There are 3 subtypes of tick-borne encephalitis virus (TBEV) distinguished, at present time: European (EU), Siberian (SIB) and Far Eastern (FE). The former one is associated with Ixodes ricinus tick, whereas the latter two - with I. persulcatus. The circulation of TBEV in nature is mediated by the non-viraemic transmission, between infected and. uninfected ticks co-feeding on the same hosts and. it was shown that transmission rate of «Hypr» strain (EU subtype) is much higher than rate of «Vasilchenko» strain (SIB subtype) - 60 and 5 % respectively. To reveal the viral determinants of transmission efficacy, we constructed the series of recombinant viruses with gradually exchanged genes coding structural (str) and. non-structural (ns) viral proteins. The recombinant virus Hypr[str]Vs[ns] achieved the rate of non-viraemic transmission of 70 %. The introduction of separate structural genes of Hypr into Vs infectious clone has enhanced the transmission efficacy as well, though not to such extent as entire structural region but up to 33 % only. Thus, it was shown that efficacy of non-viraemic transmission of TBEV depends from properties of viral structural proteins mainly.

Background: Tick-borne encephalitis virus (TBEV) is transmitted to humans by Ixodid ticks causing >10,000 cases of disease annually. The risk of human infection relates to the efficiency of virus transmission between infected and uninfected ticks. Here we identify specific mutations in the viral envelope protein that affect transmission efficiency of TBEV between ticks. Methods: The genomes of 4 field isolates of TBEV deficient in haemagglutination, were sequenced and recreated by site-directed mutagenesis, in a TBEV infectious clone. They were then compared with the wild-type infectious clone in mice, porcine kidney PS cells and adult and nymphal I. ricinus ticks. Results: Sequence analysis revealed unique amino acid substitutions D67G, E122G or D277A in the envelope glycoprotein. Each mutation resulted in an increase of net charge and hydrophobicity on the virion surface. When introduced individually into the TBEV infectious clone (IC), each substitution inhibited haemagglutination and reduced mouse neuroinvasiveness from 65% to 15-30%. Antibody production in infected mice was 1.5-3 times lower for IC-E122G and IC-D277A suggesting lower levels of viraemia and/or deficient immune stimulation induced by these viruses. All mutants demonstrated delayed growth in PS cells during the first 24hpi; however, mutant IC-D67G exhibited significantly better growth characteristics than IC-E122G and IC-D277A. The reproduction of IC-E122G and IC-D277A in fasting ticks was similar to that of control HA positive virus whereas the titres of IC-D67G were significantly lower (2.5-3 vs. 1-2 log10PFU/ml, respectively). In feeding ticks, the titre of IC-E122G increased approximately 1000-fold and IC-D277A and IC-D67G - approximately 300-fold, whereas for control virus the increase was about 10-fold. Non-viraemic transmission efficiency from infected to uninfected ticks was increased by each individual substitution in nymphal I. ricinus (Figure 1). Figure 1 Tick-to-tick transmission rate (clear bars) is expressed as the proportion of infected I.ricinus nymphs. Black triangles show the average virus titres in individually infected recipient nymphs as determined by plaque assay. Conclusion: We hypothesize that the mechanism of adaptation of TBEV to its host utilizes the shift of charge/hydrophobicity at several critical aminoacid residues exposed on the virion surface. This shift results in different biological consequences depending on the localisation of certain aminoacid residue. The results provide valuable information concerning the maintenance in nature and the emergence of pathogenic variants of TBEV. Abstracts for SupplementInternational Journal of Infectious DiseasesVol. 14Preview Full-Text PDF Open Archive

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.

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.

Ticks are obligatory blood-feeding arthropods that secrete various immunomodulatory molecules to antagonize host inflammatory and immune responses. Cytokines play an important role in regulating these responses. We investigated the extent to which ticks interact with the sophisticated cytokine network by comparing the effect of salivary gland extracts (SGE) of 3 ixodid tick species, Dermacentor reticulatus, Amblyomma variegatum and Ixodes ricinus, all of which are important vectors of tick-borne pathogens. Using specific ELISAs, anti-cytokine activity was demonstrated with 7 cytokines: IL-8, MCP-1, MIP-1α, RANTES, eotaxin, IL-2 and IL-4. The results varied between species, and between adult males and females of the same species. Relatively high activity levels were detected in saliva of female D. reticulatus, confirming that the observed anti-cytokine activities are an integral part of tick saliva secreted into the host. Results with fractionated SGE indicated that from 2 to 6 putative cytokine binding molecules are produced, depending on species and sex. Binding ability of SGE molecules was verified by cross-linking with radio-isotope labelled MIP-1α. By targeting different cytokines, ixodid ticks can manipulate the cytokine network, which will greatly facilitate blood-feeding and provide a gateway for tick-borne pathogens that helps explain why ticks are such efficient and effective disease vectors.

The Saguenay-Lac St-Jean population of Quebec is relatively isolated and has genealogical records dating to the 17th-century French founders. In 120 extended families with at least one sib pair affected with early-onset hypertension and/or dyslipidemia, we analyzed the genetic determinants of hypertension and related cardiovascular and metabolic conditions. Variance-components linkage analysis revealed 46 loci after 100,000 permutations. The most prominent clusters of overlapping quantitative-trait loci were on chromosomes 1 and 3, a finding supported by principal-components and bivariate analyses. These genetic determinants were further tested by classifying families by use of LOD score density analysis for each measured phenotype at every 5 cM. Our study showed the founder effect over several generations and classes of living individuals. This quantitative genealogical approach supports the notion of the ancestral causality of traits uniquely present and inherited in distinct family classes. With the founder effect, traits determined within population subsets are measurably and quantitatively transmitted through generational lineage, with a precise component contributing to phenotypic variance. These methods should accelerate the uncovering of causal haplotypes in complex diseases such as hypertension and metabolic syndrome.

