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CCR5 Deficiency Is a Risk Factor for Early Clinical Manifestations of West Nile Virus Infection but not for Viral Transmission - pdf attached
  The Journal of Infectious Diseases Jan 15 2010;201:178-185
Jean K. Lim,1 David H. McDermott,1 Andrea Lisco,3 Gregory A. Foster,4 David Krysztof,4 Dean Follmann,2 Susan L. Stramer,4 and Philip M. Murphy1
1Molecular Signaling Section, Laboratory of Molecular Immunology, 2Biostatistics Research Branch, National Institute of Allergy and Infectious Diseases, 3Section on Intercellular Interactions, Laboratory of Cellular and Molecular Biology, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, and 4American Red Cross, Gaithersburg, Maryland
LINKS: CCR5 Deficiency Is a Risk Factor for Early Clinical Manifestations of West Nile Virus Infection but not for Viral Transmission
Jean K. Lim, David H. McDermott, Andrea Lisco, Gregory A. Foster, David Krysztof, Dean Follmann, Susan L. Stramer, and Philip M. Murphy
Abstract-Full Text-PDF Version (505 kB)
Background. West Nile virus (WNV) is a neurotropic flavivirus transmitted to humans by mosquito vectors. Homozygosity for CCR5Δ32, a complete loss-of-function mutation in CC chemokine receptor 5 (CCR5), has been previously associated with severe symptomatic WNV infection in patients who present with clinical disease; however, whether it acts at the level of initial infection or in promoting clinical progression is unknown.
Methods. Here, we address this gap in knowledge by comparing CCR5Δ32 distribution among US blood donors identified through a comprehensive blood supply screening program (34,766,863 donations from 2003 through 2008) as either WNV true positive (634 WNV-positive cases) or false positive (422 WNV-negative control participants). All subjects self-reported symptoms occurring during the 2 weeks following blood donation using a standardized questionnaire.
Results. No difference was observed in CCR5Δ32 homozygous frequency between the WNV-positive cases and WNV-negative control participants. However, CCR5Δ32 homozygosity was associated in cases but not controls with clinical symptoms consistent with WNV infection ( ).
Conclusions. CCR5 deficiency is not a risk factor for WNV infection per se, but it is a risk factor for both early and late clinical manifestations after infection. Thus, CCR5 may function normally to limit disease due to WNV infection in humans.
West Nile virus (WNV) is a mosquito-borne flavivirus that has caused annual outbreaks in humans since its entry into the United States in 1999 [1]. As of 18 November 2008, 28,906 confirmed cases of symptomatic WNV infection had been reported to the US Centers for Disease Control and Prevention (CDC), with 1121 (4%) fatalities ( Currently, there are no specific treatment options or licensed vaccines for humans approved by the US Food and Drug Administration [2, 3]. Since transmission of WNV can occur through blood transfusion and organ transplantation, universal screening for WNV RNA was implemented in 2003 for blood donations in the United States [4-6]. Screening data have shown that WNV infection in the general population remains rare, with seropositivity being found in 1 in 10,000 blood donors [7, 8].
It is estimated that most WNV-infected individuals remain asymptomatic, with 20%-30% developing symptoms after a 2-14-day latency period [9]. Clinical features can range from mild flu-like symptoms (West Nile fever [WNF]) to more severe West Nile neuroinvasive disease (WNND) [9]. Clinical features of WNF typically include fever, headache, fatigue, skin rash, swollen lymph glands, and eye pain ( WNND manifests primarily as meningitis, encephalitis, and/or acute-flaccid paralysis, and associated severe clinical features commonly include diarrhea and vomiting, generalized weakness, bone and joint pain, severe muscle aches, tremors, seizures, new cognitive difficulty, and changes in mental status [9]. Thus, outcome of WNV infection is heterogeneous, and identification of the risk factors that affect outcome is an important goal.
