[Show abstract][Hide abstract] ABSTRACT: Abstract 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.
[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: 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.
PLoS ONE 01/2009; 4(10):e7295. · 3.53 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Improved diagnostics are critical for optimally detecting and managing tick-borne encephalitis (TBE) infections, and therefore quality control measures are essential for both serological and molecular diagnostics. Two external quality assurance (EQA) studies were performed to assess the quality of serological and molecular diagnostics of TBE infections. For the EQA of serological diagnostics, each participating laboratory received a proficiency panel of freeze-dried human sera containing 7 TBE-positive samples, 3 negative samples, and 2 samples positive for heterologous flaviviruses. For the EQA of molecular diagnostics, panels of prepared human plasma samples comprising 8 samples spiked with RNA from different TBE virus strains of European, Siberian, and Far Eastern subtypes, 2 specificity controls with heterologous flaviviruses, and two negative control samples were distributed. A total of 46 invited expert laboratories from 28 European and non-European countries participated at least in one of these studies. Applying proficiency criteria, the number of participating laboratories who passed the minimum requirements for successful participation was 60% for the EQA of serological (both IgM and IgG detection) and 48% for the EQA of PCR diagnostics. The EQA studies provide information on the diagnostic quality of the participating laboratories and indicate that most of them need to improve their assays.
International journal of medical microbiology: IJMM 04/2008; 298:333-335. · 4.54 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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.
Applied and Environmental Microbiology 03/2008; 74(4):1289-93. · 3.95 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The speed with which horseflies (Diptera: Tabanidae) obtain a bloodmeal suggests they have potent vasodilators. We used isolated perfused rat heart to examine the vasoactivity of salivary gland extracts (SGEs) of three horsefly species, Hybomitra bimaculata Macquart, Tabanus bromius Linnaeus and Tabanus glaucopis Meigen. Administration of horsefly SGEs to the heart produced biphasic coronary responses: a decrease and subsequent increase in coronary flow (CF), characterized by initial vasoconstriction followed by prolonged vasodilation of coronary vessels. However, although SGEs of H. bimaculata induced a significant decrease in left ventricular pressure (LVP), the effect on changes in CF was not significant except at the highest dose tested. The ability to reduce LVP without significantly lowering CF, or affecting heart rate and rhythm, represents a unique set of properties that have considerable therapeutic potential if they can be reproduced by a single molecule.
Medical and Veterinary Entomology 01/2008; 21(4):384-9. · 2.21 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Abstract The speed with which horseflies (Diptera: Tabanidae) obtain a bloodmeal suggests they have potent vasodilators. We used isolated perfused rat heart to examine the vasoactivity of salivary gland extracts (SGEs) of three horsefly species, Hybomitra bimaculata Macquart, Tabanus bromius Linnaeus and Tabanus glaucopis Meigen. Administration of horsefly SGEs to the heart produced biphasic coronary responses: a decrease and subsequent increase in coronary flow (CF), characterized by initial vasoconstriction followed by prolonged vasodilation of coronary vessels. However, although SGEs of H. bimaculata induced a significant decrease in left ventricular pressure (LVP), the effect on changes in CF was not significant except at the highest dose tested. The ability to reduce LVP without significantly lowering CF, or affecting heart rate and rhythm, represents a unique set of properties that have considerable therapeutic potential if they can be reproduced by a single molecule.
Medical and Veterinary Entomology 11/2007; 21(4):384 - 389. · 2.21 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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 (Ki ∼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.
Journal of Biological Chemistry 10/2007; 282(40):29101-29113. · 4.65 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Ticks secrete a cocktail of immunomodulatory molecules in their saliva during blood-feeding, including chemokine-binding factors that help control the activity of host immunocompetent cells. Here we demonstrate differential dynamics of anti IL-8 (CXCL8), MCP-1 (CCL2), MIP-1 (CCL3), RANTES (CCL5) and eotaxin (CCL11) activities in salivary gland extracts of adult Amblyomma variegatum. Unfed male and female ticks showed activity against all the chemokines except CCL5; anti-CCL11 activity was particularly high. However, during feeding the dynamics of anti-chemokine activity differed significantly between males and females, and varied between chemokines. In males, anti-chemokine activities increased, whereas in females they declined or increased slightly as feeding progressed. The exception was anti-CCL11 activity, which declined and then increased in both males and females. Comparison of salivary gland equivalents of individual ticks prepared at various feeding intervals revealed some differences that were most pronounced between individual females fed for 8 days. These observations reflect the feeding behaviour of male and female A. variegatum. They support the concept of 'mate guarding', in which males help their mates to engorge by controlling their host's immune response, and the possibility that ticks benefit from feeding together by exploiting molecular individuality.
[Show abstract][Hide abstract] ABSTRACT: 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.
To assess an external quality assurance (EQA) program for the serologic diagnosis of TBE infections.
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).
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.
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.
Journal of Clinical Virology 04/2007; 38(3):260-4. · 3.29 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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.
Enperimental and Applied Acarology 01/2007; 41(1):147-152. · 1.85 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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.
To assess the diagnostic quality of laboratories by performing an external quality assurance (EQA) programme for the molecular detection of TBE infections.
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.
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%.
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.
Journal of Clinical Virology 01/2007; 38(1):73-7. · 3.29 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Tick vaccines derived from Bm86, a midgut membrane-bound protein of the cattle tick, Boophilus microplus, are currently the only commercially available ectoparasite vaccines. Despite its introduction to the market in 1994, and the recognized need for alternatives to chemical pesticides, progress in developing effective antitick vaccines (and ectoparasite vaccines in general) is slow. The primary rate-limiting step is the identification of suitable antigenic targets for vaccine development. Two sources of candidate vaccine antigens have been identified: 'exposed' antigens that are secreted in tick saliva during attachment and feeding on a host and 'concealed' antigens that are normally hidden from the host. Recently, a third group of antigens has been distinguished that combines the properties of both exposed and concealed antigens. This latter group offers the prospect of a broad-spectrum vaccine effective against both adults and immature stages of a wide variety of tick species. It also shows transmission-blocking and protective activity against a tick-borne pathogen. With the proliferation of molecular techniques and their application to vaccine development, there are high hopes for new and effective antitick vaccines that also control tick-borne diseases.
[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: 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.
Journal of Clinical Microbiology 07/2005; 43(6):2756-63. · 4.07 Impact Factor