Tick-Borne Encephalitis Virus Structural Proteins Are the Primary Viral Determinants of Non-Viraemic Transmission between Ticks whereas Non-Structural Proteins Affect Cytotoxicity.

Over 50 million humans live in areas of potential exposure to tick-borne encephalitis virus (TBEV). The disease exhibits an estimated 16,000 cases recorded annually over 30 European and Asian countries. Conventionally, TBEV transmission to Ixodes spp. ticks occurs whilst feeding on viraemic animals. However, an alternative mechanism of non-viraemic transmission (NVT) between infected and uninfected ticks co-feeding on the same transmission-competent host, has also been demonstrated. Here, using laboratory-bred I. ricinus ticks, we demonstrate low and high efficiency NVT for TBEV strains Vasilchenko (Vs) and Hypr, respectively. These virus strains share high sequence similarity but are classified as two TBEV subtypes. The Vs strain is a Siberian subtype, naturally associated with I. persulcatus ticks whilst the Hypr strain is a European subtype, transmitted by I. ricinus ticks. In mammalian cell culture (porcine kidney cell line PS), Vs and Hypr induce low and high cytopathic effects (cpe), respectively. Using reverse genetics, we engineered a range of viable Vs/Hypr chimaeric strains, with substituted genes. No significant differences in replication rate were detected between wild-type and chimaeric viruses in cell culture. However, the chimaeric strain Vs[Hypr str] (Hypr structural and Vs non-structural genomic regions) demonstrated high efficiency NVT in I. ricinus whereas the counterpart Hypr[Vs str] was not transmitted by NVT, indicating that the virion structural proteins largely determine TBEV NVT transmission efficiency between ticks. In contrast, in cell culture, the extent of cpe was largely determined by the non-structural region of the TBEV genome. Chimaeras with Hypr non-structural genes were more cytotoxic for PS cells when compared with Vs genome-based chimaeras.

Evaluation of chimeric yellow fever 17D/dengue viral replication in ticks.

Chimeric yellow fever 17D/DENV-1-4 viruses (CYD-1-4) have been developed as a tetravalent dengue vaccine candidate which is currently being evaluated in efficacy trials in Asia and America. While YF 17D and DENV are mosquito-borne flaviviruses, it has been shown that CYD-1-4 do not replicate after oral infection in mosquitoes and are not transmitted to new hosts. To further document the risk of environmental dissemination of these viruses, we evaluated the replication of CYD-1-4 in ticks, the vector of tick-borne encephalitis virus (TBEV), another member of the flavivirus family. Females of two hard tick species, Ixodes ricinus and Rhipicephalus appendiculatus, were inoculated intracoelomically with CYD-1-4 viruses and parent viruses (DENV-1-4 and YF 17D). Virus persistence and replication was assessed 2, 16, and 44 days post-inoculation by plaque titration and qRT-PCR. CYD-1-4 viruses were detected in I. ricinus ticks at early time points post-inoculation, but with infectious titers at least 100-fold lower than those observed in TBEV-infected ticks. Unlike TBEV, complete viral clearance occurred by day 44 in most ticks except for CYD-2, which had a tendency to decline. In addition, while about 70% of TBEV-infected I. ricinus nymphs acquired infection by co-feeding with infected tick females on non-viremic hosts, no co-feeding transmission of CYD-2 virus was detected. Based on these results, we conclude that the risk of dissemination of the candidate vaccine viruses by tick bite is highly unlikely.

Effect of saliva from horse fly Hybomitra bimaculata on kinetic properties of Na,K-ATPase: possible role in regulation of relaxation.