In a mouse model of WNV disease, chemokine receptor CCR5 was found to be a strong host defense factor, with CCR5-deficient mice displaying markedly increased viral titers in the central nervous system and experiencing a higher mortality rate [10]. We have extended these findings in mice to humans by demonstrating a strong epidemiologic association between symptomatic WNV disease and homozygosity for a common, complete loss-of-function mutation named CCR5Δ32 in the chemokine receptor gene CCR5 (p<.001; odds ratio [OR], 4.2; 95% confidence interval [CI], 2.1-8.3) [11, 12]. Although these studies showed a strong, reproducible association with WNV-positive patients with a clinical diagnosis of WNF or WNND from 4 geographically and temporally distinct US collections, they were unable to distinguish whether the association was with susceptibility to infection or with severity of clinical presentation. Our previous analyses were retrospective; they did not include individuals who remained asymptomatic or mildly symptomatic, did not record specific clinical symptoms, and were not designed to capture patients in early stages of infection. To address these questions, we retrospectively screened a nonconcurrent prospective cohort of blood donors from the American Red Cross (ARC) to identify asymptomatic and symptomatic WNV-positive individuals and compared CCR5Δ32 genotypic frequencies with infection and self-reported symptoms associated with WNV disease.
In this study, we show, using samples collected from random blood donors, that CCR5 deficiency is not associated with increased susceptibility to infection but rather with a more aggressive early clinical presentation, with a higher frequency of individual symptoms and more total symptoms, compared with CCR5-sufficient individuals. In fact, we found no individuals who were WNV positive and homozygous for CCR5Δ32 who did not report symptoms. CCR5Δ32-homozygous individuals with WNV infection were more likely to present with multisystem symptomatology, including lymphadenopathic, neurologic, and gastrointestinal symptoms. The role of CCR5Δ32 homozygosity as a predictor of severity of clinical presentation is consistent with the hypothesis that functional CCR5 is critical for control of WNV in humans.
It is important to note that the present study is a retrospective analysis of a nonconcurrent prospective cohort, with the WNV-positive cases identified before the onset of symptoms, whereas our previous studies of WNV-related symptoms were retrospective and selected for individuals seeking medical attention for clinical manifestations of WNV disease. The current study also captures a wide range of clinical presentation of initial infection, including individuals who remained healthy and those who experienced symptoms. Thus, unlike our previous study that analyzed patients with more advanced disease, the current study evaluates WNV-infected individuals at an early stage of disease. The current study design allowed us to directly compare CCR5Δ32 frequency among symptomatic WNV-positive cases to both asymptomatic WNV-positive cases and uninfected WNV-negative control participants and therefore to evaluate the role of CCR5Δ32 in both susceptibility to infection and severity of disease. Our data show no evidence for increased susceptibility or symptoms associated with CCR5Δ32 heterozygosity, because frequency of heterozygosity was similar between cases and control participants (Table 2), and individuals who were CCR5Δ32 heterozygotes were identical to individuals homozygous for wild-type CCR5 with respect to the number of symptoms experienced (Figure 2A), and individual symptom prevalence (data not shown). These data suggest that in CCR5Δ32 heterozygotes, normal CCR5 expression is in excess relative to what is needed for normal host defense against WNV. The mechanism by which CCR5 functions at the biochemical level in vivo is not known and may involve dimerization with another chemokine receptor. Potential partners, on the basis of previously reported biochemical data, include CXCR4 and CCR2 [14, 15].
The frequency of CCR5Δ32 homozygosity observed among the WNV-false-positive control participants (2.4%) was slightly elevated, compared with previous reports of the frequency of this genotype in white individuals in the United States, which range from 0.7% through 1.7% [11, 16-21]. It is important to note that the study populations tested in these previous studies were sampled from 1 region or city of the United States, whereas the control population tested in the current study is the first, to our knowledge, to evaluate the frequency of CCR5Δ32 homozygosity on the basis of large-scale sampling of the entire nation. In any case, the frequency we found is only 0.7% greater than the upper range previously reported. Other virtues of the control group used in the present study are that the participants (1) were temporally and geographically matched to the cases, (2) were accrued in the same manner as the cases, and (3) had filled out and submitted the same symptom questionnaire as the cases (Figure 3). The only other potential control group available from among the about 35 million donations is one composed of those who had negative screening results at the time of blood donation (true negatives). However, these donors would not have filled out a questionnaire, because they were nonreactive according to the initial WNV NAT screening test and thus were not enrolled in the WNV follow-up study. Even if these patients had been enrolled in the study, they would have answered the questionnaire regarding symptoms with the knowledge that they were WNV negative, which might bias the response regarding symptoms.
