Proteome dataset of mouse macrophage cell line infected with tick-borne encephalitis virus.

We report the proteomic datasets on the mouse macrophage cell line PMJ2R infected with tick-borne encephalitis virus (TBEV) for two and six days. Data were acquired using shotgun ultra-high resolution mass spectrometry. Peptide identifications were done using the Mascot version 2.4 (Matrix Science), and quantification was performed by a label-free approach. Protein profiles of early (two days) and late (six days) stages of infection were compared between each other and the respective control samples. Protein profiles of infected and control samples differed in the number of identified proteins and their relative abundances. Proteins detected in the TBEV-infected cells were involved in various processes related to the infection, including defense response against the virus, regulation of viral process, negative regulation of viral genome replication, RNA binding, or innate immune response. Also, proteins specific for the early and late stages of infection were identified.

Isolation of live Borrelia burgdorferi sensu lato spirochaetes from patients with undefined disorders and symptoms not typical for Lyme borreliosis.

Lyme borreliosis is a multisystem disorder with a diverse spectrum of clinical manifestations, caused by spirochaetes of the Borrelia burgdorferi sensu lato complex. It is an infectious disease that can be successfully cured by antibiotic therapy in the early stages; however, the possibility of the appearance of persistent signs and symptoms of disease following antibiotic treatment is recognized. It is known that Lyme borreliosis mimics multiple diseases that were never proven to have a spirochaete aetiology. Using complete modified Kelly-Pettenkofer medium we succeeded in cultivating live B. burgdorferi sensu lato spirochaetes from samples taken from people who suffered from undefined disorders, had symptoms not typical for Lyme borreliosis, but who had undergone antibiotic treatment due to a suspicion of having Lyme disease even though they were seronegative. We report the first recovery of live B. burgdorferi sensu stricto from residents of southeastern USA and the first successful cultivation of live Borrelia bissettii-like strain from residents of North America. Our results support the fact that B. bissettii is responsible for human Lyme borreliosis worldwide along with B. burgdorferi s.s. The involvement of new spirochaete species in Lyme borreliosis changes the understanding and recognition of clinical manifestations of this disease.

Ticks and tick-borne pathogens in South Bohemia (Czech Republic)--Spatial variability in Ixodes ricinus abundance, Borrelia burgdorferi and tick-borne encephalitis virus prevalence.

Spatial distribution of Ixodes ricinus tick host-seeking activity, as well as prevalence of Borrelia burgdorferi sensu lato and tick-borne encephalitis virus (TBEV) were studied in the TBE endemic area of South Bohemia (Czech Republic). High variability in tick abundance detected in a network of 30 study sites was most closely associated with characteristics of vegetation cover. Of 11,182 tested tick samples, 12% carried DNA of spirochete from B. burgdorferi s.l. complex. B. afzelii and B. garinii prevailed among spirochete species. The presence of B. spielmanii in the region was confirmed. The median number of borrelial genome copies in positive samples reached 6.6 × 10(3) by real-time PCR. The total prevalence of TBEV in pooled samples reached 0.32% (20,057 samples tested), at least one TBEV positive tick was present in 21 out of 30 sampling sites.

Der-p2 ( Dermatophagoides pteronyssinus) allergen-like protein from the hard tick Ixodes ricinus - a novel member of ML (MD-2-related lipid-recognition) domain protein family.

OBJECTIVE: Expression of the gene encoding Der-p2 allergen-like protein in the castor bean tick Ixodes ricinus is induced by blood intake. Tick Der-p2 allergen-like protein belongs to a diverse family of ML proteins that includes major allergens of house dust mites, human MD-2 or similar proteins from Drosophila melanogaster. In ticks, genes encoding proteins belonging to the ML protein family were identified, but their protein products have not been characterized yet. METHODS: A gene encoding tick Der-p2 allergen-like protein was amplified from cDNA of engorged I. ricinus female using the gene-specific primers designed on a basis of partial sequences of related allergen-like genes. The tissue and state specific patterns of expression of the gene were analysed. The IgE binding activity of the produced recombinant protein was studied by use of ELISA. RESULTS: Analysis of the expression pattern showed that the gene encoding the tick Der-p2 allergen-like protein is strongly induced by the bloodmeal in gut and haemolymph throughout all tick developmental stages. Der-p2 allergen-like protein possesses a putative lipid-binding site, according to the comparisons with the related proteins. The ability of tick Der-p2 allergen-like protein to bind immunoglobulin E (IgE) was revealed. DISCUSSION: The presence of a putative lipid-binding domain in Der-p2 allergen-like protein and its ability to interact with IgE might indicate the involvement of the protein in the tick's immune response.