The possible involvement of salivary gland extract (SGE) from horse flies in modifying hyperpolarization and relaxation via alterations in functional properties of sarcolemmal Na,K-ATPase in the host tissue was tested in vitro by application of various amounts of SGE from Hybomitra bimaculata.SGE in the amount of 3 µg proteins representing approximately the equivalent of one salivary gland of Hybomitra bimaculata induced a stimulatory effect on Na,K-ATPase at all ATP concentrations applied. This effect resulted from the improved ATP-binding site affinity in the Na,K-ATPase molecule, as implicated by the reduction in K(M). Increasing the amount of SGE to 6.5 µg resulted in inhibition of the enzyme, which was characterized by reduction in V(max) and also K(M). This suggests that in the presence of relatively high Hybomitra bimaculata SGE concentration some SGE components affect Na,K-ATPase, when ATP is already bound to the enzyme.Our results indicate that SGE from the horse fly Hybomitra bimaculata contain at least two different biologically active compounds modifying the acute recovery and maintenance of excitability during contractile activity in the host tissue by affecting Na,K-ATPase with opposite effects, depending on the ratio of SGE-proteins to proteins of the host tissue.

Non-hemagglutinating flaviviruses: molecular mechanisms for the emergence of new strains via adaptation to European ticks.

Tick-borne encephalitis virus (TBEV) causes human epidemics across Eurasia. Clinical manifestations range from inapparent infections and fevers to fatal encephalitis but the factors that determine disease severity are currently undefined. TBEV is characteristically a hemagglutinating (HA) virus; the ability to agglutinate erythrocytes tentatively reflects virion receptor/fusion activity. However, for the past few years many atypical HA-deficient strains have been isolated from patients and also from the natural European host tick, Ixodes persulcatus. By analysing the sequences of HA-deficient strains we have identified 3 unique amino acid substitutions (D67G, E122G or D277A) in the envelope protein, each of which increases the net charge and hydrophobicity of the virion surface. Therefore, we genetically engineered virus mutants each containing one of these 3 substitutions; they all exhibited HA-deficiency. Unexpectedly, each genetically modified non-HA virus demonstrated increased TBEV reproduction in feeding Ixodes ricinus, not the recognised tick host for these strains. Moreover, virus transmission efficiency between infected and uninfected ticks co-feeding on mice was also intensified by each substitution. Retrospectively, the mutation D67G was identified in viruses isolated from patients with encephalitis. We propose that the emergence of atypical Siberian HA-deficient TBEV strains in Europe is linked to their molecular adaptation to local ticks. This process appears to be driven by the selection of single mutations that change the virion surface thus enhancing receptor/fusion function essential for TBEV entry into the unfamiliar tick species. As the consequence of this adaptive mutagenesis, some of these mutations also appear to enhance the ability of TBEV to cross the human blood-brain barrier, a likely explanation for fatal encephalitis. Future research will reveal if these emerging Siberian TBEV strains continue to disperse westwards across Europe by adaptation to the indigenous tick species and if they are associated with severe forms of TBE.

Blackbirds and song thrushes constitute a key reservoir of Borrelia garinii, the causative agent of borreliosis in Central Europe.

Blackbirds (Turdus merula) and song thrushes (Turdus philomelos) were found to carry 95% of all spirochete-infected tick larvae among 40 bird species captured in Central Europe. More than 90% of the infections were typed as Borrelia garinii and Borrelia valaisiana. We conclude that thrushes are key players in the maintenance of these spirochete species in this region of Central Europe.

Variegin, a novel fast and tight binding thrombin inhibitor from the tropical bont tick.

Tick saliva contains potent antihemostatic molecules that help ticks obtain their enormous blood meal during prolonged feeding. We isolated thrombin inhibitors present in the salivary gland extract from partially fed female Amblyomma variegatum, the tropical bont tick, and characterized the most potent, variegin, one of the smallest (32 residues) thrombin inhibitors found in nature. Full-length variegin and two truncated variants were chemically synthesized. Despite its small size and flexible structure, variegin binds thrombin with strong affinity (K(i) approximately 10.4 pM) and high specificity. Results using the truncated variants indicated that the seven residues at the N terminus affected the binding kinetics; when removed, the binding characteristics changed from fast to slow. Further, the thrombin active site binding moiety of variegin is in the region of residues 8-14, and the exosite-I binding moiety is within residues 15-32. Our results show that variegin is structurally and functionally similar to the rationally designed thrombin inhibitor, hirulog. However, compared with hirulog, variegin is a more potent inhibitor, and its inhibitory activity is largely retained after cleavage by thrombin.