It is also important to consider potential limitations to our study. First, there may be recall bias, because all individuals were aware of their potential infection with WNV and may have been more likely to report symptoms. However, both cases and control participants answered the questionnaire before learning the results of their true WNV infection status, and thus a similar bias would be expected. Because the latency period before symptom onset has been estimated to be 2-14 days, it is possible that a donor could have tested positive for WNV but already have experienced the majority of their symptoms, causing an underreporting of symptoms on the questionnaire for this group. Second, evaluation of symptoms was based on participant response, rather than on objective measurements, and therefore could be imprecise. Finally, our results do not address the mechanism of action of CCR5 during WNV pathogenesis. Our study involving WNV in an excellent mouse model has suggested that it works, in part, at the level of leukocyte trafficking, at least for neuroinvasive disease; however, mechanisms in humans could differ [10].
WNV infection is the first human disease for which normal CCR5 has clearly been shown to be beneficial [11, 12, 22]. Prior to these studies, CCR5-deficient individuals were believed to have minimal, if any, health defects as a result of their deficiency [23]. In contrast, they were well known to have strong resistance to human immunodeficiency virus (HIV) infection, because normal CCR5 is used as a critical coreceptor for cell entry. This finding led to the development of Maraviroc, an effective CCR5 antagonist used in the treatment of HIV/AIDS [24]. Although Maraviroc had an excellent safety profile in clinical trials, because it is now marketed and used on a larger scale, it will be important to maintain a high index of suspicion in HIV-infected individuals treated with Maraviroc who present with signs and symptoms compatible with WNV infection, particularly because these patients are already immunocompromised. Our results suggest that, if a patient who is currently taking Maraviroc does test positive for WNV infection or displays the characteristic symptoms during an outbreak, that the drug should be withheld until the infection is cleared. A more general caution regarding WNV risk may apply to any CCR5 blocking agent that might be developed and any disease in which it might be used. It will also be interesting to investigate the susceptibility of CCR5-deficient individuals to other types of flaviviruses. In this regard, CCR5Δ32 homozygosity has recently been associated in 1 study with increased susceptibility to tick-borne encephalitis virus [25].
Although our study specifically highlights the importance of a single genetic risk factor in a specific infectious disease, it also illustrates the power of large-scale databases to answer important clinical questions regarding diseases in humans, particularly for rare diseases and/or rare genetic mutations.
From May 2003 through July 2008, a total of 34,766,863 blood donations were screened by the nationwide WNV blood screening program of the ARC by WNV NAT. As shown in Figure 1, initial screening revealed 1892 donations to be reactive. Because initial WNV NAT screening has a high false positivity rate, all 1892 positive donations were also subjected to serological testing and a follow-up WNV NAT to determine true or false WNV positivity. Of the 1892 donations identified as potentially positive, 1065 were confirmed as WNV true positive (WNV-positive cases, 1 in 32,645 blood donors); the remaining 827 donations were WNV false positive (WNV-negative control participants), defined as negative by both serological testing and a follow-up WNV NAT. On the basis of sample availability and the inclusion criteria for this study (defined as self-reported white/non-Hispanic, information available regarding age and sex, and completed questionnaire), we were able to analyze data from 656 unique WNV-positive cases representing 35 states and 431 unique WNV-negative control participants, also from 35 states. CCR5 genotypes were obtained for 634 WNV-positive case samples and 422 WNV-negative control samples, with an overall success rate of 96.1%.
