Jochen Süss

Epidemiology and ecology of TBE relevant to the production of effective vaccines

This review focuses on the epidemiology and ecology of the tick-borne encephalitis (TBE) virus including all the factors influencing the ecology of the TBE virus, environmental and climatic conditions, the vectors and reservoir hosts and their interactions. Consequently, the structure and the conditions of natural foci of TBE are described, as well. Special emphasis is given on data regarding the virus prevalence in ticks in the field. In the second part of this review all the epidemiological and surveillance data of TBE in the 27 European countries and in the Far East with risk areas of TBE and in China and Japan are summarised. Therefore the review is a basis for travellers and inhabitants to get background information for their personal risk assessment.

Tick-borne encephalitis (TBE) in Germany - epidemiological data, development of risk areas and virus prevalence in field-collected ticks and in ticks removed from humans.

In Germany, 100-300 autochthonous clinical TBE cases have been recorded annually. There are high-risk areas in Bavaria and Baden-Wuerttemberg and ongoing low-risk areas in Hesse, Thuringia, and the Rhineland-Palatinate and single cases in Saxony. In order to be able to evaluate the epidemiological changes described here, it must be mentioned that a new definition of TBE risk areas was introduced on the district level in 1998 in Germany and in 2001 with the new Infection Protection Act (Infektionsschutzgesetz) which states that TBE is a notifiable disease. This led to the replacement of earlier surveillance systems and to many changes to data collection. In 1998 63 country and town districts were TBE risk areas, in 2001 79 and in 2002 86. There were new risk districts within Bavaria and Baden-Wuerttemberg and outside these regions in Thuringia, Hesse and the Rhineland-Palatinate. An interesting trend was observed in TBE epidemiology. The TBE incidence in Bavaria and Baden-Wuerttemberg has been stable on a high level for years; outside these areas it has steadily been climbing (Odenwald, Thuringia). On the basis of epidemiological data on TBE from the eastern part of Germany since 1960, it is obvious that major changes in virus activity in TBE risk areas also occurred in the past, the explanation of which has remained a matter for speculation. The epidemiological situation in the different risk areas for TBE in Germany was found to vary considerably, if one considers the surveillance data of the last 40 years. 1. Establishment of completely new low-risk areas. 2. Reactivation of formerly active areas with endemic latency. 3. High-risk areas with stable viral activity over long periods. 4. High-risk areas which have expanded and merged with low-risk areas. 5. High-risk areas which have developed into endemic areas or become inactive. High-risk TBE areas from 1960-1975 (i.e. Mecklenburg-Western Pomerania) have since completely disappeared. There were, at the same time, high-risk areas in Thuringia which had only become latent and have now obviously become active again. The Odenwald demonstrated growing virus activity in the 1990s. These changes in TBE activity in German risk areas over more than the last 40 years are presented schematically. This ongoing number of risk areas is certainly linked to the notification obligation and greater public awareness. Nevertheless, any effects of ecological and climatic changes on the natural foci cannot be ruled out nor can changes in human leisure behaviour. Local weather conditions also have a major effect on the TBE incidence. Warm and dry summers may cause low tick activities, rainy summers may lead to low exposure rates of human beings. Even changes in forms of agricultural production prompted by different political structures probably have an impact as do economic constraints which may lead to lower vaccination and higher exposure rates. Regular, systematic virus prevalence measurements from 1997 to 2002 in field-collected ticks in German high-risk areas do not indicate any risk increase nor do they suggest a downward trend. Studies on virus prevalence in questing versus partially engorged ticks indicate that we neither exactly know nor understand the real quantitative relations between the virus and the host. In a first study, virus prevalence in Ixodes ricinus removed from humans was examined. Humans which were exposed in some districts near Passau in Bavaria. In the autumn of 2001, virus prevalence of unengorged free-living nymphs (n = 820) in this area was 0.38 (0.08-1.1)% and of adults (n = 90) 1.17 (0.03-6.38)%. Surprisingly, virus prevalence in partially engorged ticks from the same area collected during the same period was significantly higher (nymphs, n = 86, 6.9% and adults, n = 129, 9.3%). Virus-positive partially engorged ticks were only found in districts known as risk areas. Nucleotide and deduced amino acid sequence data of the PCR products have confirmed the presence of virus prototype Neudoerfl only.

