Proteolytic activation of tick-borne encephalitis virus by furin

Flaviviruses are assembled intracellularly in an immature form containing heterodimers of two envelope proteins, E and prM. Shortly before the virion exits the cell, prM is cleaved by a cellular enzyme, and this processing step can be blocked by treatment with agents that raise the pH of exocytic compartments. We carried out in vivo and in vitro studies with tick-borne encephalitis (TBE) virus to investigate the possible role of furin in this process as well as the functional consequences of prM cleavage. We found that prM in immature virions can be correctly cleaved in vitro by recombinant bovine furin but that efficient cleavage occurs only after exposure of the virion to mildly acidic pH. The data suggest that exposure to an acidic environment induces an irreversible structural change that renders the cleavage site accessible to the enzyme. Cleavage by furin in vitro resulted in biological activation, as shown by a 100-fold increase in specific infectivity, the acquisition of membrane fusion and hemagglutination activity, and the ability of the envelope proteins to undergo low-pH-induced structural rearrangements characteristic of mature virions. In vivo, prM cleavage was blocked by a furin inhibitor, and infection of the furin-deficient cell line LoVo yielded only immature virions, suggesting that furin is essential for cleavage activation of flaviviruses.     

Importance of localized skin infection in tick-borne encephalitis virus transmission

Arboviruses are transmitted to vertebrates by the "bite" of infected arthropods. Events at the site of virus deposition are largely unknown despite increasing evidence that blood-sucking arthropods immunomodulate their skin site of feeding. This question is particularly relevant for ixodid ticks that feed for several days. To examine events under conditions mimicking tick-borne encephalitis (TBE) virus transmission in nature (i.e., infected and uninfected Ixodes ricinus ticks feeding on the same animal), infected adult and uninfected nymphal ticks were placed in one retaining chamber (skin site A) and uninfected nymphs were placed within a second chamber posteriorly (skin site B) on two natural host species, yellow-necked field mice (Apodemus flavicollis) and bank voles (Clethrionomys glareolus). Virus transmission from infected to uninfected cofeeding ticks was correlated with infection in the skin site of tick feeding. Furthermore, virus was recruited preferentially to the site in which ticks were feeding compared with uninfested skin sites. Viremia did not correspond with a generalized infection of the skin; virus was not detected in an uninfested skin site (C) of 12/13 natural hosts that had viremia levels > or = 2.0 log10 ic mouse LD50/0.02 ml blood. To characterize infected cells, laboratory mouse strains were infested with infected ticks and then explants were removed from selected skin sites and floated on culture medium. Numerous leukocytes were found to migrate from the skin explants of tick feeding sites. Two-color immunocytochemistry revealed viral antigen in both migratory Langerhans cells and neutrophils; in addition, the migratory monocyte/macrophages were shown to produce infectious virus. The results indicate that the local skin site of tick feeding is an important focus of viral replication early after TBE virus transmission by ticks. Cellular infiltration of tick feeding sites, and the migration of cells from such sites, may provide a vehicle for transmission between infected and uninfected cofeeding ticks that is independent of a patent viremia. The data support the hypothesis that viremia is a product, rather than a prerequisite, of tick-borne virus transmission.     

Adjuvant/carrier activity of inactivated tick-borne encephalitis virus

This study was designed to measure the electroencephalogram (EEG) after exercise with increasing intensity. In a field test with increments in running velocity a 2-min EEG was recorded, together with blood lactate concentration and heart rate, after each stage. An individual protocol was used, with up to six stages of running to ensure comparability of exercise intensity among the subjects, in each of 19 athletes (17 men, 2 women) experienced in leisure-time running. The exercise consisted initially of three running stages of aerobic exercise intensity without blood lactate accumulation followed by stages with an increase of lactate concentration. The protocol of the field test led to a progressive increase in cortical activity directly after the stages without blood lactate accumulation mainly in the delta frequency band, followed by theta and alpha-1 frequency band, and less pronounced in the alpha-2 and in the beta frequency bands. After the stages with an onset and further increase of blood lactate accumulation significant decreases in the beta-2, beta-1 and alpha-1 frequency bands occurred predominantly in temporal (T3, T4, T5, and T6) and occipital (O1, and O2) electrode positions, indicating a stage-by-stage decrease of activity. This decrease may be explained by feed-back from working muscle, via afferents to the cortex from intero- and proprio-receptors and affective processes. This could suggest that through a higher running intensity indicated by an onset of blood lactate accumulation metabolic and mechanical changes led to alterations within the afferent systems influencing the level of cortical activity.     

Typifying foci of tick-borne encephalitis in the pre-Baikal

The existence of tick-borne encephalitis foci of several landscape types was revealed in the Prebaikalye. Of particular interest were foci of the forest-steppe type, in which virus strains of the Western serological type circulated along with the Eastern serological type strain of the tick-borne encephalitis virus. It was shown that infection of man was possible not only by transmissive, but also by the almientary route--through cow milk.     

