Emergence of tick-borne encephalitis in new endemic areas in Austria: 42 years of surveillance.

Human infections with tick-borne encephalitis (TBE)virus are a public health concern in certain regions of Europe, central and eastern Asia. Expansions of endemic areas and increased incidences have been associated with different factors including ecological changes supporting tick reproduction, socioeconomic changes increasing human outdoor activities and climatic changes favouring virus circulation in natural foci. Austria is among the most strongly affected countries in Central Europe, but the annual number of cases has strongly declined due to vaccination. Here,we have analysed changes of the incidence of TBE in the unvaccinated population of all federal states of Austria over a period of 42 years. The overall incidence in Austria has remained constant, but new strongly affected endemic regions have emerged in alpine valleys in the west of Austria. In parallel, the incidence in low-land regions in the north-east of the country is decreasing. There is no evidence for a shift to higher altitudes of infection sites in the traditional TBE zones,but the average altitudes of some newly established endemic areas in the west are significantly higher. Our analyses underscore the focal nature of TBE endemic areas and the potential of TBE virus to emerge in previously unaffected regions.

Retrospective identification of human cases of West Nile virus infection in Austria (2009 to 2010) by serological differentiation from Usutu and other flavivirus infections.

There is increasing evidence for the spread of West Nile virus (WNV) in southern, eastern and central Europe. In parallel, another flavivirus, the antigenically closely related Usutu virus, was introduced from Africa and first detected in Austria (2001), followed by Spain (2003), Hungary (2005), Italy (2006), Switzerland (2006) and Germany (2007). In Austria, human WNV infections have not previously been documented, although the virus was isolated from birds and detected in mosquitoes in 2008 and 2009. We therefore conducted a retrospective search for human cases of WNV infection using serum and cerebrospinal fluid samples collected from patients with central nervous system (CNS) disease in the summers of 2009, 2010 and 2011. Although all samples were negative for WNV by polymerase chain reaction, quantitative evaluation of standardised antibody assays with purified flavivirus antigens (including Usutu virus, which cross-reacts with WNV even in neutralisation assays) provided serological evidence for three autochthonous WNV infections in Austria: two in 2009 and one in 2010. Our data highlight the importance of raising awareness of WNV infections in Austria and neighbouring countries and suggest including testing for this infection in routine diagnostic practice of CNS diseases.

Humoral and cellular immune response to tick-borne-encephalitis (TBE) vaccination depends on booster doses in patients with Juvenile Idiopathic Arthritis (JIA).

Juvenile Idiopathic Arthritis (JIA) patients living in areas with high prevalence of tick-borne-encephalitis-virus-(TBEV)-infection are recommended for administration of inactivated TBE-vaccination. However, there are serious concerns regarding protective vaccine-induced immune responses against TBEV in immunocompromised patients. The present study aimed to analyze the humoral and cellular immune response to TBE-vaccination in previously TBE-vaccinated JIA patients compared to healthy controls (HC) including investigation of IgG-anti-TBEV avidity, neutralization capacity, cellular reactivity by IFNgamma-ELISPOT and cytokine secretion assays. Similar IgG-anti-TBEV antibody concentrations, neutralization titers and cellular reactivity were found between JIA and HC. The number and the early timing of booster vaccinations after primary vaccination had the most prominent effect on neutralizing antibodies in JIA and on IgG-anti-TBEV concentrations in both JIA and HC. Administration of booster vaccinations made it more likely for JIA patients to have IgG-anti-TBEV concentrations ≥165 VIEU/ml and avidities >60%. TNF-alpha inhibitors had a positive and MTX administration a negative effect on humoral immune responses. In conclusion, irrespective of having JIA or not, vaccinated children showed similar humoral and cellular immunity against TBEV several years after primary TBE-vaccination. However, in JIA, booster vaccinations mounted a significantly higher humoral immune response than in JIA without boosters. Our results highlight the need for timely administration of boosters particularly in JIA. Although immunosuppressive treatment at vaccinations in diagnosed JIA had a negative effect mainly on TBEV-specific cellular immunity, most JIA patients mounted a favorable humoral immune response which was maintained over time. Thus, successful TBE-vaccination seems highly feasible in JIA patients with immunosuppressive regimens.

West Nile virus lineage 2 infection in a blood donor from Vienna, Austria, August 2014.

Eastern Austria is neighbouring regions with ongoing West Nile virus (WNV) transmissions. Three human WNV infections had been diagnosed during the past decade in Austria. The Austrian Red Cross Blood Service (ARC-BS) started a first voluntary screening for WNV in blood donors from Eastern Austria by Nucleic Acid Testing (NAT) in June 2014. This is also the most extensive WNV surveillance programme in humans in Austria so far. In August 2014, one autochthonous WNV infection was detected in a blood donor from Vienna. By now, one in 67,800 whole blood donations was found to be positive for WNV RNA.

