From a set of 86 candidate genes, we identified variants in three loci (IRF3
, and OAS1
) that were associated with WNV disease (). For two of these loci, IRF3
, no association with WNV disease has been reported previously in humans. OAS1
and one of its murine orthologues, Oas1b
, have been associated with modulating WNV disease in humans and mice, respectively 
Summary of significant results.
encodes a member of the interferon regulatory transcription factor family involved in the upregulation of type 1 IFN genes as well as other pathway genes. However, IRF-3 has been reported to protect mice from WNV-induced disease by both interferon-dependent and independent mechanisms 
. After a peripheral WNV infection, Irf3−/−
mice exhibited increased mortality, earlier viral entry into the CNS, and increased virus levels in the brain and spinal cord compared to wild-type mice 
mice also exhibit enhanced WNV infection in macrophages in vivo
and ex vivo 
. Increased replication of WNV in macrophages in the periphery would be expected to enhance disease symptoms consistent with the observed association of IRF3
variation with human symptomatic WNV infections. Accordingly, IRF3
is a compelling candidate for influencing the risk of symptomatic WNV infection in humans.
belongs to the MX (myxovirus resistance) family of interferon-induced proteins that are GTPases with antiviral functions 
. Upon viral infection, a host cell secretes type 1 interferons that, in turn, induce the production of MX proteins that diminish viral replication. In mice, Mx1
confers resistance to orthomyxoviruses including influenza viruses, but has not been demonstrated to confer resistance to flaviviruses 
. However, it is possible that MX1
may have an effect on flavivirus infections in humans.
The products of OAS1
, and their downstream effector RNASEL
each influence host defense by blocking viral replication 
. Evidence from human cell culture indicates that OAS gene products have an antiviral effect on flavivirus infections 
. The products of the Oas1b
alleles differentially affect susceptibility to flavivirus-induced disease in mice, but by an RNase L independent mechanism 
. In the present study, a single variant in OAS1
, SNP rs34137742 located in intron 2, was identified as a risk factor for human WNV disease progression, although no association with symptomatic infection as a whole was identified.
rs10774671 was previously reported to be associated with risk of WNV infection 
; however, we were unable to replicate this finding using our asymptomatic controls or the control samples of Lim et al. 
. In contrast, an association between another SNP, OAS1
rs34137742, and risk for WNE/P was found using multiple, different control populations (1. WNF/M plus asymptomatic WNV-positive cases, 2. RBD, 3. WNF/M plus RBD, and 4. WNF/M plus asymptomatic WNV-positive cases and RBD). Furthermore, we found an association between OASL
rs3213545 and risk for WNE/P, symptomatic disease, and infection, but only with the control samples of Lim et al. 
. The observation that OASL
is associated with WNV disease confirms a previously reported association discovered in a very small sample of cases (n
27) that failed replication in a second study using the same control samples as included here, but different WNV-positive samples 
We did not use the conventional phenotypic classification method of WNND and WNF. Instead, we grouped West Nile patients presenting with fever and/or meningitis into a single category, WNF/M, and categorized patients presenting with encephalitis and/or paralysis as WNE/P. Our decision was based on two observations. First, WNV patients presenting with meningitis are clinically more similar to WNF patients than they are to WNE/P patients. Second, patients classified as WNF are likely to include individuals with unconfirmed meningitis 
. Therefore, patients with meningitis may be included in both the WNF and WNND categories. When removing West Nile meningitis patients from the analysis, the association of OAS1
rs34137742 with WNE/P remained significant, but our power to detect this association decreased. However, if WNND patients were compared to WNF plus asymptomatic controls, no association with OAS1
rs34137742 and WNND was detected (Table S20
). These results illustrate the importance of accurate phenotypic classification, as the associations are contingent upon the classification system. The difference in our phenotypic classification of WNV-positive samples compared to that used by Lim et al.
could explain why we detected an association between OASL
rs3213545 and risk for WNE/P whereas the Lim et al.
study did not detect an association between this SNP and WNND 
. However, this difference in the classification system cannot explain our inability to replicate the association for OAS1
rs10774671 and WNV infection, as this analysis compared all WNV-positive cases to WNV-negative controls.
It has been shown that the host response to viral infections does not depend on host factors alone. Rather, the virulence of the infecting virus strain may impact the clinical course of infection and could therefore influence the likelihood of disease progression from asymptomatic infection to more severe forms of a disease. For example, particular strains of Dengue virus have been shown to be more virulent than others 
. Analysis of the specific WNV viral strains infecting each of the participants in this study was beyond the scope of this research. However, it is important to point out that varying degrees of virulence between infecting strains may have impacted WNV pathogenesis in our cohort. Furthermore, differences in strain virulence may also account for the discrepant results obtained in our study compared to previous work.
We searched for loci influencing risk for symptomatic WNV disease by comparing symptomatic WNV cases to asymptomatic, WNV-positive individuals. This approach likely is more robust than using random blood donors with unknown WNV exposure history or WNV-negative individuals as controls. Groups of RBDs with an unknown WNV exposure are likely to include previously uninfected individuals with the potential to develop either WNF/M or WNE/P, and therefore may reduce the power to detect a difference between groups. By considering only asymptomatic, WNV-positive blood donors, the probability of including individuals who might develop WNF/M or WNE/P was eliminated. This strategy could explain the observation that the significant associations between IRF3
and WNV disease were not replicated when RBD of unknown WNV infection status were used for comparison. Similarly, using WNV-negative controls rather than WNV-positive asymptomatic controls could confound risk factors for infection and symptomatic disease. This could explain, in part, our ability to replicate the association between CCR5Δ32
and symptomatic infection reported by Glass et al.
when we used sero-negative controls but not when WNV-positive asymptomatic blood donors were used as a control group 
The composition of the asymptomatic control cohort also appeared to influence the power to detect variants associated with risk for WNE/P. For example, inclusion of RBD in the control group with WNF/M or WNF/M and asymptomatic infection diminished the ORs and the level of significance of the association between OAS1 rs
34137742 and WNE/P. As a corollary, since some of the asymptomatic individuals may have had mild symptoms 
, comparing both asymptomatic WNV-positive cases combined with WNF/M would mitigate errors introduced by the misclassification of asymptomatic cases.
Age is a known independent risk factor for WNV disease progression with elderly patients at the highest risk for encephalitis and death 
. Ages were not available for many of the asymptomatic WNV-positive blood donors (233 out of 331) so that we were unable to control for age as a covariate. To test whether the analyses of WNV disease progression were robust to confounding by age, the effect of age was tested by comparing the results of a logistic regression using the subset of cases for which age information was available to those obtained for the entire set of samples. Age was available for 94% of WNE/P cases, 96% of WNF/M cases, and 30% of asymptomatic cases. The results were virtually the same, suggesting that age is not a major confounder of our analyses. Furthermore, in order for age to confound the analysis, it would have to be associated with the SNPs tested here. This could happen via settlement of different ethnic groups over the relevant time period in the regions from which cases and controls were collected. However, adjusting for ethnicity eliminated this potential issue.
In summary, we have identified two novel loci, IRF3 and MX1, associated with risk of WNV disease, and a SNP in OAS1 associated with WNE/P. Each of the SNPs responsible for the observed association signal is located in an intron and none is in strong linkage disequilibrium with known functional variants in their respective genes. Each of these loci is involved in either an interferon regulatory pathway or is an effector of the interferon response. Accordingly, these findings provide further evidence, albeit indirect, that the interferon pathway MAY play an important role in modulating human WNV disease, influencing both the risk of symptomatic infection and disease progression.