Development of Kozhevnikov''s epilepsy in tick-borne encephalitis

The examination was carried out in 787 patients with macrofocal myocardial infarction. The most frequently encountered variant of intraventricular block in males was the right bundle branch block, in females--the left bundle branch block. The rarest variant of intraventricular conductivity disorders in myocardial infarction was the left-posterior hemiblock. The prognostically severest variant of bilateral block consists in a combination of the right bundle branch block with the left-posterior hemiblock. The leading causes of death among the patients with myocardial infarction and intraventricular blocks were acute (cardiogenic shock, pulmonary oedema) and chronic cardiac insufficiency. In patients with bilateral blocks the frequent causes of death were, along with cardiac insufficiency, also arrhythmias (ventricular fibrillation, asystole).     

The distribution of tick-borne encephalitis virus-infected ticks along a linear census route

An individual study of Ixodes persulcatus Schulze, 1930, established that the distribution of infected ticks differs from the normal distribution and it is most close to the Poisson distribution. There is a tendency to deviation from the uniform-random distribution associated with higher infected tick contact rates in some parts of the route.     

The prevention of tick-borne encephalitis (author's transl)

Tick-borne encephalitis is one of the most frequently-encountered virus diseases of the central nervous system in Europe. Hence, prophylactic measures for the prevention of tick-borne encephalitis are of particular importance, especially since it is virtually impossible to influence the course of the manifest disease by means of current therapeutic procedures. Moreover, complications (paralysis and other sequelae) are frequent. A recently-developed vaccine (killed virus) is capable of providing easy and long-lasting protection for a large part of the population. The safety and efficacy of the vaccine was proved in 30,000 persons who were vaccinated in a field trial. Wide-scale implementation may be recommended mainly for persons under regular exposure, but also for the protection of people remaining in an endemic area for a short time only. However, if immediate protection of non-immunised persons is required, i.e. persons who discover ticks on their body or who show the presumptive signs of an existing virus infection, passive immunisation with tick-borne encephalitis immune globulin is recommended.     

Spontaneous and engineered deletions in the 3' noncoding region of tick-borne encephalitis virus: construction of highly attenuated mutants of a flavivirus

The flavivirus genome is a positive-strand RNA molecule containing a single long open reading frame flanked by noncoding regions (NCR) that mediate crucial processes of the viral life cycle. The 3' NCR of tick-borne encephalitis (TBE) virus can be divided into a variable region that is highly heterogeneous in length among strains of TBE virus and in certain cases includes an internal poly(A) tract and a 3'-terminal conserved core element that is believed to fold as a whole into a well-defined secondary structure. We have now investigated the genetic stability of the TBE virus 3' NCR and its influence on viral growth properties and virulence. We observed spontaneous deletions in the variable region during growth of TBE virus in cell culture and in mice. These deletions varied in size and location but always included the internal poly(A) element of the TBE virus 3' NCR and never extended into the conserved 3'-terminal core element. Subsequently, we constructed specific deletion mutants by using infectious cDNA clones with the entire variable region and increasing segments of the core element removed. A virus mutant lacking the entire variable region was indistinguishable from wild-type virus with respect to cell culture growth properties and virulence in the mouse model. In contrast, even small extensions of the deletion into the core element led to significant biological effects. Deletions extending to nucleotides 10826, 10847, and 10870 caused distinct attenuation in mice without measurable reduction of cell culture growth properties, which, however, were significantly restricted when the deletion was extended to nucleotide 10919. An even larger deletion (to nucleotide 10994) abolished viral viability. In spite of their high degree of attenuation, these mutants efficiently induced protective immune responses even at low inoculation doses. Thus, 3'-NCR deletions represent a useful technique for achieving stable attenuation of flaviviruses that can be included in the rational design of novel flavivirus live vaccines.     

Protein E 98-113 sequence is a fusion site of tick-borne encephalitis virus with cellular membrane

The synthetic peptide with the conservative 98-113 sequence of protein E of tick-borne encephalitis virus was studied in order to elucidate its role in the functioning of flaviviruses. The peptide was shown to inhibit the in vitro infection of macrophages with the virus. An antibody that specifically binds this peptide was found among the set of monoclonal antibodies produced against protein E. This antibody was found to prevent penetration of the virus into liposomes. A correlation was found between our results and data on the spatial structure of protein E and its interspecies homology. The protein E 98-113 sequence of the tick-borne encephalitis virus was found to be the fusion site of the viral envelope with a cellular membrane.     

Passive immunization reduces immunity that results from simultaneous active immunization against tick-borne encephalitis virus in a mouse model

Concomitant administration of an antigen and antibodies of the respective specificity has been shown to result in reduced levels of actively produced antibodies. This has also recently been observed in a clinical trial on simultaneous passive and active immunization against tick-borne encephalitis virus (TBEV). In the current study the influence of simultaneous passive and active immunization on vaccine induced protective immunity against TBEV has been evaluated in a mouse model. Two immunizations with licensed whole-killed TBEV vaccines gave close to complete protection. Administration of human or mouse TBEV antibodies together with the first vaccine dose resulted in a significant reduction of vaccine induced protection against TBEV challenge. This effect was even more pronounced than that observed earlier on the levels of vaccine induced antibody.     