A randomized controlled study of pergolide in patients with restless legs syndrome.

BACKGROUND: Open clinical trials indicate that low doses of pergolide, a long-acting D1 and D2 dopamine agonist, lead to a reduction in the symptoms of restless legs syndrome (RLS) with subjective improvement in sleep quality. OBJECTIVE: To assess the therapeutic efficacy of pergolide in improving sleep and subjective measures of well-being in patients with idiopathic RLS using polysomnography and clinical ratings. METHODS: In a randomized, double-blind, placebo-controlled crossover design we enrolled 30 patients with idiopathic RLS according to the criteria of the International RLS Study Group. All patients were free of psychoactive drugs for at least 2 weeks before the study. Patients were monitored using polysomnography, clinical ratings, and sleep diaries at baseline and at the end of a 4-week pergolide or placebo treatment period. The initial dosage of 0.05 mg pergolide was increased to the best subjective improvement paralleled by 20 mg domperidone tid. RESULTS: At a mean dosage of 0.51 mg pergolide as a single daily dose 2 hours before bedtime, there were fewer periodic leg movements per hour of time in bed (5.7 versus 54.9, p < 0.0001), and total sleep time was significantly longer (373 versus 261 minutes, p < 0.0001). Ratings of subjective sleep quality, quality of life, and severity of RLS were improved significantly without relevant adverse events. CONCLUSION: Pergolide given as a single low-to-medium bedtime dose in combination with domperidone provides a well-tolerated and effective treatment of sensorimotor symptoms and sleep disturbances in patients with primary RLS.

Long-term effects of pergolide in the treatment of restless legs syndrome.

An open follow-up of a controlled study in patients with restless legs syndrome (RLS) shows that the beneficial effect of pergolide on RLS symptoms persists throughout at least 1 year. Twenty-two patients of 28 (78.6%) continued to take pergolide. Polysomnographic measurements showed a persistent improvement of PLM index, PLMS arousal index, total sleep time, and sleep efficiency (p = 0.0001). Side effects, in particular nausea, were common but were well controlled by domperidone in most patients.

Treatment of idiopathic restless legs syndrome (RLS) with the D2-agonist cabergoline--an open clinical trial.

STUDY OBJECTIVES: To define the effective dose of cabergoline and to evaluate the tolerability and efficacy of cabergoline in patients with restless legs syndrome (RLS). DESIGN: Treatment of idiopathic RLS patients with cabergoline in a 12-week open label trial. Patients on levodopa therapy were allowed to either stop levodopa prior to study entry or to continue, taper or discontinue levodopa during the study. Efficacy parameters were assessed by polysomnography and subjective ratings at baseline and at week 12. Primary efficacy parameters were the number of PLM and total sleep time. SETTING: Dept. of Neurology, Sleep Disorders Center PATIENTS: Nine patients with moderate to severe RLS (age 38.1 to 64.3 years, mean 54.1 years) who had experienced insufficient benefit under levodopa therapy and/or in part developed daytime augmentation participated. At study entry five patients were still under levodopa therapy (400-800 mg). INTERVENTIONS: Up-titration of cabergoline (single evening dose) until RLS symptoms clearly improved. Initial comedication with domperidone 20 mg t.i.d. MEASUREMENTS AND RESULTS: At the endpoint all patients were on cabergoline monotherapy (mean dosage 2.1 mg, range 1 to 4 mg). Domperidone was stopped in all patients due to good tolerability. Polysomnographic data showed a significant reduction of the number of periodic leg movements (PLM) (195.8+/-109.1 (baseline) vs. 26.4+/-40.2 (12 weeks cabergoline monotherapy; p=0.002), PLM arousals (51.7+/-42.3 vs. 6.4+/-11.2; p=0.017) and PLM awakenings (10.4+/-7.8 vs. 1.0+/-1.7; p=0.001). Total sleep time was prolonged (302.7+/-50.7 vs. 379.4+/-59.8 min; p=0.018), sleep latency shortened (42.4+/-49.1 vs. 16.3+/-22.8 min; p=0.214) and sleep efficiency increased (63.1+/-10.5 vs. 79.1+/-12.5%; p=0.017). All patients reported a impressive relief or became free of RLS symptoms. CONCLUSION: Cabergoline is effective and well tolerated in restless legs syndrome especially in patients with severe RLS and those who developed augmentation under levodopa therapy.

L-dopa therapy of uremic and idiopathic restless legs syndrome: a double-blind, crossover trial.

