Comparison of whole genomes of tick-borne encephalitis virus from mountainous alpine regions and regions with a lower altitude.

Tick-borne encephalitis (TBE) has been a notifiable disease in Germany since 2001. Its causative agent, the TBE virus (TBEV), is the most important arbovirus in Europe and Northern Asia. The illness, caused by the European Subtype usually displays flu-like symptoms, but can result in sequelae and, in 2 % of all cases, in death. Over the last few decades, the virus has spread into new habitats, such as higher altitudes in the Alpine region. For this study, it was hypothesized that the environmental challenges that the virus might be exposed to at such altitudes could lead to the selection of viral strains with a higher resilience to such environmental factors. To determine whether strains identified at higher altitudes possessed different genetic traits compared to viruses from lower altitudes, an analysis of viral genomes from higher Alpine altitudes (> 500 m above sea level) (n = 5) and lower altitudes (< 500 m above sea level) (n = 4) was performed. No common phylogenetic ancestry or shared amino acid substitutions could be identified that differentiated the alpine from the lowland viral strains. These findings support the idea of many individual introductions of TBEV into the alpine region and the establishment of foci due to non-viral specific factors such as favorable conditions for vector species and host animals due to climate change.

Tick-Borne Encephalitis Virus Nonstructural Protein 1 IgG Enzyme-Linked Immunosorbent Assay for Differentiating Infection versus Vaccination Antibody Responses.

Tick-borne encephalitis virus (TBEV) is an important central nervous system (CNS) infection in Europe and Asia. It is a flavivirus in the tick-borne group. Effective vaccines against TBE are available in the affected countries. However, diagnosing TBE is challenging due to cross-reactive antibodies between different viruses of the genus Flavivirus, family Flaviviridae. Differentiation between infection-induced and vaccine-induced antibodies can be difficult and in many cases impossible, due to the increasing vaccination rate against TBEV. We present a new approach to detect antibodies against the TBEV nonstructural protein 1 (NS1) as a diagnostic marker, which is exclusively indicative for virus replication in natural infection, on the basis of an enzyme-linked immunosorbent assay (ELISA). A total of 188 anonymous serum samples from the National Consultant Laboratory for TBEV were included in our study. The assay was validated according to the European Laboratory Norm DIN EN ISO 15189 for diagnostic use. The ELISA for the detection of TBEV NS1 specific IgG class antibodies has demonstrated a sensitivity of >94% and a specificity of >93% in broadly cross-reacting sera from patients with vaccinations against flaviviral diseases and single or multiple flavivirus infections, respectively. The detection of anti-NS1 antibodies is feasible and facilitates reliable differentiation between different flavivirus infections, TBEV infection, and TBE vaccination.

A cluster of two human cases of tick-borne encephalitis (TBE) transmitted by unpasteurised goat milk and cheese in Germany, May 2016.

In May 2016, two cases of tick-borne encephalitis (TBE) were confirmed by serology (positive IgM and IgG antibodies against TBE virus (TBEV) in serum), with a possible link to raw milk and cheese from a goat farm in a region in Baden-Württemberg, Germany not previously known as TBE-endemic. The outbreak investigation identified 32 consumers of goat dairy products (29 consumers, one farm employee, two owners) of whom none had IgM antibodies against TBEV 3-8 weeks after consumption. Of the 27 notified TBE cases in the State, none reported consumption of raw goat milk or cheese from the suspected farm. Five of 22 cheese samples from 18 different batches were RT-qPCR-positive for TBEV -genome, and two of the five samples were confirmed by virus isolation, indicating viability of TBEV in the cheese. Nine of the 45 goats had neutralising TBEV antibodies, two of them with a high titre indicating recent infection. One of 412 Ixodes ricinus was RT-qPCR-positive, and sequencing of the E gene from nucleic acid extracted from the tick confirmed TBEV. Phylogenetic analyses of tick and cheese isolates showed 100% amino acid homology in the E gene and a close relation to TBEV strains from Switzerland and Austria.

[Threat of transmission of infectious pathogens by Ixodes ricinus ticks in Germany]. / Gefahren der Übertragung von Krankheitserregern durch Schildzecken in Deutschland.