Gene organization of a novel defensin of Ixodes ricinus: first annotation of an intron/exon structure in a hard tick defensin gene and first evidence of the occurrence of two isoforms of one member of the arthropod defensin family.

Antimicrobial peptides (defensins) are effectors of the immune system. Herein, we describe a novel Ixodes ricinus defensin gene(s), analyse its structure and compare it with other known antimicrobial peptides from different tick species. For the first time, an intron/exon structure is discovered in a hard-tick defensin gene. The intron/exon genomic organization of the gene is similar to the organization in Ornithodoros moubata, but not to that of the intronless defensins of Dermacentor variabilis and Ixodes scapularis. The analysis of the deduced amino acid sequences of different recombinants from the I. ricinus cDNA library reveals the presence of two defensin isoforms with three amino acid substitutions. Whether or not these substitutions affect the biological properties of the peptides is currently unknown. The expression of the defensin gene is strongly induced in the tick midgut after infection with Borrelia burgdorferi.

Lectin-binding characteristics of a Lyme borreliosis spirochete Borrelia burgdorferi sensu stricto.

Borrelial glycoconjugates were localized by labeled lectins on ultrathin cryosections and on surfaces of intact negatively stained bacteria. Protein-saccharide complexes in these glycoconjugates were partially characterized by means of enzyme deglycosylation and mild alkali pretreatment of cryosections. The results of labeling were examined by transmission electron microscopy. Statistically evaluated results (relative labeling index, chi2 test) of gold labeling indicated that surfaces of Borrelia burgdorferi strain B31 and external (outer) membrane vesicles (MVs) were covered with glycoconjugates containing O-glycosidically linked N-acetyl-D-galactosamine (GalNAc) and N-glycosidically linked N-acetyl-D-glucosamine (GlcNAc). The presence of N-linked GalNAc, sialic acid, mannose and fucose on the surfaces of outer membranes and MVs was probably due to an adherence of BSK-H medium components, especially rabbit serum, to Borrelia surfaces.

Improved method of detection and molecular typing of Borrelia burgdorferi sensu lato in clinical samples by polymerase chain reaction without DNA purification.

A simple assay by polymerase chain reaction was used for the of detection of Borrelia burgdorferi, causative agent of Lyme borreliosis (LB). It involves no DNA purification and is based on the amplification of a specific region of ospA gene of B. burgdorferi, followed by direct detection of the PCR product with SYBR Green I by agarose gel electrophoresis. The method was used to analyze samples from patients with LB diagnosis, with presumable infection with the LB spirochete, those with unclear clinical symptoms and after the course of an antibiotic treatment. Spirochetal DNA was detected by PCR even in contaminated samples in which B. burgdorferi was overgrown by fungi and other bacteria. Spirochetal DNA was detected and borrelia species was identified in cerebrospinal fluid of two patients hospitalized with the diagnosis "fever of unknown origin". Western blot and ELISA were negative in both cases. Total analysis of 94 samples from the hospital in Ceské Budejovice (South Bohemia, Czechia) showed infection with B. burgdorferi sensu stricto in 11% and B. garinii in 15% of cases. The highest prevalence was found for B. afzelii (43%). Co-infection was confirmed in 24 % of the analyzed symplex; 7% of samples that were B. burgdorferi sensu lato positive gave no results in DNA amplification with B. burgdorferi sensu stricto-, B. garinii- and B. afzelii-specific primers. The proposed reliable, rapid, unexpensive and specific technique could form the basis of laboratory tests for routine detection and identification of Lyme-disease spirochete in different samples.

Tick-borne encephalitis virus-specific RT-PCR--a rapid test for detection of the pathogen without viral RNA purification.

Among diseases transmitted by ticks in the Czech Republic, tick-borne encephalitis (TBE) caused by Tick-borne encephalitis virus (TBEV) and Lyme disease caused by Borrelia burgdorferi spirochete are most important. We propose an effective and specific test for detection of TBEV in a single tick or a pool of ticks based on the detection of TBEV RNA using an RT-PCR technique without RNA purification. The method is very sensitive with the detection limit of about 14 fg TBEV RNA in total RNA obtained from brain suspension from suckling mice infected with TBEV per reaction. The primers were derived from the 5'-terminal non-coding region, a highly conserved part of the virus. The method was successfully applied to field-collected ticks in detecting TBEV RNA. This method can be used in studies of several aspects of TBEV: epidemiology, screening of natural foci, circulation and detection of virus genome sequences in clinical materials.

Distribution of E and NS1 proteins of TBE virus in mammalian and tick cells.