We report the effects of a single bedtime dose of L-dopa 100-200 mg on sleep quality, frequency of periodic leg movements (PLM) and daily living in patients with idiopathic and uremic restless legs syndrome (RLS). Seventeen patients with idiopathic and 11 with uremic (on continuous hemodialysis) RLS were evaluated comparatively by polysomnography, actigraphy and subjective ratings in a randomized, controlled and double-blind crossover trial with L-dopa and placebo for 4 weeks each. Neurophysiologic assessments showed significant reduction of the number of periodic leg movements (p = 0.003) and the PLM-index (p = 0.005) most pronounced during the first 4 hours of bedtime after L-dopa (p = 0.001). Subjective evaluation confirmed improvement of sleep quality (p = 0.002) and showed significantly higher quality of life during daytime (p = 0.030) while the patients received L-dopa therapy. We conclude that L-dopa 100-200 mg proved to be effective in idiopathic RLS and for the first time under controlled conditions in uremic RLS without any severe side effects.

The entry machinery of flaviviruses.

We have been using the flavivirus tick-borne encephalitis virus (TBEV) as a model system for investigating the molecular mechanisms underlying the membrane fusion process mediated by a class II viral fusion protein, the flavivirus envelope protein E. In the mature virion this protein exists as a metastable dimer that dissociates at the acidic pH in endosomes and is converted into a more stable trimeric conformation. The dimer dissociation step liberates an internal fusion peptide that interacts with the target endosomal membrane, and then further conformational changes are believed to drive membrane fusion. Although flavivirus fusion appears to be a more facile and efficient process than that of alphaviruses, which also possess a class II viral fusion protein, the fusion mechanism in both viral systems involves structurally related interactions with lipids, specifically the 3beta-hydroxyl group at C3 of cholesterol. The class II viral fusion machineries are structurally different from those involving class I viral fusion proteins, such as those found in orthomyxoviruses, paramyxoviruses, retroviruses, and filoviruses, but have certain similarities in common with bacterial pore-forming proteins.

The interactions of the flavivirus envelope proteins: implications for virus entry and release.

Viral membrane proteins play an important role in the assembly and disassembly of enveloped viruses. Oligomerization and proteolytic cleavage events are involved in controlling the functions of these proteins during virus entry and release. Using tick-borne encephalitis virus as a model we have studied the role of the flavivirus envelope proteins E and prM/M in these processes. Experiments with acidotropic agents provide evidence that the virus is taken up by receptor-mediated endocytosis and that the acidic pH in endosomes plays an important role for virus entry. The envelope glycoprotein E undergoes irreversible conformational changes at acidic pH, as indicated by the loss of several monoclonal antibody-defined epitopes, which coincide with the viral fusion activity in vitro. Sedimentation analysis reveals that these conformational changes lead to aggregation of virus particles, apparently by the exposure of hydrophobic sequence elements. None of these features are exhibited by immature virions containing E and prM rather than E and M. Detergent solubilization, sedimentation, and crosslinking experiments provide evidence that prM forms a complex with protein E which prevents the conformational changes necessary for fusion activity. The functional role of prM before its endoproteolytic cleavage by a cellular protease thus seems to be the protection of protein E from acid-inactivation during its passage through acidic trans Golgi vesicles in the course of virus release.

Quantitative determination of IgM antibodies reduces the pitfalls in the serodiagnosis of tick-borne encephalitis.

BACKGROUND: Tick-borne encephalitis (TBE) is the most important arbovirus disease in parts of Europe and Asia. Its laboratory diagnosis depends on the detection of specific IgM antibodies which can be impeded by (1) long-time persistence of IgM antibodies after infection, (2) vaccine-induced IgM antibodies, and (3) cross-reactive IgM antibodies from other flavivirus infections. OBJECTIVES: To assess the extent of interference factors in the serodiagnosis of TBE that might lead to the false positive assignment of a recent infection. STUDY DESIGN: We quantified TBE virus-specific IgM and IgG antibodies in sera collected at different time points from cohorts of (1) 61 TBE patients, (2) 131 TBE vaccinees, and (3) 42 patients with recent dengue or West Nile virus infections. RESULTS: All of the TBE patients were IgM- and IgG-positive upon hospitalization and 87% of acute TBE sera had IgM antibody titers of >500 Arbitrary Units (AU). These titers rapidly declined and only 16% of TBE patients had low IgM titers ≥9 months after infection. Vaccine-induced as well as flavivirus cross-reactive IgM antibodies were rarely detectable and of low titer. CONCLUSIONS: Most of the potential problems of TBE serodiagnosis can be resolved by the quantification of IgM antibodies in a single serum sample taken upon hospitalization. High IgM values (>500 AU in our assay) are indicative of a recent infection. Lower IgM values, however, may require the analysis of a follow-up sample and/or a specific neutralization assay to exclude the possibilities of IgM persistence, vaccine-induced IgM antibodies or heterologous flavivirus infections.

