibodies alone. After 24 h, the animals were infected with a NA or SA EEEV strain. No significant differences in survival were observed between the sham control, poly(I-C) plus antibodies, and antibody-treated cohorts infected with NA and SA strains, suggesting that the protection observed after poly(I-C) treatment alone was IFN mediated. When the concentrations of poly(I-C) and antibodies were increased to 4 μg and 20,000 U, respectively, a more rapid mortality was observed in both antibody and antibody plus poly(I-C)-treated groups compared to controls. These results indicated that treatment of mice with large doses of antibodies that neutralize IFN can decrease resistance to EEEV (Fig. 6), again suggesting the importance of IFN in survival after EEEV infection.
Infection of IFN-/ receptor-deficient mice. To evaluate EEEV-induced viremia and mortality in animals with a defect in the IFN response, a cohort of 10 129 SV/EV IFN-/ receptor-deficient (or knockout [KO]) mice and age-matched, congenic wild-type controls were subcutaneously infected with 1,000 PFU of various strains. Figure 7 summarizes the viremia and survival results. Most EEEV strains produced higher titer viremia at some time points and faster mortality in the IFN-/ receptor-deficient than in wild-type mice. The viremia levels in the IFN-/ receptor-deficient mice were significantly higher than in wild-type mice for NA strain 792138 (P < 0.05) and SA strains GML903836 and BeAr300851 (P < 0.001; Fig. 7), and survival of the KO mice was significantly shorter following infection with strains 792138 and BeAr300851 (P < 0.05). However, the IFN-/ receptor deficiency had no major effect on survival after infection with SA strain GML. The IFN-/ receptor-deficient mice infected with the NA strain FL93-939 did not show any appreciable difference in viremia titers (Fig. 7C), and no major effect on survival compared to the wild-type mice (Fig. 7D). Overall, these results suggested that mice lacking a functional IFN-/ response are impaired to various degrees in their ability to clear EEEV and generally succumb faster to fatal disease (Fig. 7A).
Infection of IFN- receptor-deficient mice. To evaluate the effects on viremia, disease progression, and mortality in animals with a defect in the IFN- response, three 129 SV/EV IFN- receptor-deficient (KO) mice were subcutaneously infected with 1,000 PFU of the NA strain 792138 or SA strain GML903836 (only two EEEV strains were used due to the limitation in KO mouse availability), and the viremia and mortality compared to the results obtained with the wild-type group (n = 10). No significant differences in viremia titers or mortality were observed between IFN- receptor-deficient and wild-type mice infected with either NA or SA strains (Fig. 8). However, the IFN- receptor-deficient mice exhibited slightly shorter survival (NA strain MST of 7 days versus 6 days for wild-type mice and of 7 days versus 8.5 days for the SA strain). These results suggested that IFN- plays a smaller role than IFN-/ in vivo in protecting against fatal EEE.
DISCUSSION
Sensitivity to IFNs. To explain the difference in human virulence between NA and SA strains of EEEV we tested the hypothesis that innate immune mechanisms are more effective in controlling infection by SA than NA strains by evaluating sensitivity to and induction of IFN by using human/primate and murine systems. When we evaluated EEEV sensitivity to human IFN-/ and IFN-, our data indicated that NA strains are generally more resistant. Interestingly, the yields of both NA and SA strains were inhibited in the presence of IFN-, -, and - at 24 h p.i.. However, at 48 and 72 h, NA strains reached similar titers in IFN-treated and sham-treated controls, whereas yields of SA strains remained depressed in the treated cultures. Because IFN was replaced daily, these differences are most likely due to the greater ability of the NA strains to counteract the inhibitory effects of IFN on virus replication rather than to IFN degradation. Previous studies have shown that IFN-/ is responsible for protection against fatal alphavirus infection (25, 29, 34, 41, 43, 57) and our in vitro results support these conclusions. IFN- has at least 23 different variants, and further studies are needed to determine which are important in protection.
In contrast to IFN-/, IFN- is believed to have a lesser role in protecting against alphavirus infection. This conclusion was supported by studies with IFN- receptor-deficient mice, which failed to show differences in disease or survival compared to normal animals infected with Semliki Forest virus (SFV) (36). However, other studies demonstrated that IFN- increases the expression of major histocompatibility complex class I and class II antigens in primary brain cells infected with SFV (49). The SFV infection itself does not augment class I antigen presentation, nor does it affect levels of expression in IFN-treated cells. More recent studies demonstrated that recovery from viral encephalitis involves immune-mediated, noncytolytic clearance from neurons, an activity that requires both CD4+ and CD8+ T cells, and that T cells mainly require IFN- for clearing virus from certain neuron populations (7). Our in vitro results also suggest that IFN- plays a role in modulating EEEV replication, but our studies with the IFN- receptor-deficient mice revealed little difference in viremia or survival compared to normal animals. Further studies with EEEV are necessary to determine whether a defect in the IFN- response has any effect on virus replication in other target organs, such as the brain, before ruling out any role in protection in vivo.