The characteristics of the study subjects, including symptoms experienced within 2 weeks after donation, are shown in Table 1. Cases and control participants were similar in age, but cases were more likely to be male, as observed elsewhere [9]. The frequency of individuals who reported no symptoms on the questionnaire after donation was significantly greater among cases than control participants (75.6% vs 33.6%; p<.001; OR, 5.42; 95% CI, 4.15-7.09). When each symptom was evaluated separately, cases were more likely than control participants to report symptoms (Table 1), as expected and as reported elsewhere [13].
To test whether CCR5Δ32 is associated with susceptibility to WNV infection, we compared genotypic frequencies of cases with control participants. Within these groups, the CCR5 genotypes were in Hardy-Weinberg equilibrium (p=.13 and .16, respectively). Among control samples, 10 (2.4%) of 422 were CCR5Δ32 homozygotes (Table 2). This value was identical to the frequency of CCR5Δ32 homozygotes observed in WNV-positive cases (15 of 634 cases; 2.4%). Thus, CCR5Δ32 homozygosity is not associated with WNV infection per se. It is important to note that there is a fundamental difference in the way participants were identified in the present study versus in our previous studies that reported an association between CCR5Δ32 homozygosity and symptomatic WNV disease. The previous studies selected for individuals seeking medical attention for symptomatic disease, whereas the ARC donors in the present study were accrued as voluntary blood donors independent of symptoms. Therefore, we next hypothesized that CCR5Δ32 homozygosity is associated with symptoms in early WNV infection, which the design of the present study uniquely allowed us to test by examining the distribution of symptoms according to genotype. The symptoms evaluated here are based on a previously published and validated follow-up questionnaire [13] that was administered to all individuals whose samples were reactive on the initial WNV NAT screening test (both cases and control participants) prior to learning their WNV infection status. As shown in Figure 2A, patients who were homozygous for CCR5Δ32 experienced more symptoms (5.47 symptoms), on average, than did those patients who were heterozygous (2.81 symptoms) or homozygous (3.05 symptoms; p=.002) for wild-type CCR5. This increase in symptom number was not observed among the participants who were homozygous for CCR5Δ32 identified in the WNV-negative control group (p=,49 ; Figure 2A) and was not related to age. The mean ages (± SD) for WNV-seropositive CCR5 patients with wild-type alleles (48.5+/-14), heterozygous for CCR5Δ32 (48.0+/-13), and homozygous for CCR5Δ32 (47.5+/-11) were very similar. To further understand the relationship between CCR5Δ32 homozygosity and symptoms, we stratified the ARC cases into 2 groups, those with symptoms and those without symptoms. As shown in Table 2, we identified no patients homozygous for CCR5Δ32 among the 169 asymptomatic WNV-positive cases, which was less than expected on the basis of the overall frequency observed in the ARC cohort (4.4 cases homozygous for CCR5Δ32 were expected) and significantly decreased when compared with the frequency observed among the 280 asymptomatic WNV-negative control participants, where 7 patients homozygous for CCR5Δ32 were identified (<.05). Compared with cases who developed symptoms (465 patients), a significant difference was observed in the frequency of this genotype (0% vs 3.2%; p=.015) (Figure 2B). No significant differences were observed when the asymptomatic and symptomatic control participants were compared (p>.99), or when symptomatic WNV-positive cases were compared with symptomatic WNV-negative control participants (p=.78). However, a logistic regression analysis plotting the predicted probability curve of CCR5Δ32 homozygosity as a function of the number of symptoms identified a significant positive correlation (p=.002) for WNV-positive cases (Figure 2C) but not for WNV-negative controls (not shown).
To understand whether any specific symptoms were associated with CCR5Δ32 homozygosity, we evaluated the symptom prevalence among the cases homozygous for CCR5Δ32 versus cases with a wild-type allele. As shown in Table 3, significant increases were found for patients homozygous for CCR5Δ32 who reported painful eyes (p=.003), swollen glands (p=.018), generalized weakness (p=.013), vomiting or diarrhea (p=.035), and abdominal pain (p=.032). Of note, the prevalence of painful eyes (which could indicate photophobia), headache, tremors, and new cognitive difficulty, all of which could be associated with neurologic disease, were increased, although only the first reached statistical significance.
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