Affinity labelling of the tick-borne encephalitis virus RNA replicase proteins by 4-N-exo-base-substituted photoreactive CTP analogs

4-N-exo-base-substituted photoreactive analogs of CTP were designed and synthesized. Two flavivirus proteins NS5 and NS3 are shown to be labelled after RNA synthesis in the presence of the analogs, irradiation by UV-light (313 nm) and subsequent [alpha-32P]NTP incorporation.     

The laboratory diagnosis of spring-summer infections in Yekaterinburg in the epidemic season for tick-borne encephalitis

The authors propose a comprehensive approach to laboratory diagnosis of seasonal transmissible infections, based on modern methods permitting etiological deciphering of disease. A universal diagnostic algorithm notably accelerated the laboratory diagnosis due to cutting the period between collection of material from a patient and consecutive screening for antibodies to agents of tick-borne encephalitis, Lyme disease, and California encephalitis.     

The morphological characteristics of cell death in different forms of acute tick-borne encephalitis

The study was aimed to identify different types of cell death in monkey brain in flavivirus experimental encephalitis. 10 brain areas most vulnerable ("indicator") of the disease in its different forms (symptomless, intermediate and severe) were examined in animals infected intracerebrally with viruses of tick-borne encephalitis. Cells of ectodermal and mesenchymal origin displayed apoptosis that was most pronounced in intermediate form of the severity of the disease. Apoptosis was not characteristic for the symptomless form of tick-borne encephalitis. Two types of apoptosis morphological manifestations in nerve cells were described.     

Little known pages from the history of discovering tick-borne encephalitis

The purpose of this study was to evaluate the clinical usefulness of three-dimensional CT angiography (3D-CTA) for the diagnosis of cerebral aneurysm using a new reconstruction method called Multi-Angle Reconstruction Plan (MARP). The threshold values, region of interest and six angles of view were preset for the MARP method on the basis of our experience with cerebral 3D-CTA. Spiral CT angiography was performed in 27 patients with 29 aneurysms. The six images reconstructed with the MARP method were generated from the volumetric CT data. Two radiologists independently reviewed the six 3D-CT images for the presence of aneurysm. It took about five minutes to semiautomatically reconstruct the six 3D-CT images with the MARP method. There were no discrepancies in the evaluation of the 3D-CT images. Twenty-six aneurysms of 24 patients were demonstrated on the six 3D-CT images. Three aneurysms in 3 patients were not depicted on the 3D-CT images by the MARP method. In them, the additional 3D-CT images which were generated after evaluating the 2D-CT images clearly showed the aneurysms. The MARP method is considered to be a useful three-dimensional reconstruction technique for the diagnosis of cerebral aneurysm.     

An analysis of the stability of the antigenic structure in productive strain 205 of the tick-borne encephalitis virus during prolonged passage

The stability of the antigenic structure of tick-borne encephalitis (TBE) virus strain 205 in the process of passage through the brain of BALB/c mice has been studied. Its relationships with other viruses of the TBE complex have been analyzed with the use of monoclonal antibodies to virus proteins E and NS3. The stability of the protein structure of the virus has been determined by the immunofluorescence test and the enzyme immunoassay.     

Ultrastructural changes in the central nervous system of mice in tick-borne encephalitis

Tick-borne encephalitis virus is localized in nerve cells and intracellular spaces. According to the nature of ultrastructural changes, the pathological process in nerve cells may be divided into three stages connected with the stages of virus reproduction. In the first stage, the protein-synthesizing systems of the cell are degraded. In the second stage specific virus inclusions form in the cell cytoplasm as aggregates of mature and immature virions alongside with destruction of ultrastructures and growth of smooth membranes. In the third stage degradation of neurons is accompanied by necrobiosis and discomplexation of the surrounding glial elements, processes, cells of the inflammatory infiltrate and capillary walls. A certain combination of nonspecific changes in organelles of various cells with viral inclusions makes the ultrastructural picture of the focus of lesions in tick-borne encephalitis sufficiently characteristic for differential pathological diagnosis.     

Status of the tick-borne diseases in sheep and goats in Turkey

A patient treatment is presented to demonstrate a technique using visible-light-cured (VLC) resin to create a characterized denture base for an interim denture. This indication for characterization is routinely seen with African-Americans with dark-pigmented gingiva. Using a combination of blue VLC tray resin and pink VLC denture base resin, the mixture achieves a darker-colored denture base. This technique was used for the fabrication of an interim denture for a patient who required an immediate interim denture. The prosthesis was worn during the healing phase and allowed the patient to function while the definitive prosthesis was being fabricated.