Clinical data on restless legs syndrome: a dose-finding study with cabergoline.

Current treatment options for restless legs syndrome (RLS), based on the American Academy of Sleep Medicine practice parameters, favor dopaminergic agents. The drug of first choice is levodopa, which is now licensed for RLS in two European countries. However, the short duration of action and augmentation of symptoms under therapy may limit the clinical use of levodopa, especially in severely affected patients. An open pilot study shows that the long-acting dopamine agonist cabergoline is a promising new tool in the treatment of RLS. Results of a double-blind, controlled trial are pending.

[Therapy of idiopathic and uremic restless legs syndrome]. / Therapie des idiopathischen und urämischen Restless-legs-Syndroms.

Sensory and motor symptoms of the limbs, motor restlessness and an urge to move only at rest are the characteristics of the restless legs syndrome (RLS), which often leads to severe sleep disturbances. The clinical diagnosis can be made on the basis of the typical history, normal neurological findings and, in some cases, a positive family history, and can be confirmed by polysomnography. The indication for treatment depends on the patient's discomfort and the severity of the sleep disturbances. L-DOPA is the treatment of first choice both in idiopathic and uremic RLS. A bedtime dose of 100-200 mg L-DOPA standard plus decarboxylase inhibitor is effective against mild and moderate sleep disturbances in RLS. Titration of the dosage and additional treatment with sustained-release preparations of L-DOPA should be applied individually. Opioids and dopamine agonists are effective alternative treatments in idiopathic RLS. Benzodiazepines are indicated only in individual cases. Besides L-DOPA, uremic RLS patients can be treated with opioids and benzodiazepines. Various approaches in the treatment of idiopathic and uremic RLS are reviewed and the practical management of therapy is outlined.

[Restless legs syndrome and periodic limb movements in sleep]. / Restless-legs-Syndrom und "periodic limb movements in sleep".

Restless legs syndrome is one of the most common neurological disorders, with a prevalence of 2% to 9% in the elderly population. Sensory and motor symptoms of the legs and an urge to move that occur at rest may lead to severe sleep disturbances and are part of the syndrome. Typical history and normal neurological examination lead to the clinical diagnosis. Additional laboratory and neurophysiological investigations are necessary to rule out associated diseases. The indication for polysomnography to record periodic limb movements in sleep must be discussed in individual cases. Treatment strategies will be recommended individually according to the disease severity. In this article we present an overview of the clinical symptomatology and include recommendations on diagnosis and treatment of RLS and differentiation of RLS from periodic limb movement disorder. To this purpose, the Motor System and Sleep Work Group of the German Society of Sleep Medicine presents modified guidelines for diagnosis and treatment of RLS according to recent recommendations of the American Sleep Disorder Association.

Tick-borne encephalitis virus envelope protein E-specific monoclonal antibodies for the study of low pH-induced conformational changes and immature virions.

A set of ten monoconal antibodies (mabs) specific for the tick-borne encephalitis (TBE) virus envelope protein E were prepared and characterized with respect to their functional activities, the location of their binding sites on protein E and the involvement of their epitopes in acid pH-induced conformational changes and interactions with the precursor to the membrane protein (prM) in immature virions. The majority of these mabs mapped to the previously defined antigenic domain A. All of the mabs recognize parts of the E protein which undergo low pH-induced structural rearrangements believed to be necessary for the fusion activity of the virus, and six of the mabs define epitopes which are affected by the prM-E interaction in immature virions. They are therefore of potential value as specific reagents for studying the structure and function of protein E, as well as the function of the prM-E association. Five of the mabs exhibited neutralizing activity, and can therefore be used for the selection of escape mutants.

Structural requirements for low-pH-induced rearrangements in the envelope glycoprotein of tick-borne encephalitis virus.

The exposure of the flavivirus tick-borne encephalitis (TBE) virus to an acidic pH is necessary for virus-induced membrane fusion and leads to a quantitative and irreversible conversion of the envelope protein E dimers to trimers. To study the structural requirements for this oligomeric rearrangement, the effect of low-pH treatment on the oligomeric state of different isolated forms of protein E was investigated. Full-length E dimers obtained by solubilization of virus with the detergent Triton X-100 formed trimers at low pH, whereas truncated E dimers lacking the stem-anchor region underwent a reversible dissociation into monomers without forming trimers. These data suggest that the low-pH-induced rearrangement in virions is a two-step process involving a reversible dissociation of the E dimers followed by an irreversible formation of trimers, a process which requires the stem-anchor portion of the protein. This region contains potential amphipathic alpha-helical and conserved structural elements whose interactions may contribute to the rearrangements which initiate the fusion process.