Tick-borne encephalitis in childhood - Consensus 2004

SummaryTick-borne encephalitis (TBE) is a communicable disease caused by a flavi-virus, ticks being the main vectors. The nervous system is affected, four clinical features of different severity are observed: meningitis, meningoencephalitis, meningoencephalomyelitis, meningoradiculoneuritis. TBE is a preventable disease, which is rapidly becoming a growing public health problem in Europe. So far, no causal treatment is possible but an efficient, safe vaccination is available. During the 6th meeting of the International Scientific Working Group on TBE with the main conference issue Tick-borne encephalitis in childhood an international consensus was achieved. In countries where TBE is endemic – and not prevented by immunization –, both children and adults are affected. The disease in children is generally milder, although severe illness may occur and even lead to permanent impairment of the quality of life due to neuropsychological sequelae. Therefore, immunization should be offered to all children living in or traveling to endemic areas.ZusammenfassungDie Frühsommer-Meningo-Enzephalitis ist eine durch Zecken übertragbare Krankheit des Zentralnervensystems. Vier wesentliche Krankheitsbilder unterschiedlichen Schweregrades sind bekannt: Meningitis, Meningoenzephalitis, Meningoenzephalomyelitis und Meningoradiculoneuritis. FSME als eine vermeidbare Erkrankung stellt ein zunehmendes Problem für die Volksgesundheit dar. Es gibt keine kausale Therapie, aber eine effiziente und sichere Impfung. im Rahmen der 6. Konferenz der International Scientific Working Group on TBE mit dem zentralen Thema FSME in der Kindheit wurde ein internationaler Konsensus erzielt. In den Ländern, wo FSME endemisch vorkommt – und nicht durch Impfung verhindert wird –, sind Kinder und Erwachsene betroffen. Der Krankheitsverlauf ist bei Kindern generell milder, es gibt aber sehr wohl schwere Verläufe. Unter Umständen kommt es sogar zu neuropsychologischen Spätschäden mit einer entsprechenden Verschlechterung der Lebensqualität. Deshalb sollte die Schutzimpfung allen Kindern, die in einem endemischen Gebiet wohnen oder dorthin reisen, ermöglicht werden.

A nested RT-PCR for the detection of tick-borne encephalitis virus (TBEV) in ticks in natural foci

We have developed a sensitive nested reverse-transcriptase polymerase chain reaction assay (n RT-PCR) for the detection of the tick-borne encephalitis virus (TBEV) RNA, especially in ticks. The primer pairs were selected from the 5-terminal noncoding region, a highly conserved part of the virus. The specificity was tested by computer homology searches of sequences as well as by the sequencing of the first and second amplificate, by Southern blot hybridization with a DIG-labelled oligonucleotide probe, and by restriction enzyme analysis. The method has proved to be very sensitive. The detection limit is about 20 fg of TBEV RNA per PCR run (25 microliters), or a single positive tick, i.e. (adult or nymph). The method can be used for comparative studies of the epidemiological situation, as well as for the screening of natural foci for the presence and circulation of TBEV or for the detection of TBEV-genome-sequences in clinical materials.

Characterization of tick-borne encephalitis (TBE) foci in Germany and Latvia (1997–2000)