Although our in vitro results suggest a difference in sensitivity to human IFN between NA and SA strains, the IFN-/ receptor KO mice experiments did not corroborate the in vitro results and raised additional questions about the role of IFN in protection. IFN response-deficient mice succumbed more rapidly to infection with some but not all NA and SA EEEV strains. Larger studies with more mice are needed to increase the statistical power of detecting minor viremia and survival differences suggested by some experiments.
Induction of IFNs. We also analyzed the possibility that NA versus SA EEEV strains differ in their ability to induce IFN in vivo. In mice, we observed low and variable levels of IFN induction; NA strain 792138 induced significantly lower levels of IFN than did all other EEEV strains we tested. However, NA strain FL93-939 induced the highest IFN levels, suggesting that IFN induction is an unlikely explanation for the human virulence difference between NA and SA strains. The NA strains are highly conserved genetically (9, 56), and thus the dramatic difference in IFN induction observed between two NA strains could be exploited to investigate the nucleotide and/or amino acid differences responsible for the IFN induction phenotypes. When compared to murine IFN induction by other alphaviruses such as VEEV (26, 57), EEEV induced 200-fold-lower levels, suggesting that it is a generally poor IFN inducer or is highly efficient in suppressing induction. Another explanation could be that replication of EEEV is lower relative to VEEV, reducing the quantity of the inducer. These findings should be pursued in primate systems to investigate their possible importance in human EEE pathogenesis.
We also demonstrated that artificial IFN induction prior to infection can protect mice in a dose-dependent manner against EEE. Although poly(I-C) conferred up to 100% protection when administered prior to infection, its effective use as a therapeutic agent (or IFN treatment) is unlikely because it is only effective against alphaviral disease when administered before or soon after infection (8, 22, 59, 60). Early treatment is generally impossible because the alphavirus incubation period is usually 24 h or longer. However, based on previous studies (17), combination treatments of poly(I-C) and EEEV antibodies might protect against alphaviral disease even if administered after the virus reaches the brain and therefore should be tested with EEEV.
When the combined results of the IFN induction experiment and the IFN-/ receptor KO mice infections were analyzed, three distinct patterns were observed. (i) NA strain FL93-939 induced the highest levels of IFN-/, but IFN-/-deficient mice infected with this strain had little or no difference in viremia or mortality compared to wild-type mice. These results suggested that IFN-/ plays little or no role in protection against this strain. (ii) NA strain 792138 induced the lowest IFN levels, and the differences in viremia and mortality between KO and wild-type mice infected were the most pronounced. This suggests that weak IFN induction and/or IFN suppression enhanced viral replication and EEE pathogenesis, and that IFN-/ plays a significant role. (iii) The SA strains induced higher levels of IFN, and significant differences in viremia and in some cases mortality were observed between IFN-/ KO and wild-type mice, suggesting that IFN plays an important role in protection against SA viruses.
Studies with other alphaviruses such as VEEV, SINV, and SFV (20, 26, 41, 57) reported earlier (usually 2 to 3 days) mortality in IFN-/ KO compared to wild-type mice. In our study the MSTs of KO mice were 4 to 7 days, suggesting that EEEV may be less sensitive than other alphaviruses to the antiviral action of IFN-/. Differences in the ages of the animals used in our studies (10 to 13 weeks) compared to those used in others (5 to 7 weeks) may also have contributed to these differences.
In contrast to our results with IFN-/ KO mice, in which differences in viremia levels and survival were observed compared to wild types, IFN- receptor KO mice did not show any major difference in viremia or mortality compared to wild-type mice, supporting the conclusions of previous studies that showed no difference in disease outcome in IFN- receptor-deficient mice infected with SFV (36). However, the small number of animals used in our experiment may have limited statistical power, and additional experiments with more animals are needed to fully elucidate the role of IFN- in controlling murine EEE.
Although some differences in human IFN sensitivity in vitro were observed between NA and SA EEEV strains, these differences were not evident in our animal studies, perhaps due to the fact that mice are similarly susceptible to both NA and SA strains. This deficiency of the murine model represents the main limitation of our study, and future research should evaluate differences in neuroinvasion and central nervous system replication levels between the strains that might yield results more consistent with human infection.
Determinants of EEEV strain virulence. The genetic determinants that confer high human IFN sensitivity phenotypes exhibited by SA strains of EEEV are unknown. The NA and SA strains differ by ca. 20 to 29% in their nucleotide sequences and by ca. 5 to 10% in structural protein amino acids (9). The 5' untranslated genome region and glycoproteins are determinants of IFN resistance by VEEV (46, 57), and mutations in nsP2 of SINV confer IFN sensitivity and enhance induction (23). A single amino acid mutation in nsP2 of SFV disrupts its nuclear localization and results in widespread distribution of the virus in the brain of IFN-/-deficient mice (20). Whether these genes or cis-acting sequences are determinants of IFN sensitivity and induction by EEEV deserves further study.
