A Deletion in the Chemokine Receptor 5 (CCR5) Gene Is Associated with Tickborne Encephalitis

Tickborne encephalitis (TBE) is caused by the TBE virus (TBEV), a flavivirus associated with severe infection of the central nervous system (CNS) [1]. The virus is endemic in many areas of Europe and Asia and is mainly transmitted to humans by Ixodes ricinus and I. persulcatus ticks [1, 2]. Although infection with TBEV can result in mild symptoms as well as severe injuries of the CNS, serological surveys have suggested that between 70% and 95% of the infections in regions of endemicity are asymptomatic [2]. Symptomatic disease typically has a biphasic course, with an initial viremic phase characterized by an influenza-like illness followed by an asymptomatic interval. In ∼1 of 4 patients, a second phase with meningoencephalitis is seen [3, 4]. Early in the second phase, a specific antibody response is usually present [5], and virus can usually no longer be detected in either blood or cerebrospinal fluid (CSF) by polymerase chain reaction (PCR) [4]. However, in most fatal cases, TBEV can be demonstrated in brain tissue [6].

A most intriguing question is why certain individuals respond with severe clinical symptoms after infection while the majority either remains asymptomatic or develops only mild disease. Given that no specific virulence markers have been identified in TBEV infection, host immune factors have been considered a contributing factor for the disease. Our hypothesis is that an impaired immune response contributes to the severity of TBEV infection, and a prime candidate is the chemokine receptor CCR5, an important receptor affecting leukocyte migration and, hence, the host immune response [7]. The hypothesis emanated from a recent observation that a mutation in the CCR5 gene is associated with disease severity in infections with West Nile virus (WNV) [8, 9], which also is member of the Flaviviridae and shares certain similarities with TBEV [1].

Subjects, materials, and methods. To test our hypothesis, patients with TBE were recruited from a previous prospective follow-up study in Kaunas, Lithuania [10]. A total of 129 Lithuanian patients with documented TBE, 79 patients with aseptic meningoencephalitis (AME) who were antibody negative for TBE, and 134 age-matched and geographically matched control subjects with no documented TBEV infection were included in the study. All subjects were investigated for a 32-bp deletion in the CCR5gene. Informed consent was obtained from all patients. The meningoencephalitis of admitted patients was clinically classified as mild, moderate, or severe as described elsewhere [10]. Briefly, mild disease (n = 56) was that with predominantly meningeal symptoms, including fever, headache, rigidity of the neck, and nausea; moderate disease (n = 57) was that with monofocal symptoms of the CNS and/or moderate diffuse brain dysfunction; and severe disease (n = 16) was that with multifocal symptoms of the CNS and/or severe diffuse brain dysfunction. All patients with signs of encephalitis were classified as having moderate or severe disease. Encephalitic symptoms were defined as altered consciousness, ataxia, dysphasia, tremor, seizures, and mono- or multifocal symptoms. The non-TBE group consisted of consecutive patients with an acute illness suggestive of meningoencephalitis in whom lumbar puncture was performed and showed CSF pleocytosis compatible with viral etiology. Spontaneous remission occurred in these patients without antibiotic treatment, and no other etiological factor, such as drug-induced reaction, was observed. At the time of patient recruitment, no attempts to establish a diagnosis other than bacterial culture of the CSF and serological analysis for TBEV were made.

CCR5Δ32 genotyping was performed by pyrosequencing essentially, as described elsewhere [11]. Briefly, genomic DNA was purified from 200 μL of CSF (patients with menigoencephalitis) or serum (healthy control subjects) by use of a QiaAmp 96 DNA Blood Kit, in accordance with the manufacturer's instructions (Qiagen). DNA was eluted into 200 μL of AE buffer (Qiagen) and stored frozen, at −20°C, until analyzed. Ten microliters of patient DNA was used to amplify a 132-bp-long fragment of CCR5, including the deletion with primers 5′-CACCTGCAGCTCTCATTTTCC-3′ (forward) and 5′-BIOTIN-GTTTTTAGGATTCCCGAGTAGCA-3′ (reverse). Amplified PCR products were then sequenced by pyrosequencing as described elsewhere [11], using the sequencing primer 5′-CAGCTCTCATTTTCCAT-3′ and the dispension order GACAGTCAGA. Pearson's χ² test was used to compare allele frequencies between the groups (SPSS; version 13 for Mac OS X). Analyses were 2-tailed, with P < .05 considered to indicate statistical significance.

Results. All patients with TBE (n = 129) and control subjects (n = 134) as well as 76 (96.2%) of the 79 patients with AME were successfully genotyped. Both control subjects and patients with AME resided in the same geographic region as the majority of the patients with TBE. The control subjects were based on a random-sample collection from this population and were matched by age to the TBE group. The genotype in 8 samples could not be determined by pyrosequencing and were instead determined by gel electrophoresis, by separation of the 100- and 132-bp PCR fragments in agarose gel. The genotype distribution for the patients with TBE, the patients with AME, and the Lithuanian control subjects did not deviate from Hardy-Weinberg equilibrium. No difference in allele distribution was seen between the patients with AME and the Lithuanian TBE naive control subjects (P = .43), and these 2 groups were considered as one when statistical calculations were performed.

Altogether, 3 CCR5Δ32 homozygotes were found among the analyzed samples, and all of them were patients with TBE (table 1). The prevalence of CCR5Δ32 homozygotes was, thus, higher among the patients with TBE (2.3% [3/129]) than among the patients with AME (0% [0/76]), the Lithuanian TBE-naive control subjects (0% [0/134]), Swedish blood donors (0.4% [1/265]) [11], and Lithuanians in general (0.7% [2/283]) [12] (table 1), and the difference was statistically significant when patients with TBE were compared with the TBE-naive patients with AME and healthy control subjects (P = .026).