Knowledge concerning the prevalence of the tick-borne encephalitis virus (TBEV) in wild living tick populations is very important for understanding the epidemiology of the disease and for immuno prophylactic strategy. In Germany high and low risk areas of TBE exist. In the years 1997-2000, 533 autochthonous clinical TBE cases were recorded, in the high-risk areas of Bavaria and Baden-Wuerttemberg 140 and 363, and in the low risk areas in Hesse (Odenwald) and Rhineland-Palatinate 22 and 8, respectively. Corresponding to these case reports we have measured the virus prevalence in free living ticks in these four risk areas and compared these findings with the situation in high-risk areas in Latvia. In the years 1997-2000, 2,797 clinical TBE cases were recorded in Latvia. For the studies in Germany, a total of 17,398 Ixodesricinus ticks (14,860 nymphs and 2,538 adults) were collected by flagging and examined for TBEV, in Latvia the corresponding numbers were 525 I. ricinus ticks (350 adults and 175 nymphs) and 281 I. persulcatus ticks (adults only). Information concerning annual and seasonal differences of the TBEV prevalence in natural TBE foci is not available in Germany. This paper is a continuation of the study (Süss et al., 1999), starting in 1997. We investigated every year, in May and September, the virus prevalence in ticks in high risk areas of Bavaria (8 foci) and Baden-Wuerttemberg (5 foci). A total of 15,400 ticks (13,100 nymphs and 2,300 adults) were examined for TBEV. The ticks were tested for the presence of TBEV-RNA using a sensitive, nested-RT-PCR. The virus prevalence in the Bavarian foci of the whole tick population ranged from 0.3 to 2.0% during these four years, in adults between 1.2 and 5.3% and in nymphs between 0.1 and 1.4%. In the high-risk areas of Baden-Wuerttemberg, in the Black Forest, the estimated virus prevalence rates of investigated ticks varied from 0.2 to 3.4%, in adults from 0 to 4.8%, and in nymphs from 0.2 to 3.4%. Using the same methods, we have also tested the low risk areas in the Odenwald (840 nymphs, 160 adults) and in Rhineland-Palatinate (920 nymphs, 78 adults). Ticks were collected in those areas where most TBE cases were registered. The virus prevalence in the Odenwald was 0% in adults and 0.5% in nymphs, whereas in ticks from Rhineland-Palatinate we have not found any positive PCR signal. Sequence data of the PCR products have shown that all strains in Germany were closely related to the central European virus prototype Neudoerfl. In I. ricinus ticks, collected in Riga county, the following virus prevalence rates were found: in females 2.4%, in males 3.7%, and in all adults 3.0%, in nymphs 2.4% and in the I. ricinus tick population examined 2.8%. The virus prevalence in I. persulcatus, collected in the eastern parts of Latvia was 6% in females, 4% in males and 5% in all adults. All the PCR products were sequenced and a phylogenetic tree was constructed. Studies in natural foci of TBE in Latvia have shown that I. ricinus carried the central European virus subtype (prototype Neudoerfl) whereas in I. persulcatus two strains have been found, the central European virus subtype (prototype Neudoerfl) and the Siberian virus subtype (prototype Vasilchenko). Sequences of the Far Eastern subtype have not been detected yet.

Goats and sheep as sentinels for tick-borne encephalitis (TBE) virus – Epidemiological studies in areas endemic and non-endemic for TBE virus in Germany

The aim of the study was to examine grazing goats and sheep as specific sentinels for characterization of the tick-borne encephalitis virus (TBEV)-related risk in an area by means of serosurveillance tests in the German federal states Baden-Wuerttemberg, Bavaria, Thuringia, North Rhine-Westphalia, Lower Saxony, Schleswig-Holstein, and Mecklenburg-West Pomerania. A total of 3590 sheep sera and 3793 goat sera was collected in 2003 and 2006-2009 and were examined by ELISA screening and confirmed by serum neutralization test. Considerable differences in seroprevalence were seen between single flocks in districts in Baden-Wuerttemberg, Bavaria, and Thuringia with values between 0 and 43% which confirmed the patchy pattern of TBEV foci that can range in size from very small to large. The here described serological screening may be a helpful tool for an early warning system of a potential TBEV risk. Testing of 1700 ticks by real-time RT-PCR in two districts in Baden-Wuerttemberg revealed only one positive tick, thus illustrating the problems of expensive and time-consuming tick collection.

Genetic Characterization of a Porcine H1N2 Influenza Virus Strain Isolated in Germany

We examined selected influenza virus strains from various outbreaks of respiratory diseases among pigs in Germany. Besides H3N2 and H1N1 subtypes presently circulating in Europe, an H1N2 influenza virus was isolated in 2000. Sequence analysis of hemagglutinin, neuraminidase and matrix genes indicated that this virus is a reassortant of human H1N1 and human H3N2 strains with an internal gene, the M gene of avian origin. It is a result of a multiple reassortment event.

Annual and seasonal variation of tick-borne encephalitis virus (TBEV) prevalence in ticks in selected hot spot areas in Germany using a nRT-PCR: Results from 1997 and 1998