An attempt to control a natural focus of leptospirosis grippotyphosa by rodenticide--a long-term study (1977-2004).

The purpose of the study was to suppress a known natural focus of field fever exerting an influence on animal hosts of leptospires--small terrestrial mammals (s.t.m.) by rodenticide. After repeated application of the zinc phosphide rodenticide, the s.t.m. were regularly live-trapped and checked for leptospirosis by dark-field microscopy and culture of their renal tissue and serological examination. Isolated leptospira strains were typed by help of rabbit factor sera. The deratization influenced the s.t.m. structure considerably: the proportion of the dominant Microtus arvalis subjects--the main reservoirs of Leptospira kirschneri serovar Grippotyphosa--were gradually and substantially reduced and, contrarily, the percentage of the potential hosts subjects--Clethrionomys glareolus and Apodemus flavicollis--rose decisively over time. Changes in culture and serological positivity for leptospirosis of s.t.m. have also been unregistered. The highest original infestation of M. arvalis have slowly but strongly decreased while that of C. glareolus and A. flavicollis has increased decisively and reached its maximum within the last years of investigation. It is probable that these two animal species have undertaken the leading role in the maintenance of the natural focus of the field fever. In other animal species analogous trends were not registered. Based on these long-term findings, there exists the possibility to suppress only partially and temporarily the epizootic process of leptospirosis in a natural focus that can be desirable in some circumstances (building or free time activities, etc.).

First report of Anaplasma phagocytophilum and its co-infections with Borrelia burgdorferi sensu lato in Ixodes ricinus ticks (Acari: Ixodidae) from Republic of Moldova.

We examined 198 questing Ixodes ricinus ticks collected in Chisinau City, Republic of Moldova by PCR assays for Anaplasma phagocytophilum, Borrelia burgdorferi sensu lato and co-infection of both pathogens, which were detected in 9%, 25.2% and 2.5% of tested ticks, respectively. B. burgdorferi s.l. genotyping revealed the presence of five genospecies with dominance of B. garinii. Our preliminary study provides evidence about occurrence of both pathogens in this populated area, which represent a potential health risk for inhabitants.

Quality control assessment for the serological diagnosis of tick borne encephalitis virus infections.

BACKGROUND: The diagnosis of tick borne encephalitis (TBE) is mainly based on the demonstration of specific antibodies in serum when neurological disease is manifested. Improving diagnostics is the most important step in detecting and dealing with these pathogens. Quality control measures are essential for TBE diagnosis. OBJECTIVE: To assess an external quality assurance (EQA) program for the serologic diagnosis of TBE infections. STUDY DESIGN: A panel of 12 serum samples was sent out to be tested for the presence of TBE virus-specific IgM and IgG. This panel contained seven TBE-positive samples for IgM and/or IgG; three negative samples; two samples positive either for West Nile virus (WNV) or Dengue virus (DENV). RESULTS: Fourty-two laboratories from 25 European and 2 non-European countries participated in this EQA. The correct answer by each laboratory for all samples ranked between 58 and 96% and sera with IgM antibody positive for TBE were correctly recognized by 46-88% of the laboratories. Sera with IgG antibody positive for TBE were correctly recognized by 83-95% of the laboratories. False TBE-positive results were obtained with DENV, WNV or negative sera only for IgG-based assays. CONCLUSION: Correct results for at least 90% of the samples were obtained by 33 of 40 participating laboratories for IgM and for 16 of 42 laboratories for IgG.

Quality control assessment for the PCR diagnosis of tick-borne encephalitis virus infections.