Four monoclonal antibodies recognizing TBEV proteins were prepared. Three of them (2/H6, 8/A10, 5/F9) specifically recognize the structural E protein and the fourth binds to the nonstructural NS1 protein. These antibodies were used for an immunofluorescence study of TBEV protein distribution in infected mammalian host and tick vector tissue culture cells. Any differences in the distribution of the E and NS1 proteins were revealed. In porcine PS cell line the proteins were localized in the cytoplasm with a distinct increase of the signal in the perinuclear area 16 h post-infection (p.i.). The area gradually expanded and at 24 h p.i. covered the major part of the cytoplasm. The localization of the proteins in tick RA-257 cells revealed a uniform spread of the proteins in whole cytoplasm of the infected cells. The signal was very weak at 16 h p.i. and gradually increased during progressing time p.i., although, the distribution of the proteins did not exhibit any changes. Difference in viral protein distribution in the two cell types points to the possibility that the maturation process of TBEV exhibits different features in mammalian and tick cells.

Tick lectins: structural and functional properties.

Few papers have been published on tick lectins so far, and therefore more data are needed to complete the mosaic of knowledge of their structural and functional properties. Tissue-specific lectin/haemagglutinin activities of both soft and hard ticks have been investigated. Some tick lectins are proteins with binding affinity for sialic acid, various derivatives of hexosamines and different glycoconjugates. Most tick lectin/haemagglutinin activities are blood meal enhanced, and could serve as molecular factors of self/non-self recognition in defence reactions against bacteria or fungi, as well as in pathogen/parasite transmission. Dorin M, the plasma lectin of Ornithodoros moubata, is the first tick lectin purified so far from tick haemolymph, and the first that has been fully characterized. Partial characterization of other tick lectins/haemagglutinins has been performed mainly with respect to their carbohydrate binding specificities and immunochemical features.

Exposed and hidden lectin-binding epitopes at the surface of Borrelia burgdorferi.

The presence of surface- and subsurface-located lectin-binding epitopes of Borrelia burgdorferi was examined by electron microscopy using a variety of gold-labeled lectins. Concanavalin A reacted predominantly with extracellular material adjacent to the spirochetes. Wheat germ agglutinin bound weakly to the surface of borreliae; however, alterations of the outer membrane by preincubation in 100 ppm Triton X-100 or boiling uncovered numerous periplasmic sites recognized by the lectin. The periplasmic flagella liberated by some cells after detergent treatment were labeled with concanavalin A, wheat germ agglutinin and Ulex europaeus agglutinin UEA-I. No surface-exposed or periplasmic epitopes for the lectins from Glycine max, Dolichos biflorus or Helix pomatia were detected.

Isolation and characterization of Dorin M, a lectin from plasma of the soft tick Ornithodoros moubata.

A lectin with high hemagglutinating activity, which we have named Dorin M, was identified in the plasma of the soft tick Ornithodoros moubata. The activity of the plasma lectin could be efficiently inhibited by sialic acid, N-acetyl-D-hexosamines and sialoglycoproteins. Dorin M was purified to homogeneity using two different isolation systems: affinity chromatography on a column of bovine submaxillary mucin conjugated to Sepharose 4B with specific elution by N-acetyl-D-glucosamine and chromatography on Blue-Sepharose followed by anion exchange FPLC on a MonoQ column. The purified lectin is a glycoprotein which, in the native state, forms aggregates with molecular mass of about 640 kDa. Non-reducing SDS PAGE revealed that the lectin consists of two noncovalently bound subunits migrating closely around 37 kDa. Dorin M is a glycoprotein, probably modified by N-type glycosylation. After chemical deglycosylation, only one band of about 32 kDa was detected. Dorin M is the first lectin purified from ticks.

Characterization of an alpha-macroglobulin-like glycoprotein isolated from the plasma of the soft tick Ornithodoros moubata.

We report the identification of the first representative of the alpha-2-macroglobulin family identified in terrestrial invertebrates. An abundant acidic glycoprotein was isolated from the plasma of the soft tick Ornithodoros moubata. Its molecular mass is about 420 kDa in the native state, whereas in SDS/PAGE it migrates as one band of 190 kDa under nonreducing conditions and a band of 92 kDa when reduced. Chemical deglycosylation reveals that it is composed of two different subunits, designated A and B. The N-terminal amino-acid sequence of subunit A is similar to the N-terminus of invertebrate alpha-2-macroglobulin. Sequence analysis of several internal peptides confirms that the tick protein belongs to the alpha-2-macroglobulin family, and the protein is therefore referred to as tick alpha-macroglobulin (TAM). Functional analyses strengthen this assignment. TAM inhibits trypsin and thermolysin cleavage of the high-molecular-weight substrate azocoll in a manner similar to that of bovine alpha-2-macroglobulin. This effect is abolished by pre-treatment of TAM with methylamine. In the presence of TAM, trypsin is protected against active-site inhibition by soybean trypsin inhibitor. We cloned and sequenced a PCR product containing sequences from both subunits and spanning the N-terminus of subunit B and the putative 'bait region' (a segment of alpha-2-macroglobulin which serves as target for various proteases). This indicates that the two subunits are generated from a precursor polypeptide by post-translational processing.