The prevalence of tick-borne encephalitis virus (TBEV) in Ixodes ricinus tick populations in endemic areas of Germany with the highest TBE risk is unknown. Annual and seasonal differences in TBEV prevalence have also not been studied. Against this background, in May 1997 we started a systematic virus surveillance programme in ticks collected in locations known to have a high incidence of autochthonous TBE cases. These were 5 locations in Baden-Württemberg (Black Forest) and 8 locations in Bavaria (surrounding Passau). Field-collected ticks were randomly assigned to pools of 10 adults or 20 nymphs, respectively. The tick pools were tested for the presence of TBEV-RNA using a newly developed, sensitive nested reverse transcriptase polymerase chain reaction assay (nRT-PCR). The primer pairs were selected from the 5-terminal noncoding region, a highly conserved part of the virus. The specificity was tested by computer homology searches of sequences, as well as by sequencing of the first and the second amplificates, by Southern blot hybridisation with a DIG-labelled oligonucleotide probe, and by restriction enzyme analysis. The method has proved to be very sensitive, with a detection limit of 20 fg of TBEV RNA per PCR run, or a single positive tick. Based on biostatistical considerations a sample size of at least 1000 ticks per estimation point was chosen. The estimated TBEV prevalence and confidence intervals (CI) were calculated from the nRT-PCR results of pooled samples (10 adults or 20 nymphs) using appropriate formulae for pooled testing. In order to identify the estimated TBEV prevalence as well as to assess the influence of annual and seasonal factors on TBEV prevalence, ticks were sampled twice a year (May and September) in 1997 and 1998 at exactly identical sites. These sites were selected because they were known to have had the highest incidence of autochthonous TBE cases during the previous 10 years. On sampling days, relevant local meteorological data were also noted. In total, 8500 I. ricinus ticks were investigated in this study, 4270 (3540 nymphs, 730 adults) from the Black Forest habitats, and 4230 (3680 nymphs, 550 adults) from the Bavarian locations. In the foci near Freiburg (Black Forest), the estimated virus prevalence was relatively high in the whole tick population, during 1997 with only slight seasonal differences [3.4% (confidence interval, CI, 2.3-4.8%) in May and 2.9% (CI 1.7-4.5%) in September]. In contrast, in 1998, in the same foci the estimated TBEV prevalence was considerably lower [1.1% (CI 0.5-2.0%) in May and 0.6% (CI 0.2-1.4%) in September]. Thus, while the seasonal differences again remained low, the annual variation was marked. In the Bavarian foci in 1997, the estimated virus prevalence of the whole tick population studied was lower than in the Black Forest foci and the seasonal fluctuations were low: in May 1997 0.9% (CI 0.4-1.8%) of the ticks were positive, in September 1.1% (CI 0.5-1.9%). In 1998, in May 2.0% (CI 1.1-3.3%) of the ticks were positive, and in September 1.1% (CI 0.5-2.1%). For the whole study period, every 50th to 100th I. ricinus nymph or adult in the Passau region was calculated to give a positive signal in the nRT-PCR. The TBEV prevalence data indicate that residents and visitors of areas in Germany known for high endemic activity take a significant risk of contracting TBEV infection, if bitten by ticks. In addition, the data suggest that annual fluctuations may exist in the whole tick population studied. Seasonal fluctuations of the virus prevalence in ticks were small.

Molecular Epidemiology of Porcine H3N2 Influenza A Viruses Isolated in Germany between 1982 and 2001

We examined influenza virus strains of the subtype H3N2 from outbreaks of respiratory diseases in swine herds in Germany. Four different clusters can be distinguished when comparing parts of the HA1 gene from porcine H3N2 isolates analyzed between 1982 and 2001. Comparison between these clusters reveals a bp homology of less than 90%. In contrast, the homology within the clusters is between 93.7 and 100%. Each of these clusters was confined to a specific time period. For the NA gene an additional cluster is formed by the porcine H1N2 isolate. The findings that different subtypes and drift variants are circulating in the German pig population explain the emergence of new influenza virus variants and the need for continued surveillance of swine.

Tick-Borne Encephalitis Virus Habitats in North East Germany: Reemergence of TBEV in Ticks after 15 Years of Inactivity

The incidence of tick-borne encephalitis has risen in Europe since 1990 and the tick-borne encephalitis virus (TBEV) has been documented to be spreading into regions where it was not previously endemic. In Mecklenburg-West Pomerania, a federal state in Northern Germany, TBEV was not detectable in over 16,000 collected ticks between 1992 and 2004. Until 2004, the last human case of TBE in the region was reported in 1985. Following the occurrence of three autochthonous human cases of TBE after 2004, however, we collected ticks from the areas in which the infections were contracted. To increase the chance of detecting TBEV-RNA, some of the ticks were fed on mice. Using nested RT-PCR, we were able to confirm the presence of TBEV in ticks for the first time after 15 years. A phylogenetic analysis revealed a close relationship between the sequences we obtained and a TBEV sequence from Mecklenburg-East Pomerania published in 1992 and pointed to the reemergence of a natural focus of TBEV after years of low activity. Our results imply that natural foci of TBEV may either persist at low levels of activity for years or reemerge through the agency of migrating birds.