BACKGROUND: Reverse transcriptase-polymerase chain reaction (RT-PCR) is an efficient method for the early detection of tick-borne encephalitis virus (TBEV) RNA in blood and serum samples taken prior to the appearance of antibodies. Improved diagnostics are critical for optimally detecting and managing TBE infections and quality control measures are therefore essential. OBJECTIVE: To assess the diagnostic quality of laboratories by performing an external quality assurance (EQA) programme for the molecular detection of TBE infections. STUDY DESIGN: A panel of 12 prepared human plasma samples were distributed and tested for the presence of TBEV-specific RNA. The panel comprised eight samples spiked with different TBEV strains of European, Siberian and Far Eastern subtypes, and included a 10-fold dilution series. Two specificity controls consisted of a sample with Louping ill virus (LIV) and a sample with a pool of four other flaviviruses, and two negative control samples were further included. RESULTS: Twenty-three laboratories from 16 European and 2 non-European countries participated in this EQA programme. Only two participants correctly identified all samples. Nine laboratories correctly identified 75.0-91.7% of the samples; seven laboratories correctly identified 54.5-66.7% and five laboratories correctly identified < or =50%. CONCLUSIONS: The EQA programme provides information on the quality of the RT-PCR methods used by the participating laboratories and indicates that most of these need to improve sensitivity and specificity of their molecular assays for TBEV.

Borrelia afzelii gene expression in Ixodes ricinus (Acari: Ixodidae) ticks.

Spirochetes belonging to the Borrelia burgdorferi sensu lato (s. l.) complex have evolved remarkable ability to survive in diverse ecological niches during transmission cycles between ticks and vertebrate hosts by variable gene expression. To understand the events during spirochete transmission from feeding ticks to hosts, mRNA levels of selected B. afzelii genes (bbk32, dbpA, ospA, ospC and vlsE) were measured by quantitative real-time SYBR Green PCR. B. afzelii infected Ixodes ricinus nymphs fed on laboratory BALB/c mice for 0, 24, 48, and 72 hours. The mRNA levels of the constantly expressed flagellin gene were used for the relative quantification of selected genes. Differences in gene expression profiles were observed in unfed ticks and during tick feeding. mRNA levels of bbk32 and dbpA showed distinctive decreasing patterns during the first 24 hours post-attachment, while ospC and vlsE mRNA levels increased significantly during the feeding process. In contrast, ospA levels decreased for the 48 hours of tick feeding and slightly increased by 72 hours. More detailed and comprehensive studies on regulation of gene expression in different Borellia genospecies on the vector-host interface would aid to develop effective strategies in preventing pathogen transmission.

An antivector vaccine protects against a lethal vector-borne pathogen.

Vaccines that target blood-feeding disease vectors, such as mosquitoes and ticks, have the potential to protect against the many diseases caused by vector-borne pathogens. We tested the ability of an anti-tick vaccine derived from a tick cement protein (64TRP) of Rhipicephalus appendiculatus to protect mice against tick-borne encephalitis virus (TBEV) transmitted by infected Ixodes ricinus ticks. The vaccine has a "dual action" in immunized animals: when infested with ticks, the inflammatory and immune responses first disrupt the skin feeding site, resulting in impaired blood feeding, and then specific anti-64TRP antibodies cross-react with midgut antigenic epitopes, causing rupture of the tick midgut and death of engorged ticks. Three parameters were measured: "transmission," number of uninfected nymphal ticks that became infected when cofeeding with an infected adult female tick; "support," number of mice supporting virus transmission from the infected tick to cofeeding uninfected nymphs; and "survival," number of mice that survived infection by tick bite and subsequent challenge by intraperitoneal inoculation of a lethal dose of TBEV. We show that one dose of the 64TRP vaccine protects mice against lethal challenge by infected ticks; control animals developed a fatal viral encephalitis. The protective effect of the 64TRP vaccine was comparable to that of a single dose of a commercial TBEV vaccine, while the transmission-blocking effect of 64TRP was better than that of the antiviral vaccine in reducing the number of animals supporting virus transmission. By contrast, the commercial antitick vaccine (TickGARD) that targets only the tick's midgut showed transmission-blocking activity but was not protective. The 64TRP vaccine demonstrates the potential to control vector-borne disease by interfering with pathogen transmission, apparently by mediating a local cutaneous inflammatory immune response at the tick-feeding site.