A putative host cell receptor for tick-borne encephalitis virus identified by anti-idiotypic antibodies and virus affinoblotting.

Anti-idotypic monoclonal antibodies (anti-ID MAbs) were made against two mouse MAbs that neutralize the infectivity of the tick-borne encephalitis (TBE) virus. Three of the anti-ID MAbs (1) inhibited the binding of respective idiotypic MAb to the TBE virus antigen, (2) inhibited the infectivity of TBE virus when preincubated with virus-susceptible cells, and (3) bound to the surface of virus-susceptible but not virus-nonsusceptible cells. They recognized a 35-kD protein in immunoblotting analysis. Identification of this protein as a component of a putative TBE virus receptor was supported by the viroblot technique. In this assay, two polypeptide signals of 35 and 18 kD were obtained after incubation of blotted cell membrane proteins with the TBE virion antigen.

Lectin-like sequences in genome of Borrelia burgdorferi.

Using degenerative primers designed on the basis of known sequences of lectin genes from different sources a fragment of genomic DNA of Borrelia burgdorferi (strain B31) that contained a lectin-like sequence was isolated, cloned and sequenced. The presence of an open reading frame of 268 amino acids (position 1501-2304 bp) and the computer analysis of the predicted amino acid sequence showed 37% of identity and 75% of homology over region of 25 amino acids with the legume lectin proteins, including erythroagglutinating phytohemagglutinin (PHA-E) and leucoagglutinating phytohemagglutinin (PHA-L). The further analysis of the predicted amino acid sequence showed the presence of another two domains (positions 198-211 and 215-226 aa) consisting of the characteristic conserved sequence motifs for legume lectin proteins. Hemagglutinating activity was detected in lysate of B. burgdorferi (strain B31) spirochete and the affinity to fetuin was determined in a hemagglutination inhibition test. Hemagglutinating activity was also found in a crude lysate of the recombinant clones carrying the fragment of B. burgdorferi genomic DNA. The inhibition of agglutinating activity by fetuin, D-galactosamine and D-mannosamine was determined using the standard procedure of hemagglutination inhibition test with native rabbit red blood cells (RBC).

Animal virus receptors.

The term 'receptor' is generally accepted as the cell-surface component that participates in virus binding and facilitates subsequent viral infection. Recent advances in technology have permitted the identification of several virus receptors, increasing our understanding of the significance of this initial virus-cell and virus-host interaction. Virus binding was previously considered to involve simple recognition and attachment to a single cell surface molecule by virus attachment proteins. The classical concept of these as single entities that participate in a lock-and-key-type process has been superseded by new data indicating that binding can be a multistep process, often involving different virus-attachment proteins and more than one host-cell receptor.

Lectins and tick-pathogen interactions: a minireview.

Lectins and their glycosylated receptors in a system of the tick-transmitted pathogen are the addressed topics which the minireview is dealing with. They participate in the reciprocal protein-saccharide interactions in the transmission of the causative agents of the tick-borne encephalitis and Lyme borreliosis by the ticks. Functional significance of the tick tissue specific lectins as well the lectins/aggulutinis of the transmitted pathogens in molecular ecology of the tick borne diseases has been shown.

Two hemocyte populations in Triatoma infestans: ultrastructural and lectin-binding characterization.

Two distinct hemocyte populations are determined in the hemolymph of the triatomine bug Triatoma infestans Klug, oenocytoids and plasmatocytes, and their independent origin from separate stem cells is shown. Both hemocyte populations differ considerably in their morphology, ultrastructure and lectin-binding properties. While oenocytoids are quite uniform with easily definable cells which do not to bind any assayed lectin, the plasmatocytes are a very polymorphic population possessing several morphological types and displaying a positive reactivity with lectins.

Differences in haemagglutination activity in autogenous and anautogenous members of the Culex pipiens mosquito complex.

Hemagglutination activity was studied in whole body extracts of seven laboratory strains of Culex pipiens s.l. (Culex pipiens molestus and Culex pipiens quinquefasciatus). No significant differences in titer between females were observed, but hemagglutination activity in males differed depending on the feeding regime of the mothers of the males.