Currently, an inactivated EEEV vaccine is available only for researchers and military personnel, and a similar vaccine is administered to equines and other domestic animals. These vaccines are poorly immunogenic and require frequent boosters. Due to the potential use of EEEV as a biological weapon (27), a more effective vaccine is needed. Identification of the genetic determinants of human IFN sensitivity by SA strains of EEEV could be exploited in the development of a live-attenuated vaccine.
ACKNOWLEDGMENTS
We thank Mardelle Susman for editing the manuscript. We thank the National Institute of Health (NIAID Reference Reagent Repository) for providing the IFN reagents.
P.V.A. was supported by the James W. McLaughlin Fellowship Fund. S.P. was supported by NIH K08 grant AI059491. This research was also supported by the Region VI Center for Biodefense and Emerging Infections (NIH grant U54 AI 57156) and by NIH contract NOI-AI30027.
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Department of Microbiology and Immunology
Department of Pathology, University of Texas Medical Branch, Galveston, Texas 77555-0609
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-, -, and - 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-/ receptors than in wild-type mice, suggesting that IFN-/ is important in controlling replication. In contrast, IFN- 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- 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.
INTRODUCTION
Eastern equine encephalitis virus (EEEV) is an arthropod-borne virus (arbovirus) in the family Togaviridae, genus Alphavirus. The virus possesses a single-stranded, messenger-sense RNA genome of ca. 11.7 kb that encodes four nonstructural proteins (nsP1 to -4) and three main structural proteins (capsid and glycoproteins E1 and E2) (47). The virus is an important veterinary and human pathogen and causes periodic outbreaks in the Americas associated with high morbidity and mortality among humans and equines. In humans, EEEV infection is characterized mainly by fever, lethargy, vomiting, convulsions, and coma. Surviving patients, especially children, are often profoundly affected by neurological and physical sequelae that include mental retardation, hemiparesis, aphasia, emotional instability, convulsions, hemiplegia, strabismus, impaired vision, partial deafness, and speech disorders (45). In equines, pheasants, chickens, sandhill cranes, emus, and pigs, EEEV infection also causes severe and often fatal disease (54).
EEEV was first isolated in the United States from horses during an epizootic in coastal regions of Virginia, Maryland, and Delaware in 1933 (5, 48) and in South America (SA) in 1930 from a horse in Argentina (40). In North America (NA), the enzootic transmission cycle occurs in freshwater swamp foci and involves passerine birds and the ornithophilic mosquito vector, Culiseta melanura. Sporadic epizootic or epidemic transmission involves spillover from the avian swamp cycle and is probably mediated by bridge vectors such as Aedes canadensis, Aedes sollicitans, Aedes vexans, and Coquillettidia perturbans (24, 35, 54). Enzootic transmission in Central and SA is not well understood but probably involves mosquitoes in the Culex (Melanoconion) subgenus and rodent and/or avian reservoir hosts (44, 54).
Antigenic studies have shown that NA strains of EEEV differ from those found in SA and that these two geographic variants also differ in biological and epidemiological characteristics (12-14, 38). Genetic and antigenic studies (9, 38, 55) revealed that NA strains are highly conserved, constituting a unique major lineage, whereas SA strains constitute three major lineages with more antigenic and genetic variation. All strains isolated in NA and most from the Caribbean belong to the NA subtype (lineage I), whereas isolates from Central and SA constitute three SA subtypes (lineages II to IV) (9, 11, 14). Importantly, the NA subtype causes severe disease in both humans and equines, with mortality rates of 30 to 80% in humans and 90 to 95% in equines. In contrast, infection by the SA EEEV subtypes can be fatal in equines, but human infections have rarely been recognized and in most cases were identified only by serosurveys with no evidence of disease. Causey and Theiler (16) demonstrated seroprevalence as high as 25% in children and 31% in adults living in the Amazon basin, and a single human encephalitis case was also reported in Brazil (1). Although EEEV was implicated in equine epizootics in Braganca, Para State, no neurological disease in humans was reported during these outbreaks (15, 50, 53). The EEEV was also isolated repeatedly in Argentina from horses between 1930 and 1958. However, no human neurological disease was reported despite active surveillance and human seroprevalence levels of up to 66% in some locations (42).
Recently, EEEV was isolated from Culex (Melanoconion) pedroi mosquitoes in Puerto Almendras, Iquitos, Peru (M. Turell, USAMRIID [unpublished data]), providing evidence that EEEV circulates throughout the Amazon basin. However, again, no fatal or neurologic human disease associated with EEEV has been recognized in Peru despite active surveillance there for febrile diseases. These epidemiological studies provide strong evidence that, unlike their NA counterparts, SA strains of EEEV generally do not produce severe disease but can infect humans. The reason for this apparent difference in human virulence is unknown.
