Three cohorts of patients, the Colorado WNV-seropositive cohort, the Arizona WNV-seropositive cohort, and the Arizona WNV-seronegative cohort, were identified that had symptomatic illness in which WNV was considered in the differential diagnosis. Inclusion criteria for patients in these cohorts are defined in the Materials and methods section and the clinical characteristics are given in . Genotypes were defined for 247 of the 261 WNV-seropositive Arizona cases (94.6%); for 148 of the 155 WNV-seropositive Colorado cases (95.5%); for 143 of 152 and 72 of 74 of the self-reporting Caucasian subsets of the Arizona and Colorado WNV-seropositive cohorts, respectively; for all WNV-seronegative random blood donors (n = 1,318); and for 145 of 154 WNV-seronegative subjects from Arizona (94.2%).
Characteristics of study subjects
The genotype distribution observed for the combined Arizona and Colorado WNV-seropositive cohorts deviated strongly from Hardy-Weinberg equilibrium (P = 0.013), mainly because of increased frequency of CCR5Δ32
homozygotes. 17 (4.3%) of the 395 informative cases in the combined Arizona and Colorado seropositive cohorts unstratified by race were CCR5Δ32
homozygotes (). This value greatly exceeds the percentage of CCR5Δ32
homozygotes reported for the general US Caucasian population (0.8–1.1%; reference 18
). To quantitate the significance of this genotype–disease association, we have used as a reference the group of 1,318 healthy Caucasian random blood donors from the US, in which 1.0% were CCR5Δ32
homozygotes. Compared with this value, the increased frequency of CCR5Δ32
homozygotes in both the Arizona and Colorado WNV-seropositive cohorts was highly statistically significant (). When all genotyped WNV-seropositive cases unstratified by race were compared with the US random blood donors, the odds ratios (OR) for this genetic factor were 4.7 for the Arizona cohort (95% confidence interval [CI], 2.1–10.6, P < 0.0001) and 4.2 for the Colorado cohort (95% CI, 1.6–11.3, P = 0.0018). It is important to note that this is a conservative analysis because the frequency of CCR5Δ32
homozygotes is certain to be lower in non-Caucasian racial groups from these cohorts ().
Association of the CCR5Δ32 allele with symptomatic WNV infection in Arizona and Colorado
CCR5Δ32 homozygotes have increased risk of symptomatic WNV infection
Consistent with this, when the data were stratified to include only self-reporting Caucasians in both the WNV-seropositive cohorts (n = 215), the strength of the association increased by 30% (12 CCR5Δ32 homozygotes, 5.6%). When the self-reporting Caucasian portions of the two WNV-seropositive cohorts were compared separately to the random blood donor benchmark, the OR was 4.4 in the Arizona cohort (95% CI, 1.6–11.8, P = 0.0013), and 9.1 in the Colorado cohort (95% CI, 3.4–24.8, P < 0.0001).
Although unlikely, it is possible that the large increase in frequency of CCR5Δ32 homozygotes in these cohorts may be caused by an atypical geographic distribution of this allele in the general population of Arizona and Colorado. To directly address this possibility, we analyzed the distribution of CCR5Δ32 in a symptomatic Arizona WNV-seronegative cohort (n = 145). Because the CCR5Δ32 allele frequency in this cohort (7.2%) is only slightly lower than in the Arizona WNV-seropositive cohort (10.7%) and the US Caucasian random blood donors (8.7%), it is likely that this cohort contains only a slightly smaller percentage of Caucasians than the other two. Thus, given the size of the Arizona WNV-seronegative group, there is sufficient power at this allele frequency to meaningfully analyze the distribution of genotypes. Only one CCR5Δ32 homozygote was identified in the cohort (0.7%). Moreover, unlike the WNV-seropositive cohort, the observed CCR5Δ32 genotypic frequencies in the WNV-seronegative cohort did not deviate from Hardy-Weinberg equilibrium (P > 0.99). This strongly suggests that the general population in Arizona does not have an anomalous genetic structure with regard to the CCR5 locus, with a skewed increase in CCR5Δ32 homozygotes that could account for the high frequencies we observed in the WNV-seropositive cohorts. When the CCR5Δ32 homozygote genotype frequency was compared between the race-unstratified Arizona WNV-seronegative and -seropositive cohorts, an OR of 6.7 was determined (95% CI, 0.86–52.6, P = 0.07).
