Results of the 20-year study of tick-borne encephalitis in Crimea

The history of tick-borne encephalitis (TBE) study in the Crimea was retraced and the possibility of introduction of TBE virus to the Crimea in the process of the acclimatization of vertebrates from areas enzootic for TBE is discussed. The foci of TBE were found throughout the whole of the mountain forest zone of the Crimea and coincided with the habitat area of lxodes ricinus, the main vector of TBE. Such ticks as Dermacentor reticulatus, D. marginatus and Hyalomma marginatum were also found to take part in the circulation of the virus. Among the residents of the mountain forest zone, 13.9% were found to have immunity to TBE, testifying to the wide contact of the population with the pathogen. TBE morbidity had pronounced seasonal character and the morbidity rate was low with the prevalence of mild clinical forms. The characteristic feature of the virus population was polymorphism: strains identical to the Far-Eastern strains Sofyin (about 60-70%) and strains differing in their antigenic structure circulated here.     

Are Patients with Erythema Migrans Who Have Leukopenia and/or Thrombocytopenia Coinfected with Anaplasma phagocytophilum or Tick-Borne Encephalitis Virus?

Lyme borreliosis (LB), tick-borne encephalitis (TBE) and human granulocytic anaplasmosis (HGA) are endemic in central part of Slovenia. We tested the hypothesis that patients with erythema migrans (EM) from this region, who have leukopenia and/or thrombocytopenia (typical findings in HGA and in the initial phase of TBE but not in patients with LB) are coinfected with Anaplasma phagocytophilum and/or with TBE virus, i.e. that cytopenia is a result of concomitant HGA or the initial phase of TBE. Comparison of clinical and laboratory findings for 67 patients with EM who disclosed leukopenia/thrombocytopenia with the corresponding results in sex- and age-matched patients with EM and normal blood cell counts revealed no differences. In addition, patients with typical EM and leukopenia and/or thrombocytopenia tested negative for the presence of IgM and IgG antibodies to TBE virus by ELISA as well as for the presence of specific IgG antibodies to A. phagocytophilum antigens by IFA in acute and convalescent serum samples. Thus, none of 67 patients (95% CI: 0 to 5.3%) with typical EM (the presence of this skin lesion attests for early Lyme borreliosis and is the evidence for a recent tick bite) was found to be coinfected with A. phagocytophilum or had a recent primary infection with TBE virus. The findings in the present study indicate that in Slovenia, and probably in other European countries endemic for LB, TBE and HGA, patients with early LB are rarely coinfected with the other tick-transmitted agents.     

森林脑炎自然疫源地样本的监测及病毒的分离研究

为了解森林脑炎疫源地的分布变化趋势及样本分离病毒的特性,采集了森林脑炎高发区周边的森林全沟硬蜱、血蜱样本及森林脑炎患者的脑组织样本,用小白鼠脑内接种法检测、分离病毒分离的病毒经鉴别试验证明为森林脑炎病毒：蜱、脑两种标本检测的阳性率分别为50％和100％、结果表明森林脑炎的疫区有从林区向农业区扩散的趋势,且全沟硬蜱的带毒率较高;森脑患者的脑组织样本与蜱标本病毒的性状育差异     

Enhancement of tick-borne encephalitis virus transmission by tick salivary gland extracts

Abstract. To investigate the role of ticks in TBE virus transmission, salivary gland extract (SGE) was derived from partially fed female Ixodes ricinus, Dermacentor reticulatus and Rhipicephalus appendiculatus ticks. Guinea-pigs were infested with uninfected R.appendiculatus nymphs and inoculated with a mixture of TBE virus and SGE or with virus alone. The number of ticks which on average acquired virus from feeding on animals inoculated with TBE virus and SGE from partially fed ticks was 4-fold greater than the number that became infected by feeding on animals inoculated with virus alone or virus plus SGE from unfed I.ricinus. Viraemia was detected in 67% of guinea-pigs inoculated with virus plus SGE compared to 30% of guinea-pigs inoculated with virus alone. Virus titres in the blood were similar for both groups of animals [range 2.0-2.8 log₁₀ plaque-forming units (PFU)/ml of blood]; however, the number of ticks that became infected was significantly higher on animals inoculated with virus plus SGE from partially fed ticks. No significant difference was observed with respect to the tick species used to derive SGE. The results indicate that TBE virus transmission is enhanced by factor(s) associated with the salivary glands of feeding ticks, and that these factor(s) may facilitate efficient transmission of TBE virus between infected and uninfected ticks even when they feed on hosts that have no detectable viraemia.     