Interferons (IFNs) have long been recognized as essential components of the innate immune response to viral infections (3, 4, 30). Specifically, IFN- and - are known to be important in protecting against alphaviral disease (26, 46). Previous studies showed a correlation between alphavirus virulence and resistance to IFN- and -. Some studies with Venezuelan equine encephalitis virus (VEEV) suggested that the IFN- and - resistance or sensitivity phenotype correlated with epizootic potential and equine virulence (31, 46), although others observed little or no difference (2). Although an important role of IFN-/ in the pathogenesis of several alphaviruses, including Sindbis virus (SINV) and Semliki Forest virus (29, 36, 41), has been described, little information is available regarding the role of IFN-, -, and especially of IFN- in the pathogenesis of other alphaviruses, including EEEV. We therefore hypothesized that NA strains of EEEV are more virulent for humans because of a greater resistance to IFN than SA strains and/or because they induce lower levels of IFN. To test these hypotheses, the effects of IFN on EEEV replication were evaluated in Vero cells treated with human IFN-/ and -. Vero cells were chosen because they respond efficiently to human IFN but are unable to produce IFN, eliminating any possibility of autocrine or paracrine effects. For the in vivo studies, the mouse model was used because it allowed us to study the effect of ablating the IFN response on EEEV infection and disease outcome.
MATERIALS AND METHODS
Viruses. Virus isolates (Table 1) were provided by the University of Texas Medical Branch World Reference Center for Emerging Viruses and Arboviruses. All strains were isolated in Vero (African green monkey kidney) cells from sentinel hamsters, monkeys, or mosquitoes and were chosen due to their low-passage histories. Stocks were prepared in mice to avoid selection for attenuated alphavirus mutants that occur after passage in cells expressing glycosaminoglycans (6, 10, 28, 32). One- to three-day-old mice were inoculated intracranially with each virus strain, and a 10% brain suspension was prepared after morbidity or mortality was observed. Virus stock titers were determined by plaque assay in Vero cells. The nucleotide sequence differences among SA strains range from ca. 1 to 2% (e.g., GML903836 versus C-49; all NA strains) to 5% (BeAn5122 versus BeAr300851) for members of the same lineage. All SA strains differed from the NA strains by 18 to 23% at the nucleotide and 5 to 10% at the amino acid level.
Previous studies showed that the amount of IFN induced in the sera and brains of neonatal mice infected with alphaviruses is on the order of 20,000 to 215,000 IU/ml (51, 52). Therefore, to avoid possible confounding effects of IFN in the homogenized brain tissue, virus stocks were diluted ca. 1,000,000-fold in all experiments.
IFN sensitivity assays. Human IFN preparations were kindly provided by the National Institutes of Health, Bethesda, Md. Both human IFN- and IFN- were obtained from human cells by induction with Sendai virus or treatment with polyinosinic polycytidylic acid [poly(I-C)], whereas the human IFN- was a recombinant preparation. Vero cells were seeded in 12-well plates, and 2 days later were pretreated with human IFN-, -, or - and incubated for 24 h. Culture media were then removed, the cells washed three times with phosphate-buffered saline (PBS), and 1,000 PFU of EEEV were added to each well. Negative controls consisted of infected cells not exposed to IFN. At 24, 48, and 72 h postinfection (p.i.), the supernatant fluid was completely removed, the cells were washed twice with PBS, and new media containing the appropriate amount of IFN were added. Virus in the supernatant fluid at selected times was titrated on Vero cells by plaque assay.
Infection of mice. Five- to six-week-old NIH Swiss mice (Harlan Laboratories, Indianapolis, Ind.) were maintained under specific-pathogen-free conditions. The animals were allowed to acclimate to the laboratory for 1 week and then were placed into cohorts of five for subcutaneous infection with 1,000 PFU of EEEV. The animals were monitored daily for clinical signs, including lethargy, paralysis, or death and bled at 24, 48, 72, and 96 h p.i. to determine viremia levels by plaque assay and IFN production.
IFN assays. To quantify levels of IFN-/ produced after EEEV infection of mice, a vesicular stomatitis virus (VSV) plaque reduction assay was used. Serum was exposed to UV light to inactivate EEEV and inactivation was confirmed by a cell culture assay. Serially diluted sera were applied to L929 cells grown in 96-well plates. Controls included the addition of known amounts of mouse IFN-, -, and - (obtained from murine cells induced with Newcastle disease virus) instead of serum samples, and UV-inactivated EEEV was used to determine whether virus particles alone could induce IFN. After a 24-h incubation, cells were washed three times with PBS, and 50 PFU of VSV were added to each well. Negative controls included mock-treated cells infected with VSV. After 1 h of incubation, 1% methylcellulose in minimal essential medium was added. When plaques were visible, the cells were stained with 1% crystal violet and the IFN- and/or IFN- titer was calculated as the highest serum dilution that inhibited 50% of VSV plaques.