Vasotab, a vasoactive peptide from horse fly Hybomitra bimaculata (Diptera, Tabanidae) salivary glands.

Horse flies feed from superficial haematomas and probably rely heavily on the pharmacological properties of their saliva to find blood. Here we describe the first evidence of vasodilators in horse fly Hybomitra bimaculata (Diptera, Tabanidae) salivary gland extract and clone and express one of the active peptides (termed vasotab). Physiological tests using crude salivary gland extracts and reverse-phase HPLC fractions demonstrated positive inotropism in isolated rat hearts, vasodilatation of coronary and peripheral vessels, and Na, K-ATPase inhibition. One of the vasoactive fractions was analysed by N-terminal Edman degradation and a 47-amino-acid sequence obtained. A full-length cDNA encoding the peptide was cloned from a phage library using degenerate primer PCR and the peptide expressed in insect cells. A 20-amino-acid signal sequence precedes the mature 56-amino-acid vasotab peptide, which is a member of the Kazal-type protease inhibitor family. The peptide has a unique 7-amino-acid insertion between the third and fourth cysteine residues. The recombinant peptide prolonged the action potential and caused positive inotropism of isolated rat heart myocytes, and may be an ion channel modulator.

Central European Dobrava Hantavirus isolate from a striped field mouse (Apodemus agrarius).

Dobrava virus (DOBV) is a hantavirus that causes hemorrhagic fever with renal syndrome (HFRS) in Europe. It is hosted by at least two rodent species, Apodemus flavicollis and A. agrarius. According to their natural hosts they form the distinct genetic lineages DOBV-Af and DOBV-Aa, respectively. We have now established a DOBV isolate named Slovakia (SK/Aa) from an A. agrarius animal captured in Slovakia. The complete S and M and partial L segment nucleotide sequences of the new isolate were determined. Phylogenetic analyses showed that the SK/Aa isolate clustered together with the other DOBV-Aa sequences amplified from A. agrarius before and can be taken as the representative of this genetic lineage. SK/Aa, in comparison with a DOBV-Af isolate, was used for serotyping neutralizing antibodies of HFRS patients in Central Europe. Most patients' sera exhibited a higher endpoint titer when probed with our new isolate, suggesting that DOBV-Aa strains are responsible for most of the DOBV-caused HFRS cases in this region.

First molecular identification of human Dobrava virus infection in central Europe.

Viral RNA was amplified by reverse transcription-PCR from a patient suffering from hemorrhagic fever with renal syndrome (HFRS) in Germany. The virus strain could be assigned to the Dobrava hantavirus (DOBV). This is the first molecular identification of human infection by DOBV in central Europe and the first proof that a virus strain related to the DOBV-Aa lineage, carried by Apodemus agrarius rodents, is able to cause HFRS.

Association of Borrelia garinii and B. valaisiana with songbirds in Slovakia.

In Europe, 6 of the 11 genospecies of Borrelia burgdorferi sensu lato are prevalent in questing Ixodes ricinus ticks. In most parts of Central Europe, B. afzelii, B. garinii, and B. valaisiana are the most frequent species, whereas B. burgdorferi sensu stricto, B. bissettii, and B. lusitaniae are rare. Previously, it has been shown that B. afzelii is associated with European rodents. Therefore, the aim of this study was to identify reservoir hosts of B. garinii and B. valaisiana in Slovakia. Songbirds were captured in a woodland near Bratislava and investigated for engorged ticks. Questing I. ricinus ticks were collected in the same region. Both tick pools were analyzed for spirochete infections by PCR, followed by DNA-DNA hybridization and, for a subsample, by nucleotide sequencing. Three of the 17 captured songbird species were infested with spirochete-infected ticks. Spirochetes in ticks that had fed on birds were genotyped as B. garinii and B. valaisiana, whereas questing ticks were infected with B. afzelii, B. garinii, and B. valaisiana. Furthermore, identical ospA alleles of B. garinii were found in ticks that had fed on the birds and in questing ticks. The data show that songbirds are reservoir hosts of B. garinii and B. valaisiana but not of B. afzelii. This and previous studies confirm that B. burgdorferi sensu lato is host associated and that this bacterial species complex contains different ecotypes.