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Figure 1

CCR5Δ32 allele prevalence among patients with TBE, stratified by severity of disease.

The CCR5Δ32 allele prevalence was also higher among the patients with TBE (35/258) than among the TBE-naive patients with AME (16/152) and the healthy control subjects (22/268) (P = .065), as well as compared with that among the Swedish [11] and Lithuanian [12] populations (53/530 and 65/566, respectively) in general (table 1). This suggests that not only homozygote but also heterozygote carriers of mutated CCR5 are predisposed for TBE. Furthermore, the CCR5Δ32 allele frequencies of the Lithuanian control subjects (0.082) and the patients with AME (0.105) were in concordance with earlier reports [11–13], which altogether strengthen the accuracy of our observed allele frequencies. Further support for accuracy is provided bythe observation that the total allele prevalence among the investigated individuals was 0.108 (73/678), compared with 0.115 among a Lithuanian population investigated previously by Libert et al. [12] (table 1). The population investigated by Libert et al. was, however, not screened for TBE and thus includes both TBE-naive and -positive individuals; therefore, it can be compared with our total Lithuanian population.

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Table 1

Prevalence of the CCR5Δ32 allele among patients with tickborne encephalitis (TBE), patients with aseptic meningoencephalitis (AME), and population control subjects.

When the patients with TBE were stratified by severity of disease, the allele prevalence increased with the severity of TBE (figure 1), but no statistical differences were found between the 3 groups. The allele prevalence was 0.116 (13/112) in patients with mild disease, 0.140 (16/114) in patients with moderate disease, and 0.188 (6/32) in patients with severe disease. All 3 CCR5Δ32 homozygote patients were found in the group with moderate disease.

Discussion. To our best knowledge, this is the first attempt to investigate a role for host genetic factors in TBEV infections. We have found an association between the prevalence of the CCR5Δ32 allele and TBEV infection among Lithuanians. Among the patients with meningitis (TBE and AME) and the TBE-naive control subjects, CCR5Δ32 homozygotes were, surprisingly, found only among the patients with TBE, and allele prevalence was higher among the patients with TBE than the patients with AME (0.136 vs. 0.105), indicating that CCR5Δ32 does not predispose for meningitis in general. Additional support for this conclusion is that no statistical difference was found between the CCR5Δ32 allele frequencies among the patients with AME and the healthy control subjects. The CCR5Δ32 allele prevalence among the patients with AME (0.105) was in concordance with that among Swedish blood donors (0.100) and Lithuanians (0.115) (table 1); thus, it is unlikely (although it cannot be excluded) that the higher prevalence of the deletion among the patients with TBE is not associated with meningoencephalitis per se but rather with meningoencephalitis caused by TBEV.

When the patients were stratified by severity of disease, the allele prevalence increased with the severity of TBE, indicating that possession of the defective allele may predispose for more severe disease. However, all 3 CCR5Δ32 homozygote patients were found in the group with moderate disease and not in the severe group, as would be expected if CCR5Δ32 were associated entirely with severity of TBE. Also, no statistical differences were found between the 3 groups. Factors other than CCR5Δ32 must also contribute to the clinical severity of TBEV infection.

The 32-bp deletion is in the region corresponding to the second extracellular loop and produces a truncated protein that is not expressed on the cell surface [14]. CCR5 is a chemokine receptor and, thus, plays a central role in the host immune defence, which is why having at least 1 functional receptor could be important for viral clearance [8]. CCR5 is also a coreceptor for the entry of macrophage-tropic strains of HIV-1 into host cells, and a CCR5 deficiency has been shown to be protective against HIV infection. CCR5Δ32 homozygosity is strongly associated with resistance to HIV-1, and heterozygotes show slower rates of disease progression to AIDS [14].

The CCR5Δ32 allele prevalence has been found to vary in different parts in Europe, with a clear north to south gradient [15]. The highest frequencies are found in Scandinavia, the Baltic states, Russia, and Central Europe (with allele frequencies >0.1), whereas the allele is almost absent in the southern Europe and in Africa [15].

A recent study found that CCR5Δ32 increases the risk of symptomatic WNV infection. The prevalence of CCR5Δ32 homozygotes was increased among WNV-seropositive patients compared with healthy control subjects, and the difference was highly statistically significant [9]. WNV is also a member of the Flavivirus genus [1], but, in contrast to TBEV, its vectors are mosquitoes, mainly bird-feeding species. Furthermore, WNV causes similar symptoms as does TBEV, with ∼20% of infected individuals developing a febrile illness, with a progression to meningitis and/or encephalitis in >30% of cases [8]. It has recently been shown in a mouse model that CCR5 promotes trafficking of leukocytes into the WNV-infected brain, and, thus, a functional CCR5 receptor may be important for clearance of WNV [8]. It is thus tempting to speculate that CCR5 is required to promote leukocyte migration to the CNS to assist in viral clearance and limit disease severity. Supportive of this hypothesis is not only the geographical colocalization of CCR5Δ32 allele prevalence and TBEV infections but also that WNV and TBEV are both members of the Flaviviridae and share neurotropism as a key property.

In conclusion, we report that patients with TBE are more frequently CCR5Δ32 homozygotes (2.3%) than are TBE-naive patients with AME and control subjects (0%) (P = .026) and that the prevalence of the deleted allele is higher among patients with TBE (P = .065). Together, this indicates that the CCR5Δ32 allele may predispose for TBE; however, studies with a larger number of patients are needed to confirm this observation.

• Received July 10, 2007.
• Accepted August 27, 2007.

• © 2008 by the Infectious Diseases Society of America