Poly(I-C) treatment. Five- to six-week-old NIH Swiss mice were placed into cohorts of five, and two groups were each pretreated intraperitoneally with 0.5, 1, 2, 4, 10, 65, or 100 μg of poly(I-C) (Amersham Biosciences, NJ), a synthetic, double-stranded RNA that induces IFN by mimicking the genetic material of some viruses or the by-products of some viral infections. Control animals were mock treated with diluent. At 24 h posttreatment, animals were inoculated subcutaneously with 1,000 PFU of EEEV and monitored daily for up to a month for clinical signs of illness and mortality. To confirm that the protection observed with the poly(I-C) treatment was IFN-mediated, mice were treated intraperitoneally with 1 μg of poly(I-C) along with 2,000 U of antibodies to murine IFN-, IFN-, and IFN- (NIH) and with 4 μg of poly(I-C) plus 50,000 U of antibodies. Controls included animals treated with antibodies alone. After 24 h, animals were infected with the NA strain 792138 and the SA strain GML903836, and mortality was recorded daily.
Infection of IFN-/ and IFN- receptor-deficient mice. Ten- to thirteen-week-old strain 129 Sv/Ev (wild-type) mice were purchased from Jackson Laboratories (Bar Harbor, ME), and breeding pairs of the 129 Sv/Ev IFN-/ receptor –/– mice were generously provided by Herbert Virgin (Washington University, St. Louis, MO) and allowed to breed under pathogen-free conditions. Strain 129 Sv/Ev IFN- receptor –/– mice were purchased from Jackson Laboratories. After 1 week of acclimation to the laboratory, mice were subcutaneously inoculated with 1,000 PFU of EEEV and bled 8, 24, 32, 48, 56, 72, and 96 h p.i. for viremia determination. The animals were observed daily for up to a month for clinical signs of illness and mortality, and the median survival time (MST) was determined.
Statistical analysis. Statistical comparisons were performed by using a one-way analysis of variance with Dunn's and Tukey's multiple comparison test to identify significant differences among samples. For survival comparisons, the log-rank test or Mantel-Haenszel test (two-tailed P values) included in the GraphPad Prism program (San Diego, CA) was used. Medians were used to present some data because they are less sensitive to extreme values than are means (37). The MST was calculated as the time at which 50% of the animals succumbed to infection. If the survival curve was horizontal at 50% survival, the MST was calculated as the average of the first and last times at which survival was 50%. If the survival exceeds 50% at the longest time point, then the median survival was not computed. P values of 0.05 were considered significant.
RESULTS
To determine the potential role of IFNs in limiting EEEV infection and to evaluate the hypothesis that NA strains are more virulent for humans because of a greater resistance to IFNs than SA strains, Vero cells were treated with human IFNs. Previous studies have shown that Vero cells respond efficiently to human IFNs but are unable to produce IFN (18, 21), thereby eliminating the confounding effects of autocrine and/or paracrine responses (19).
Sensitivity to human IFN- in vitro. Vero cells were pretreated for 24 h with 50 IU of human IFN-/ml prior to infection with EEEV strains at a multiplicity of infection (MOI) of 0.1. At 24 and 48 h p.i., yields of both NA and SA strains were 10 to 25-fold lower in the IFN-treated cells than in the sham-treated control cells (Fig. 1A) (P < 0.001). At 48 h p.i., the viral yields for the SA strains in the treated cells were again significantly lower (ca. 10- to 25-fold than in control cells that produced in average 7.6 log10 PFU of virus/ml) (P < 0.001). In contrast, viral yields for the NA strains were similar in the treated and sham-treated cells, with viral yields of 8.35 log10 PFU/ml (0-5-fold reduction) (P > 0.05), indicating that IFN- did not have a major effect on the yield of the NA strains at the later time points (Fig. 1B). At 72 h p.i., the yields of SA strains in IFN-treated cells were still lower (10- to 18-fold) than in sham-treated cells (average viral yields 6.5 log10 PFU/ml). In contrast, IFN treatment had no significant effect on viral yields by the NA strains at this time point (Fig. 1C). These results suggested that SA strains were more sensitive to IFN- than the NA strains of EEEV.
Sensitivity to human IFN- in vitro. Differences in sensitivity to 5 U of human IFN- were also detected among EEEV strains. At 24 h p.i., the yield of both NA and SA strains was 8- to 24-fold lower in cells treated with IFN than in sham-treated control cells (Fig. 2A). However, no sustained effect of IFN was observed at 48 h p.i. for the NA strains, which produced similar virus yields in the treated and sham-treated cells (P > 0.05). In contrast, the yield of most of the SA strains remained suppressed at 48 h in IFN-treated cells compared to the control group (P < 0.001), except for strains BeAr300851 and TVP 8512, which exhibited little or no yield reduction like the NA strains (Fig. 2B). Similarly, at 72 h p.i., no difference in virus yield was observed in IFN--treated cells versus sham-treated cells infected with NA strains. In contrast, yields of most of the SA strains (with the exception of strains BeAr300851 and TVP 8512) remained lower in IFN-treated cells than in sham-treated control cells (Fig. 2C).
These results demonstrated that NA strains were generally more resistant than most SA strains to the antiviral effects of IFN-.