Tick-transmitted diseases are of great importance for the general health of the German population. Several viruses, such as tick-borne encephalitis virus (TBEV), Uukuniemi virus, Tribec virus, Eyach virus or bacteria, such as Borrelia, Rickettsiae, Francisella tularensis, Anaplasma phagocytophilum, Candidatus Neoehrlichia mikurensis (CNM) and Coxiella burnetii were detected in the most prominent tick in Germany, the hard tick Ixodes ricinus. While infections, such as TBE and Lyme disease are well known, other infections are hardly known even among experts. Although there have been a few descriptions of isolated cases in Germany, a systematic investigation regarding the distribution and the pathogenic potential of these pathogens is still lacking. In particular elderly people and people with underlying diseases seem to be mostly affected. The importance of new infectious disease agents, such as Candidatus Neoehrlichia mikurensis but also of long known pathogens, such as Rickettsiae still remains unclear, while some of them could be detected in 20 % of investigated ticks. Whether climate change contributes to the further distribution of these infectious agents remains unclear and requires further investigation. The increasing initiatives to create natural environments and the trend towards spending more time in nature for recreational activities will increase the danger of coming into contact with ticks and the respective infectious agents. Considering these circumstances an increase of diseases caused by these pathogens is to be expected.

Relation of genetic phylogeny and geographical distance of tick-borne encephalitis virus in central Europe.

Tick-borne encephalitis virus (TBEV) is the most important arboviral agent causing disease of the central nervous system in central Europe. In this study, 61 TBEV E gene sequences derived from 48 isolates from the Czech Republic, and four isolates and nine TBEV strains detected in ticks from Germany, covering more than half a century from 1954 to 2009, were sequenced and subjected to phylogenetic and Bayesian phylodynamic analysis to determine the phylogeography of TBEV in central Europe. The general Eurasian continental east-to-west pattern of the spread of TBEV was confirmed at the regional level but is interlaced with spreading that arises because of local geography and anthropogenic influence. This spread is reflected by the disease pattern in the Czech Republic that has been observed since 1991. The overall evolutionary rate was estimated to be approximately 8×10(-4) substitutions per nucleotide per year. The analysis of the TBEV E genes of 11 strains isolated at one natural focus in zdár Kaplice proved for the first time that TBEV is indeed subject to local evolution.

Chikungunya fever in two German tourists returning from the Maldives, September, 2009.

This report describes the first isolation and molecular characterisation of a chikungunya virus from two German tourists who became ill after a visit to the Maldives in September 2009. The virus contained the E1 A226V mutation, shown to be responsible for an adaptation to the Asian tiger mosquito Aedes albopictus. The E1 coding sequence was identical to chikungunya virus isolates from Sri Lanka and showed three nt-mismatches to the only available E1 nt sequence from the Maldives.

Tick-borne encephalitis virus vaccination breakthrough infections in Germany: a retrospective analysis from 2001 to 2018.

OBJECTIVES: There are few data available regarding the clinical course of tick-borne encephalitis virus (TBEV) vaccination breakthrough infections. The published studies suggest that vaccination breakthrough infections may have a more severe course than native TBEV infection in unvaccinated individuals-potentially due to antibody-dependent enhancement. Here we report a large analysis of vaccination breakthrough infections. METHODS: This retrospective analysis was based on a national surveillance dataset spanning the years 2001-2018. Variables reflecting disease severity, such as 'CNS symptoms', 'myelitis', 'fatal outcome' and 'hospitalization' were analysed as well as general epidemiological variables. Cases were categorized as 'unvaccinated' or 'ever vaccinated', the latter category including cases with at least one dose of a TBEV vaccine. RESULTS: A total of 6073 notified TBEV infection cases were included in our analysis. Sufficient data on vaccination status were available for 95.1% of patients (5777/6073); of these, 5298 presented with a native infection. A total of (334/5777) cases developed an infection despite having been vaccinated at least once. Comparing unvaccinated patients with those with at least one vaccination, we find an odds ratio (OR) 2.73, (95% confidence interval (CI) 0.79-9.50) regarding the variable fatal outcome that did not reach statistical significance. Analysing the clinical variables 'CNS symptoms' and 'myelitis', there is no difference between these groups (OR 0.86, 95% CI 0.68-1.08; and OR 1.30, 95% CI 0.74-2.27 respectively). Patients who were vaccinated and had an assumed protection at symptom onset (n = 100) had a higher risk for the development of myelitic symptoms (OR 2.21, 95% CI 1.01-4.86]) than unvaccinated patients. CONCLUSION: Our findings could neither verify that vaccination breakthrough infections might cause a more severe disease than native infections nor prove a clear antibody-dependent enhancement phenomenon. It remains unclear whether the increased myelitis risk in a subgroup of vaccinated patients is a true effect or confounded.