Preclinical investigations of the safety, immunogenicity and efficacy of a purified, inactivated tick-borne encephalitis vaccine

A new tick-borne encephalitis (TBE) vaccine for human use has been developed. TBE virus (TBEV) was propagated in primary chick embryo cells, inactivated by formalin and purified by continuous-flow density gradient centrifugation. The TBE vaccine was tested for innocuity, immunogenicity and protective capacity in a series of laboratory tests. The results indicated that the vaccine is outstandingly well tolerated, highly immunogenic in various laboratory animals, and induces protective immunity in mice. These data suggest that this new vaccine should be studied in clinical trials.     

Characterization of monoclonal antibodies against Hokkaido strain tick-borne encephalitis virus

A tick-borne encephalitis (TBE) patient was found in Hokkaido in 1993, and TBE viruses were isolated from animals and ticks in our previous studies. To develop a diagnostic reagent to identify TBE viruses, monoclonal antibodies (Mabs) were produced against the TBE virus strain Hokkaido (Oshima 5-10). Seven Mabs were obtained which reacted with the envelope protein of the Oshima 5-10 strain. These Mabs were flavivirus genus-specific, TBE virus complex-specific or TBE virus type-specific. The Mabs are applicable for identification of TBE virus strains.     

Breakdown of the blood-brain barrier during tick-borne encephalitis in mice is not dependent on CD8+ T-cells

Tick-borne encephalitis (TBE) virus causes severe encephalitis with serious sequelae in humans. The disease is characterized by fever and debilitating encephalitis that can progress to chronic illness or fatal infection. In this study, changes in permeability of the blood-brain barrier (BBB) in two susceptible animal models (BALB/c, and C57Bl/6 mice) infected with TBE virus were investigated at various days after infection by measuring fluorescence in brain homogenates after intraperitoneal injection of sodium fluorescein, a compound that is normally excluded from the central nervous system. We demonstrate here that TBE virus infection, in addition to causing fatal encephalitis in mice, induces considerable breakdown of the BBB. The permeability of the BBB increased at later stages of TBE infection when high virus load was present in the brain (i.e., BBB breakdown was not necessary for TBE virus entry into the brain), and at the onset of the first severe clinical symptoms of the disease, which included neurological signs associated with sharp declines in body weight and temperature. The increased BBB permeability was in association with dramatic upregulation of proinflammatory cytokine/chemokine mRNA expression in the brain. Breakdown of the BBB was also observed in mice deficient in CD8(+) T-cells, indicating that these cells are not necessary for the increase in BBB permeability that occurs during TBE. These novel findings are highly relevant to the development of future therapies designed to control this important human infectious disease.     

Phylogenetic analysis and pathogenicity of tick-borne encephalitis virus from Japan and far-east Russia

Phylogenetic analysis of tick-borne encephalitis (TBE) virus revealed that Hokkaido strain of TBE virus evolved several hundreds years ago in far-east Russia. TBE virus strains in Irkutsk area were identified as Siberian subtype of TBE virus. BHK-cell adapted mutant of TBE virus showed lower neuro-invasive virulence in mice than parent virus. The mutant carried one amino acid substitution in envelope protein which resulted in increase of positive charge of the protein. The mutant-infected mice showed lower virus titers in bloods and spleens than the parent-infected mice. Infectious c-DNA clone of TBE virus Hokkaido strain was successfully generated and was applied to examine the neurovirulence in mice. One amino acid change in envelope protein and 2 amino acid changes in Ns5 protein showed a synergistic effect on reduced neurovirulence in mice.     

Presence of poly(A) in a flavivirus: significant differences between the 3' noncoding regions of the genomic RNAs of tick-borne encephalitis virus strains.

A poly(A) tail was identified on the 3' end of the prototype tick-borne encephalitis (TBE) virus strain Neudoerfl. This is in contrast to the general lack of poly(A) in the genomic RNAs of mosquito-borne flaviviruses analyzed so far. Analysis of several closely related strains of TBE virus, however, revealed the existence of two different types of 3' noncoding (NC) regions. One type (represented by strain Neudoerfl) is only 114 nucleotides long and carries a 3'-terminal poly(A) structure. This was also found in several TBE virus strains isolated from different geographic regions over a period of almost 30 years. The other type (represented by strain Hypr) is 461 nucleotides long and not polyadenylated. The sequence homology between the two types of TBE virus 3' NC regions terminates at a specific position 81 nucleotides after the stop codon. The second type of 3' NC region more closely resembles the common flavivirus pattern, including the potential for the formation of a 3'-terminal hairpin structure. However, it lacks primary sequence elements that are conserved among other flavivirus genomes.     