Sensitivity to human IFN- in vitro. When Vero cells were treated with 25 U of IFN-, similar results to those obtained with IFN- were observed (Fig. 3). At 24 h p.i., both NA and SA strain viral yields were suppressed 10 to 26-fold by IFN treatment (Fig. 3A). At 48 h p.i. (Fig. 3B), viral yields were ca. 10- to 11-fold lower in IFN-treated cells infected with NA strains compared to the sham-treated control cells (P < 0.05), whereas yields of the SA strains were reduced even more (18- to 38-fold) (P < 0.001). By 72 h p.i., reductions in virus yield in IFN-treated cells were only two- to eightfold compared to sham-treated cells infected with NA strains. In contrast, the antiviral effect lasted longer in the SA strains infections, with yields still significantly lower (10- to 30-fold) in IFN-treated cells (Fig. 3C). These results demonstrated that NA strains were generally more resistant than SA strains to the antiviral effects of IFN-.
Induction of IFN-/ in vivo. To test the hypothesis that SA EEEV strains induce higher levels of IFN-/ than NA strains, mice were used because they are the only inexpensive animal model with reagents available for manipulation of the innate immune response. Five- to six-week-old mice were infected subcutaneously with 1,000 PFU, and blood samples were taken daily to measure viremia and IFN-/ induction. IFN titers were determined by using the plaque reduction biological assay with VSV because of its reliability, economy, efficiency, and reproducibility (21). Neither NA nor SA strains induced detectable levels of IFN-/ by 24 h p.i., nor did UV-inactivated EEEV used as a control. However, at 48 h p.i. (Fig. 4A), some differences in the IFN-/ induction were observed among EEEV strains. Animals infected with SA strains induced variable amounts of IFN-/, ranging from 70 to 110 U/ml, whereas animals infected with NA strains induced 45 to 160 U/ml. A significant difference in IFN induction was observed between the NA strain 792138, which induced the lowest IFN levels, and the other EEEV strains (P < 0.05); however, no significant difference was observed when the other EEEV strains were compared. No significant differences in IFN induction were observed when the IFN levels were averaged and compared between NA and SA strains (mean of 87.2 for NA strains versus 88.9 for SA strains). At 72 h p.i., the levels of IFN-/ rapidly declined in all animals (Fig. 4B), with no significant differences among EEEV strains. The relative inability of NA strain 792138 to induce IFN-/ was not due to poor viral replication in the mice because all animals became viremic after infection with titers in the range of those observed for the other EEEV strains (Fig. 4C). In summary, these results indicated that IFN-/ induction in the mouse model does not appear to correlate with EEEV strain human virulence.
IFN sensitivity in vivo. To determine whether SA strains of EEEV are more sensitive to IFN than NA strains in vivo, adult mice were pretreated with several doses of poly(I-C), a potent IFN inducer and immune-modulator known to protect against viral infections (33, 39, 58). Treatment with poly(I-C) is also simpler to standardize than direct IFN treatment and less expensive, permitting larger sample sizes and greater statistical power. At 24 h posttreatment with 0.5 to 100 μg, animals were infected with SA and NA EEEV strains, and clinical signs of infection and mortality were monitored daily. All animals treated with 10 μg of poly(I-C) survived EEEV infection regardless of the strain used. When animals were pretreated with 0.5 μg of poly(I-C), some differences in survival were observed after infection with different EEEV strains (Fig. 5). No significant difference in survival was observed between mice infected with the NA strain 792138 and the SA strain GML903836 compared to sham-treated controls. However, treated mice infected with the NA strain FL93-939 and the SA strain BeAr300851 had significant increases in survival compared to control groups (P < 0.05). Interestingly, these strains induced the highest IFN levels in the animals (160 and 110 U/ml, respectively), suggesting an additive interaction between transcription factors activated by poly(I-C) and by virus-induced IFN.
When poly(I-C) treatment was increased to 1 to 4 μg, a dose-dependent protective effect was observed in most experiments. Mice treated with 1 μg of poly(I-C) and infected with the NA strain 792138 did not show any significant difference in survival compared to the control mice. In contrast, animals treated with the same poly(I-C) dose and infected with other EEEV strains showed a delay in the disease progression and a reduced mortality compared to the control groups (P < 0.05).
To determine more specifically the role of IFN in protection against EEEV infection, mice were treated with 1 μg of poly(I-C), along with simultaneous intraperitoneal administration of 2,000 U of antibodies to murine IFN-, IFN-, and IFN-. Controls included animals treated with antibodies alone. After 24 h, the animals were infected with a NA or SA EEEV strain. No significant differences in survival were observed between the sham control, poly(I-C) plus antibodies, and antibody-treated cohorts infected with NA and SA strains, suggesting that the protection observed after poly(I-C) treatment alone was IFN mediated. When the concentrations of poly(I-C) and antibodies were increased to 4 μg and 20,000 U, respectively, a more rapid mortality was observed in both antibody and antibody plus poly(I-C)-treated groups compared to controls. These results indicated that treatment of mice with large doses of antibodies that neutralize IFN can decrease resistance to EEEV (Fig. 6), again suggesting the importance of IFN in survival after EEEV infection.