Imported aseptic meningitis due to Toscana virus acquired on the island of Elba, Italy, August 2008.

We describe a case of aseptic meningitis due to Toscana Virus imported to Switzerland and discuss the epidemiological situation. To our knowledge this is the first description of this infection acquired on the Island of Elba.

[Severe tick-borne encephalomyelitis with lack of cerebrospinal fluid pleocytosis]. / Schwere Frühsommer-Meningo-Enzephalomyelitis ohne Liquor-Pleozytose.

Tick borne encephalitis (TBE) is an important viral encephalitis in central and eastern Europe. Cerebrospinal fluid (CSF) pleocytosis has been described in all published patients so far. This may be due to selection bias, however, as CSF pleocytosis is often used as a case definition parameter. The frequency of TBE without CSF pleocytosis is unknown. We report two cases who developed severe TBE without CSF pleocytosis. A normal CSF cell count should therefore not discourage from the differential diagnosis of TBE and deter from serological testing in patients with a clinical constellation suggesting TBE.

Rickettsia spp. in Ixodes ricinus ticks in Bavaria, Germany.

This study aims to provide information on the occurrence of spotted fever rickettsiae in Ixodes ricinus ticks in southern Germany. A total of 2,141 I. ricinus ticks was collected in Bavaria. Pools of 5-10 ticks were studied by a PCR targeting the rickettsial citrate synthase gene gltA. The average prevalence rate was 12% (257 of 2,141). Sequencing data exclusively identified Rickettsia helvetica DNA. Results and other data demonstrate the possible role of R. helvetica in I. ricinus as a source of human infections in southern Germany.

Complete Genome Sequence of a Low-Virulence Tick-Borne Encephalitis Virus Strain.

We report here the complete genome sequence (GenBank accession no. KX268728) of tick-borne encephalitis strain HB171/11, isolated from an Ixodes ricinus tick from a natural focus where human neurological disease is rare. The strain shows unique characteristics in neuroinvasiveness and neurovirulence.

Prevalence of infection with dengue virus among international travelers.

BACKGROUND: Dengue has been recognized as a potential hazard to tourists. A prospective, controlled study in the outpatient clinic of a German infectious disease clinic was conducted to assess the prevalence of dengue virus infection among international travelers. METHODS: Serum samples from 130 patients with signs or recent history clinically compatible with dengue (fever, headache, muscle and joint pain, or rash), 95 matched controls with diarrhea, and 26 patients who never visited a country endemic for dengue were investigated. RESULTS: Nine (6.9%) of the 130 patients with compatible symptoms and 1 (1%) of the 95 controls with diarrhea developed rising antibody titers against dengue virus. Of these 10 patients with probable dengue infection, 6 had been to Thailand, 2 to Malaysia, and 1 each to Indonesia and Brazil. CONCLUSIONS: Infection with dengue virus appears to be a realistic threat to travelers to Southeast Asia. Symptoms commonly associated with dengue, such as fever, myalgia, arthralgia, and vomiting, can be helpful for diagnosis when present, but the absence of typical symptoms does not exclude infection.

Cost reduction of unlinked testing for anti-HIV by investigation of pooled sera.

Anti-HIV-1 testing of pooled sera was found to be an excellent method of considerably reducing the costs and workload of public health surveillance programmes without the loss of important information. All 54 sera found anti-HIV-1 positive in diagnostic testing and 61 out of 70 sera (87%) found positive in surveillance studies were detected when tested at a dilution of 1:10 with anti-HIV-1-negative human sera. This acceptable loss of sensitivity could be reduced further by lowering the cut-off at which single sera of pools were tested: 96% of the above 70 anti-HIV-1-positive sera were detected. During testing of 618 pools containing 6180 sera from a low-risk population, lowering of the cut-off increased the number of pools with testing of individual sera from five (containing one confirmable anti-HIV-1-positive serum each) to only six (containing no anti-HIV-1-positive serum). Therefore, testing of pooled sera from a low-prevalence population group reduced the costs of testing about ninefold and technician time by about 25-50%.