With regard to the distribution of CCR5
genotypes in symptomatic WNV-seropositive cases as a function of clinical outcome, no significant differences were observed compared with the distribution in the overall combined group () or in the Arizona or Colorado WNV-seropositive cohorts considered separately (not depicted), with the exception of death (). A total of 19 of 395 individuals in the two cohorts died from WNV neuroinvasive disease (4.8%), which is similar to the national average of mortality reported for WNV cases from the past three years: 2002 (4,156 cases, 284 deaths, 6.8%); 2003 (9,862 cases, 264 deaths, 2.7%); and 2004 (2,539 cases, 100 deaths, 3.9%). The combined total for this time interval in the US is 16,577 reported cases with 648 deaths (3.9%; references 7
). The Arizona WNV-seropositive cohort accounted for 8 of the 19 total deaths, 7 in Caucasians and 1 in a Hispanic (). The Colorado WNV-seropositive cohort accounted for 11 deaths, 1 in a Caucasian. Two of the 19 fatalities (10.5%) in the combined cohorts were CCR5Δ32
homozygotes, both self-reporting Caucasians from the Arizona WNV-seropositive cohort. The ages of the two CCR5Δ32
homozygotes who died were 70 and 74, similar to the average age of the other 17 fatal cases (74 yr). The two CCR5Δ32
homozygote fatalities represent 25 and 29% of the race-unstratified (n
= 8) and Caucasian (n
= 7) Arizona WNV-seropositive cases who died, respectively. These values exceed the expected values based on the frequency of CCR5Δ32
homozygotes among the race unstratified and Caucasian groups in the Arizona cohort (4.5 and 4.2%, respectively), and the differences are statistically significant: OR = 8.5 (95% CI, 1.5–48.2), P = 0.04, and OR = 13.2 (95% CI, 1.9–89.9), P = 0.03, respectively (). This association was not observed in the Colorado cohort. When Caucasians from both cohorts were analyzed together, the association test gave an OR of 6.6 (95% CI, 1.2–37), P = 0.07.
Distribution of CCR5Δ32 allele as a function of clinical outcome in WNV-seropositive patients
Figure 1. CCR5 deficiency is associated with increased risk of death from WNV infection. Data presented are from the Arizona WNV-seropositive cohort and demonstrate that CCR5Δ32 homozygosity is associated with fatal outcome in both the race-unstratified (more ...)
The present study provides the first evidence that the defective CCR5
is a risk factor for symptomatic WNV infection, the first genetic risk factor identified for this disease. The association was restricted to CCR5Δ32
homozygotes and was very strong, similar in magnitude to the association we and others have previously reported for CCR5Δ32
homozygosity with the exposed, uninfected HIV resistance phenotype (e.g., OR = 6.04 [95% CI, 1.42–25.7], P = 0.02, in reference 15
). Although our results are based on retrospective analysis, they are unlikely to be because of chance for six reasons. First, the results are statistically strong (an OR greater than four). Second, the results were obtained in two independent as well as geographically and temporally distinct cohorts. Third, CCR5Δ32
is a complete loss-of-function mutation and therefore homozygous individuals completely lack functional CCR5. Fourth, WNV infection is associated with infiltration of T cells and macrophages (cell types known to express CCR5) into brains of patients infected with WNV, suggesting biologically plausible involvement of CCR5 in regulating leukocyte migration to the brains of infected patients (19
). Fifth, WNV infection of mice induces expression of the CCR5 ligand CCL5 and accumulation of CCR5+
leukocytes in the brain (12
). Sixth, WNV infection in CCR5−/−
mice is uniformly fatal (12
Together these results imply that wild-type CCR5 functions as a host defense factor in WNV infection in man. CCR5 could potentially restrict WNV infection at the level of initial infection. This is not addressed by our retrospective study design and may not be feasible to resolve prospectively by measuring the association of CCR5Δ32 homozygosity with asymptomatic WNV infection because this genotype and WNV infection are both uncommon in the general population. More likely, CCR5 restricts disease progression after initial infection so that its absence results in an increased likelihood of an infected patient coming to medical attention as a symptomatic case. Our data are consistent with this interpretation, and even suggest that a fatal outcome is more likely in the absence of CCR5 (). Additional work will be needed to more critically address the latter point because the positive association we observed between CCR5 deficiency and death in the Arizona cohort, although statistically significant, was based on only two cases, and could not be detected in the Colorado cohort, possibly because of its smaller size.
Another limitation of our study was the incomplete information about the racial background of cases (), which is needed to more precisely quantitate risk because CCR5Δ32 is primarily found in Caucasians. We have addressed this by consistently applying a conservative analysis of the data that has enabled us to test boundary conditions for quantitating the strength of the genotype–disease association. Larger prospective studies will be needed to more precisely quantitate risk as well as to further define mechanisms in man.
Our data indicate that immunocompromised patients could be particularly susceptible to symptomatic WNV infection if CCR5 function is missing or blocked. Additional studies will be needed to address this issue, which should include close monitoring of AIDS patients treated with antagonists now in advanced stages of development targeting the HIV coreceptor activity of CCR5. Such agents are intended to imitate the homozygous CCR5Δ32 genetic defect and could render AIDS patients particularly vulnerable to severe and possibly fatal WNV infection.
In summary, we have established that homozygous CCR5Δ32 is a strong host genetic risk factor for symptomatic laboratory-confirmed WNV infection, the first one identified for this disease. In contrast, the homozygous CCR5Δ32 genotype has previously been strongly associated with resistance to HIV. These genetic data imply that CCR5 plays opposite roles in HIV and WNV infection, facilitating the former and restricting the latter. Our results have important implications regarding the potential safety of CCR5-blocking agents now under development for the treatment of HIV/AIDS. Clinical care of individuals taking these medicines while residing in WNV-endemic areas may mandate strict measures to limit mosquito exposure and a high index of suspicion for symptoms consistent with WNV.