Genetic interaction between distinct Dobrava hantavirus subtypes in Apodemus agrarius and A. flavicollis in nature.

Dobrava virus (DOBV) occurs in two different rodent species, Apodemus flavicollis (DOBV-Af) and A. agrarius (DOBV-Aa). We sequenced the S and M genomic segments from sympatric DOBV-Af and DOBV-Aa strains which fell into two distinct genetic lineages. Molecular phylogenetic analyses gave evidence for genetic reassortment between S and M segments of DOBV-Af and DOBV-Aa and indicated homologous recombination events in DOBV evolution. DOBV-Af and DOBV-Aa are distinct but also subject to genetic exchanges that affect their evolutionary trajectories.

The key roles of selection and migration in the ecology of Lyme borreliosis.

The roles of selection and migration of B. burgdorferi s. l. were studied. Questing adult Ixodes ricinus ticks were collected across Europe and analysed for infection with B. burgdorferi s. l. Analysis of the genospecies in individual ticks showed that B. garinii and B. valaisiana segregate from B. afzelii. Segregation of bird- and rodent-associated Borrelia genotypes can be explained by the operation of complement-mediated selection in the midgut of the feeding tick. Phylogenetic analyses of B. burgdorferi s. l. indicate high rates of migration for bird-associated genotypes. Altogether, it is emerging that the ecology of Lyme borreliosis is largely host-driven and that selection and migration are major forces shaping the population structures of B. burgdorferi s. l.

Tick-borne encephalitis virus foci in Slovakia.

Tick-borne encephalitis (TBE) virus as a typical arbovirus relies on two types of hosts for its survival: ticks act both as virus vectors and reservoir hosts, and vertebrates amplify the virus infection by acting as a source of infection for feeding ticks. Longitudinal monitoring of TBE virus in ticks and vertebrate hosts including humans over a period of 40 years resulted in the identification of the areas of Slovakia where TBE virus is endemic. These are concentrated to the western, southern, and eastern parts of the country. Even with recently identified foci there is no evidence that the size and location of the natural TBE foci have changed significantly during the last decades. Numbers of diagnosed hospitalised cases of TBE in Slovakia vary from less than 20 to almost 100 cases annually with 54-89 cases in recent years. A part of these cases (33 cases during the last 5 years) are alimentary infections after drinking of raw goat and sheep milk.

An inhibitor of thrombin-stimulated blood platelet aggregation from the salivary glands of the hard tick Amblyomma variegatum (Acari: Ixodidae).

The tropical bont tick, Amblyomma variegatum can cause intense skin irritation and inflammation and bites that often develop into septic wounds or abscess in their host. Crude salivary gland extract (SGE) of partially engorged A. variegatum females as well as SGE protein fractions purified by three-step reverse phase HPLC procedure were tested for their anti-aggregatory effect on isolated human blood platelets stimulated with thrombin and compared with the effect of recombinant hirudin. At concentrations 10(-3) and 5 x 10(-3) microg protein/ml the following rank order of antiplatelet activity was detected: AV 16/3 (inhibitor purified from AV-III, third purification) > SGE > AV-II (fraction from first purification) > AV-III (fraction from first purification) > hirudin. The effect of all fractions tested was dose-dependent. For fraction AV 16/3, the inhibitory effect was 49 and 61% for 10(-3) and 5 x 10(-3) microg protein/ml, respectively. The results suggest that protein fractions from A. variegatum SGE possess an antithrombin effect on human blood platelets with hirudin-like activity.