Thunderclap headache caused by Erve virus?

Systematic studies of a possible human neuropathogenicity of the Erve virus have not yet been carried out. In a randomized, blind study 166 patients with viral encephalitis, 46 patients with cerebral hemorrhage, 72 patients with "thunderclap" headache, and 205 healthy blood donors were examined by indirect immunofluorescence for Erve virus antibodies. None of the patients with encephalitis, two patients with cerebral hemorrhage (4.3%), 10 patients with thunderclap headache (13.9%; p < 0.0001), and two blood donors (1.0%) exhibited antibodies against the Erve virus. These results suggest a human pathogenicity of the Erve virus for the first time.

Malignant hemangioendothelioma of the thyroid. Its immunohistochemical discrimination from undifferentiated thyroid carcinoma.

Eighteen cases of malignant hemangioendothelioma (MHE) of the thyroid and 16 cases of undifferentiated thyroid carcinoma were investigated immunohistochemically with antibodies against endothelial cell specific markers (factor VIII-related antigen, BMA 120, blood group isoantigens, Ulex europaeus agglutinin I), thyroglobulin, and the intermediate filament proteins vimentin and cytokeratin. All MHE were positive for factor VIII-related antigen and vimentin, in 14 of 18 cases for BMA 120, and in 9 of 18 cases for U. europaeus. All other markers were negative in MHE. Endothelial cell specific markers were commonly negative in undifferentiated carcinomas with one exception (one case was moderately positive for U. europaeus). Twelve of 16 undifferentiated carcinomas showed vimentin positivity, and 8 of 16 showed cytokeratin positivity. Four cases showed a vimentin/cytokeratin coexpression. It is concluded that the endothelial origin of MHE can be shown by certain endothelial cell markers in almost all cases.

Identification of molecular defects causing congenital adrenal hyperplasia by cloning and differential hybridization of polymerase chain reaction-amplified 21-hydroxylase (CYP21) genes.

Congenital adrenal hyperplasia (CAH), one of the most common autosomal recessive disorders, is caused primarily by defects in the gene encoding steroid 21-hydroxylase, CYP21B. The molecular diagnosis of CAH, important for prenatal diagnosis, carrier detection, and a better understanding of the various clinical CAH forms, is complicated by the close proximity of a highly similar pseudogene, CYP21A, containing (and probably donating, by gene conversion-like events) most of the defects underlying CAH. In this study, we describe an efficient strategy to identify molecular defects causing CAH: polymerase chain reaction-amplified CYP21 loci are cloned and hybridized to a set of oligonucleotides, allowing rapid and allele-specific identification of all known CYP21B mutations relevant to 21-hydroxylase function. Possible new mutations can be identified by subsequent nucleic acid sequencing provided they reside within the cloned CYP21B fragment (from the TATA box to the 8th of the 10 CYP21B gene exons). Using this method, the CYP21B gene mutations of a heterozygous carrier and 25 CAH patients have been identified by oligonucleotide hybridization. All disease haplotypes seem to have been generated by recombinational events involving the CYP21A pseudogene. In 5 individuals, these data were subsequently verified by nucleic acid sequencing. The procedure can be used for diagnostic applications and may facilitate identification of new CYP21B defects.

Arboviruses causing neurological disorders in the central nervous system.

Arthropod-borne viruses are important causes of diseases of the central nervous system. In addition to the tick-borne encephalitis viruses, other arboviruses in Europe are known to cause neurological disorders. Among them are West Nile, California group, Bhanja, Erve, Kemerovo group, Eyach, and Thogoto viruses. The ecologies and epidemiologies of these viruses are presented and their medical importance as travel-related diseases is discussed.

First Report of Littleleaf Disease Caused by Phytophthora cinnamomi on Pinus occidentalis in the Dominican Republic.