Infection of IFN-/ receptor-deficient mice. To evaluate EEEV-induced viremia and mortality in animals with a defect in the IFN response, a cohort of 10 129 SV/EV IFN-/ receptor-deficient (or knockout [KO]) mice and age-matched, congenic wild-type controls were subcutaneously infected with 1,000 PFU of various strains. Figure 7 summarizes the viremia and survival results. Most EEEV strains produced higher titer viremia at some time points and faster mortality in the IFN-/ receptor-deficient than in wild-type mice. The viremia levels in the IFN-/ receptor-deficient mice were significantly higher than in wild-type mice for NA strain 792138 (P < 0.05) and SA strains GML903836 and BeAr300851 (P < 0.001; Fig. 7), and survival of the KO mice was significantly shorter following infection with strains 792138 and BeAr300851 (P < 0.05). However, the IFN-/ receptor deficiency had no major effect on survival after infection with SA strain GML. The IFN-/ receptor-deficient mice infected with the NA strain FL93-939 did not show any appreciable difference in viremia titers (Fig. 7C), and no major effect on survival compared to the wild-type mice (Fig. 7D). Overall, these results suggested that mice lacking a functional IFN-/ response are impaired to various degrees in their ability to clear EEEV and generally succumb faster to fatal disease (Fig. 7A).
Infection of IFN- receptor-deficient mice. To evaluate the effects on viremia, disease progression, and mortality in animals with a defect in the IFN- response, three 129 SV/EV IFN- receptor-deficient (KO) mice were subcutaneously infected with 1,000 PFU of the NA strain 792138 or SA strain GML903836 (only two EEEV strains were used due to the limitation in KO mouse availability), and the viremia and mortality compared to the results obtained with the wild-type group (n = 10). No significant differences in viremia titers or mortality were observed between IFN- receptor-deficient and wild-type mice infected with either NA or SA strains (Fig. 8). However, the IFN- receptor-deficient mice exhibited slightly shorter survival (NA strain MST of 7 days versus 6 days for wild-type mice and of 7 days versus 8.5 days for the SA strain). These results suggested that IFN- plays a smaller role than IFN-/ in vivo in protecting against fatal EEE.
DISCUSSION
Sensitivity to IFNs. To explain the difference in human virulence between NA and SA strains of EEEV we tested the hypothesis that innate immune mechanisms are more effective in controlling infection by SA than NA strains by evaluating sensitivity to and induction of IFN by using human/primate and murine systems. When we evaluated EEEV sensitivity to human IFN-/ and IFN-, our data indicated that NA strains are generally more resistant. Interestingly, the yields of both NA and SA strains were inhibited in the presence of IFN-, -, and - at 24 h p.i.. However, at 48 and 72 h, NA strains reached similar titers in IFN-treated and sham-treated controls, whereas yields of SA strains remained depressed in the treated cultures. Because IFN was replaced daily, these differences are most likely due to the greater ability of the NA strains to counteract the inhibitory effects of IFN on virus replication rather than to IFN degradation. Previous studies have shown that IFN-/ is responsible for protection against fatal alphavirus infection (25, 29, 34, 41, 43, 57) and our in vitro results support these conclusions. IFN- has at least 23 different variants, and further studies are needed to determine which are important in protection.
In contrast to IFN-/, IFN- is believed to have a lesser role in protecting against alphavirus infection. This conclusion was supported by studies with IFN- receptor-deficient mice, which failed to show differences in disease or survival compared to normal animals infected with Semliki Forest virus (SFV) (36). However, other studies demonstrated that IFN- increases the expression of major histocompatibility complex class I and class II antigens in primary brain cells infected with SFV (49). The SFV infection itself does not augment class I antigen presentation, nor does it affect levels of expression in IFN-treated cells. More recent studies demonstrated that recovery from viral encephalitis involves immune-mediated, noncytolytic clearance from neurons, an activity that requires both CD4+ and CD8+ T cells, and that T cells mainly require IFN- for clearing virus from certain neuron populations (7). Our in vitro results also suggest that IFN- plays a role in modulating EEEV replication, but our studies with the IFN- receptor-deficient mice revealed little difference in viremia or survival compared to normal animals. Further studies with EEEV are necessary to determine whether a defect in the IFN- response has any effect on virus replication in other target organs, such as the brain, before ruling out any role in protection in vivo.
Although our in vitro results suggest a difference in sensitivity to human IFN between NA and SA strains, the IFN-/ receptor KO mice experiments did not corroborate the in vitro results and raised additional questions about the role of IFN in protection. IFN response-deficient mice succumbed more rapidly to infection with some but not all NA and SA EEEV strains. Larger studies with more mice are needed to increase the statistical power of detecting minor viremia and survival differences suggested by some experiments.