The skin site at which ticks attach to their hosts to feed is the critical interface between the tick and its host, and tick-borne pathogens. This site is highly modified by the pharmacologically active molecules secreted in tick saliva. For pathogens, it is an ecologically privileged niche that many exploit. Such exploitation is referred to as saliva-activated transmission (SAT) - the indirect promotion of tick-borne pathogen transmission via the actions of bioactive tick saliva molecules on the vertebrate host. Here we review evidence for SAT and consider what are the most likely candidates for SAT factors among the tick pharmacopoeia of anti-haemostatic, anti-inflammatory and immunomodulatory molecules identified to date. SAT factors appear to differ for different pathogens and tick vector species, and possibly even depend on the vertebrate host species. Most likely we are searching for a suite of molecules that act together to overcome the redundancy in host response mechanisms. Whatever they turn out to be, the quest to identify the tick molecules that mediate SAT is an exciting one, and offers new insights to controlling ticks and tick-borne diseases.

Several human diseases in Europe are caused by viruses transmitted by tick bite. These viruses belong to the genus Flavivirus, and include tick-borne encephalitis virus, Omsk haemorrhagic fever virus, louping ill virus, Powassan virus, Nairovirus (Crimean-Congo haemorrhagic fever virus) and Coltivirus (Eyach virus). All of these viruses cause more or less severe neurological diseases, and some are also responsible for haemorrhagic fever. The epidemiology, clinical picture and methods for diagnosis are detailed in this review. Most of these viral pathogens are classified as Biosafety Level 3 or 4 agents, and therefore some of them have been classified in Categories A-C of potential bioterrorism agents by the Centers for Disease Control and Prevention. Their ability to cause severe disease in man means that these viruses, as well as any clinical samples suspected of containing them, must be handled with specific and stringent precautions.

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.

Tick-borne flaviviruses are common, widespread, and successfully adapted to their mode of transmission. Most tick vectors of flaviviruses are ixodid species. These ticks are characterized by a comparatively long life cycle, lasting several years, during which the infecting virus may be maintained from one developmental stage of the tick to the next. Hence ticks act as highly efficient reservoirs of flaviviruses. Many tick-borne flaviviruses are transmitted vertically, from adult to offspring, although the frequency is too low to maintain the viruses solely in the tick population. Instead, the survival of tick-borne flaviviruses is dependent on horizontal transmission, both from an infected tick to a susceptible vertebrate host and from an infected vertebrate to uninfected ticks feeding on the animal. The dynamics of transmission and infection have traditionally been considered in isolation: in the tick, following virus uptake in the infected blood meal, infection of the midgut, passage through the hemocoel to the salivary glands, and transmission via the saliva; and in the vertebrate host, virus delivery into the skin at the site of tick feeding, infection of the draining lymph nodes, and dissemination to target organs. However, there is now compelling evidence of a complex interaction between the tick vector and its vertebrate host that affects virus transmission profoundly. The feeding site in the skin is a battleground in which the hemostatic, inflammatory, and immune responses of the host are countered by antihemostatic, anti-inflammatory, and immunomodulatory molecules (mostly proteins and peptides) secreted in tick saliva. Here we speculate that exploitation of the tick pharmacopeia, rather than development of viremia, is the key step in successful tick-borne flavivirus transmission.

ABSTRACT 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.

Salivary gland extract (SGE) of four horsefly species (Hybomitra bimaculata Macquart, Hybomitra ciureai Seguy, Tabanus bromius L., Tabanus glaucopis Meigen) and one deerfly species (Chrysops relictus Meigen) (Diptera: Tabanidae) were shown to contain vasodilatory activity. Aliquots equivalent to 1, 5 and 10 pairs of salivary glands (SG) relaxed rat femoral artery (with intact endothelium) pre-constricted with phenylephrine. Vasodilatory activity was dose-dependent. SGE of one horsefly species (Haematopota pluvialis L.) did not induce relaxation. The kinetics of vasodilation induced by SGE of four horsefly species differed from the deerfly. These results indicate that tabanid species may produce more than one type of vasodilator to aid blood feeding.

Borrelia burgdorferi sensu lato (s.l.) is maintained in nature by complex zoonotic transmission cycles, involving a large variety of vertebrates as hosts and hard ticks of the genus Ixodes as vectors. Recent studies suggest that the genospecies of B. burgdorferi s.l. and sometimes their subtypes are propagated by different spectra of hosts, mainly birds and rodents. In order to test the concept of host-association, we analysed the relationships between Borrelia genospecies, rodent hosts and I. ricinus ticks in an endemic focus of Lyme borreliosis in western Slovakia. Rodents and questing ticks were collected at a forested lowland locality near Bratislava. Tick infestation levels on rodents were determined, and spirochaete infections in ticks and in ear punch biopsies were analysed by PCR followed by genotyping. Mice were more heavily infested with ticks than bank voles, and a higher proportion of mice was infected with spirochaetes than voles. However, the infectivity of voles was much higher than that of mice. The vast majority of infections detected in the skin and in ticks feeding on the rodents represented B. afzelii. In contrast, more than half of all infections in questing ticks collected in the same region of Slovakia were identified as B. valaisiana and B. garinii. In conclusion, whilst the study reveals that mice and voles play different quantitative roles in the ecology of Lyme borreliosis, it demonstrates that B. afzelii is specifically maintained by European rodents, validating the concept of host-association of B. burgdorferi s.l.

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.