Pinus occidentalis Sw. is an endemic species of the Caribbean island of Hispaniola (Dominican Republic and Haiti). It shows an extreme ecological plasticity and grows on a wide range of soil types from 0 to 3,175 m in elevation with annual mean temperatures ranging from 6 to 25°C and annual precipitation of 800 to 2,300 mm. P. occidentalis is a major component of forests above 800 m in elevation and forms pure climax forests above 2,000 m (4). For more than 10 years, stands of P. occidentalis in the Sierra (Cordillera Central) growing on a wide range of site conditions have suffered from a serious widespread disease. Symptoms include yellowing and dwarfing of needles, a progressive defoliation and dieback of the crown, and finally, death of weakened trees often caused by attacks by secondary bark beetles. Mature stands are mainly affected, but the disease is also present in plantations and natural regeneration that is older than 10 years. Disease spread is rapid, and occurs mainly along roads and from diseased trees downslope following the path of water runoff. Initially, Leptographium serpens was isolated from necrotic roots and was thought to be the causal agent (1). However, the symptoms of the disease more closely resemble those of littleleaf disease of P. echinata and P. taeda in the southeastern United States, which is caused by the aggressive fine-root pathogen Phytophthora cinnamomi Rands (3). Moreover, spread and dynamics of the disease are similar to the diebacks of Chamaecyparis lawsoniana in Oregon and Eucalyptus spp. in western Australia, which are caused by the introduced soilborne pathogens Phytophthora lateralis and Phytophthora cinnamomi, respectively. Soil samples containing the rhizosphere and fine roots of diseased P. occidentalis trees were collected in February 2002 at five sites near Celestina and Los Montones (Dominican Republic) and transported to the Bavarian State Institute of Forestry. The pathogen was baited from the soil by floating 3- to 7-dayold leaves of Quercus robur seedlings over flooded soil and placing the leaves on selective PARPNH agar (2). Phytophthora cinnamomi was isolated from the soil of all five sites. Crossing with A1 and A2 tester strains of Phytophthora cinnamomi confirmed that all isolates belong to the A2 mating type. In cross sections of necrotic fine roots, characteristic structures of Phytophthora cinnamomi such as nonseptate hyphae and chlamydospores could be observed. Our results indicate that the disease of P. occidentalis is caused by the introduced pathogen Phytophthora cinnamomi. Because of the ecological and economical importance of P. occidentalis, the disease poses a major threat to forestry in the Dominican Republic. Future research should include the mapping of the disease, pathogenicity tests on P. occidentalis and alternative pine species, in particular P. caribaea, screening for resistance in the field, and testing of systemic fungicides such as potassium phosphonate, which is known to be effective against Phytophthora cinnamomi. References: (1) G. Dobler. Manejo y Tablas de Rendimiento de Pinus occidentalis. Plan Sierra, San José de las Matas, Dominican Republic, 1999. (2) T. Jung et al. Plant Pathol. 49:706, 2000. (3) S. W. Oak and F. H. Tainter. How to identify and control littleleaf disease. Protection Rep. R8-PR12, USDA Forest Service Southern Region, Atlanta, Georgia, 1988. (4) L. Sprich. Allg. Forst. Jagdztg. 168:67, 1997.

Clinical value of specific intrathecal production of antibodies.

The production of intrathecal antibodies is considered a highly specific marker for an infection of the central nervous system (CNS), e.g. borreliosis or tick-borne encephalitis (TBE). To investigate the validity of this assumption, we examined records of patients who had been hospitalized between 1989 and 1995, who were tested for borreliosis (n = 8003) and TBE (n = 904) and whose cerebrospinal fluid (CSF) had subsequently tested positive for intrathecal production of antibodies. The time period between the beginning of the symptoms and the time of the CSF examination ranged from one day to six weeks. Seventy-seven patients showed a production of intrathecal antibodies against Borrelia burgdorferi. Three of these patients were false positives with no history and no clinical signs of neuroborreliosis. In two cases, this was due to a non-specific cross-reaction caused by a preceding infection with syphilis. The third false positive was possibly caused by an earlier administration of immunoglobulins. Three patients showed a production of intrathecal antibodies against TBE virus. Two of these patients were false positives. In one case, we suspect that the production of intrathecal antibodies was caused by a non-specific immune reaction during an acute neuroborreliosis. One year earlier, the patient had contact with TBE virus through a vaccination against TBE. The cause of the second false positive is unclear, the clinical findings, acute encephalitis and the serological analysis suggest a cross-reaction with a virus similar to TBE. A specific intrathecal production of antibodies is not a proof for an infection of the CNS. In unclear cases, one should carry out a Western blot analysis or, if one suspects a case of TBE, a neutralization test.