Induction of IFNs. We also analyzed the possibility that NA versus SA EEEV strains differ in their ability to induce IFN in vivo. In mice, we observed low and variable levels of IFN induction; NA strain 792138 induced significantly lower levels of IFN than did all other EEEV strains we tested. However, NA strain FL93-939 induced the highest IFN levels, suggesting that IFN induction is an unlikely explanation for the human virulence difference between NA and SA strains. The NA strains are highly conserved genetically (9, 56), and thus the dramatic difference in IFN induction observed between two NA strains could be exploited to investigate the nucleotide and/or amino acid differences responsible for the IFN induction phenotypes. When compared to murine IFN induction by other alphaviruses such as VEEV (26, 57), EEEV induced 200-fold-lower levels, suggesting that it is a generally poor IFN inducer or is highly efficient in suppressing induction. Another explanation could be that replication of EEEV is lower relative to VEEV, reducing the quantity of the inducer. These findings should be pursued in primate systems to investigate their possible importance in human EEE pathogenesis.
We also demonstrated that artificial IFN induction prior to infection can protect mice in a dose-dependent manner against EEE. Although poly(I-C) conferred up to 100% protection when administered prior to infection, its effective use as a therapeutic agent (or IFN treatment) is unlikely because it is only effective against alphaviral disease when administered before or soon after infection (8, 22, 59, 60). Early treatment is generally impossible because the alphavirus incubation period is usually 24 h or longer. However, based on previous studies (17), combination treatments of poly(I-C) and EEEV antibodies might protect against alphaviral disease even if administered after the virus reaches the brain and therefore should be tested with EEEV.
When the combined results of the IFN induction experiment and the IFN-/ receptor KO mice infections were analyzed, three distinct patterns were observed. (i) NA strain FL93-939 induced the highest levels of IFN-/, but IFN-/-deficient mice infected with this strain had little or no difference in viremia or mortality compared to wild-type mice. These results suggested that IFN-/ plays little or no role in protection against this strain. (ii) NA strain 792138 induced the lowest IFN levels, and the differences in viremia and mortality between KO and wild-type mice infected were the most pronounced. This suggests that weak IFN induction and/or IFN suppression enhanced viral replication and EEE pathogenesis, and that IFN-/ plays a significant role. (iii) The SA strains induced higher levels of IFN, and significant differences in viremia and in some cases mortality were observed between IFN-/ KO and wild-type mice, suggesting that IFN plays an important role in protection against SA viruses.
Studies with other alphaviruses such as VEEV, SINV, and SFV (20, 26, 41, 57) reported earlier (usually 2 to 3 days) mortality in IFN-/ KO compared to wild-type mice. In our study the MSTs of KO mice were 4 to 7 days, suggesting that EEEV may be less sensitive than other alphaviruses to the antiviral action of IFN-/. Differences in the ages of the animals used in our studies (10 to 13 weeks) compared to those used in others (5 to 7 weeks) may also have contributed to these differences.
In contrast to our results with IFN-/ KO mice, in which differences in viremia levels and survival were observed compared to wild types, IFN- receptor KO mice did not show any major difference in viremia or mortality compared to wild-type mice, supporting the conclusions of previous studies that showed no difference in disease outcome in IFN- receptor-deficient mice infected with SFV (36). However, the small number of animals used in our experiment may have limited statistical power, and additional experiments with more animals are needed to fully elucidate the role of IFN- in controlling murine EEE.
Although some differences in human IFN sensitivity in vitro were observed between NA and SA EEEV strains, these differences were not evident in our animal studies, perhaps due to the fact that mice are similarly susceptible to both NA and SA strains. This deficiency of the murine model represents the main limitation of our study, and future research should evaluate differences in neuroinvasion and central nervous system replication levels between the strains that might yield results more consistent with human infection.
Determinants of EEEV strain virulence. The genetic determinants that confer high human IFN sensitivity phenotypes exhibited by SA strains of EEEV are unknown. The NA and SA strains differ by ca. 20 to 29% in their nucleotide sequences and by ca. 5 to 10% in structural protein amino acids (9). The 5' untranslated genome region and glycoproteins are determinants of IFN resistance by VEEV (46, 57), and mutations in nsP2 of SINV confer IFN sensitivity and enhance induction (23). A single amino acid mutation in nsP2 of SFV disrupts its nuclear localization and results in widespread distribution of the virus in the brain of IFN-/-deficient mice (20). Whether these genes or cis-acting sequences are determinants of IFN sensitivity and induction by EEEV deserves further study.
Currently, an inactivated EEEV vaccine is available only for researchers and military personnel, and a similar vaccine is administered to equines and other domestic animals. These vaccines are poorly immunogenic and require frequent boosters. Due to the potential use of EEEV as a biological weapon (27), a more effective vaccine is needed. Identification of the genetic determinants of human IFN sensitivity by SA strains of EEEV could be exploited in the development of a live-attenuated vaccine.
ACKNOWLEDGMENTS
We thank Mardelle Susman for editing the manuscript. We thank the National Institute of Health (NIAID Reference Reagent Repository) for providing the IFN reagents.
P.V.A. was supported by the James W. McLaughlin Fellowship Fund. S.P. was supported by NIH K08 grant AI059491. This research was also supported by the Region VI Center for Biodefense and Emerging Infections (NIH grant U54 AI 57156) and by NIH contract NOI-AI30027.
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