Development of an ELISA system for tick-borne encephalitis virus infection in rodents

Tick-borne encephalitis (TBE) virus causes severe encephalitis with serious sequelae in humans. An epizootiological survey of wild rodents is effective to detect TBE virus-endemic areas; however, limited serological diagnostic methods are available to detect anti-TBE virus antibodies in wild rodents. In this study, ELISAs for the detection of rodent antibodies against the TBE virus were developed using two recombinant proteins, domain III of the E protein (EdIII) and subviral particles (SPs), as the antigens. As compared with the neutralization test, the ELISA using EdIII had 77.1% sensitivity and 80.0% specificity, and the ELISA using SPs had 91.4% sensitivity and 100% specificity. Furthermore, when the ELISAs were applied to the epizootiological survey in the TBE virus-endemic area, both of the ELISAs was able to detect wild rodents with TBE virus-specific antibodies. This is the first study to show that ELISAs using recombinant antigens can be safe and useful in the detection of TBE virus-infected wild rodents in epizootiological research.     

Replication complex of tick-borne encephalitis complex. I. Identification of a nuclear fraction protein responsible for the initiation of RNA synthesis using affinity labeling

The pig embryo kidney cells infected by tick-borne encephalitis virus were fractionated into nuclear-associated, cytoplasmic and membrane fractions. The main part of the virus replicase activity was associated with the nuclei. The replication complex is able to synthesize full-length viral RNAs in vitro. To identify proteins involved in the initiation of the replication at the late stages of the infection, the highly specific affinity labelling technique was used. It was shown that with aldehyde-containing derivatives of ATP, ADP and AMP and [alpha-32P]GTP the target of labelling is a polypeptide having molecular weight about 69 kDa. The same protein is immunostained with TBE virus specific antibodies after blotting onto nitrocellulose. The conclusion is made that nonstructural protein NS3 takes part in virus replication at the late stage of the infection.     

Incorporation of tick-borne encephalitis virus replicons into virus-like particles by a packaging cell line

RNA replicons derived from flavivirus genomes show considerable potential as gene transfer and immunization vectors. A convenient and efficient encapsidation system is an important prerequisite for the practical application of such vectors. In this work, tick-borne encephalitis (TBE) virus replicons and an appropriate packaging cell line were constructed and characterized. A stable CHO cell line constitutively expressing the two surface proteins prM/M and E (named CHO-ME cells) was generated and shown to efficiently export mature recombinant subviral particles (RSPs). When replicon NdDeltaME lacking the prM/M and E genes was introduced into CHO-ME cells, virus-like particles (VLPs) capable of initiating a single round of infection were released, yielding titers of up to 5 x 10(7)/ml in the supernatant of these cells. Another replicon (NdDeltaCME) lacking the region encoding most of the capsid protein C in addition to proteins prM/M and E was not packaged by CHO-ME cells. As observed with other flavivirus replicons, both TBE virus replicons appeared to exert no cytopathic effect on their host cells. Sedimentation analysis revealed that the NdDeltaME-containing VLPs were physically distinct from RSPs and similar to infectious virions. VLPs could be repeatedly passaged in CHO-ME cells but maintained the property of being able to initiate only a single round of infection in other cells during these passages. CHO-ME cells can thus be used both as a source for mature TBE virus RSPs and as a safe and convenient replicon packaging cell line, providing the TBE virus surface proteins prM/M and E in trans.     

Capsid protein C of tick-borne encephalitis virus tolerates large internal deletions and is a favorable target for attenuation of virulence

Deletions ranging in size from 4 to 21 amino acid residues were introduced into the capsid protein of the flavivirus tick-borne encephalitis (TBE) virus. These deletions incrementally affected a hydrophobic domain which is present at the center of all flavivirus capsid protein sequences and part of which may form an amphipathic alpha-helix. In the context of the full-length TBE genome, the deletions did not measurably affect protein expression and up to a deletion length of 16 amino acid residues, corresponding to almost 17% of mature protein C, viable virus was recovered. This virus was strongly attenuated but highly immunogenic in adult mice, revealing capsid protein C as a new and attractive target for the directed attenuation of flaviviruses. Apparently, the larger deletions interfered with the correct assembly of infectious virus particles, and this disturbance of virion assembly is likely to be the molecular basis of attenuation. However, all of the mutants carrying large deletions produced substantial amounts of subviral particles, which as judged from density gradient analyses were identical to recombinant subviral particles as obtained by the expression of the surface proteins prM and E alone. The structural and functional flexibility of protein C revealed in this study and its predicted largely alpha-helical conformation are reminiscent of capsid proteins of other enveloped viruses, such as alphaviruses (N-terminal domain of the capsid protein), retroviruses, and hepadnaviruses and suggest that all of these may belong to a common structural class, which is fundamentally distinct from the classical beta-barrel structures of many icosahedral viral capsids. The possibility of attenuating flaviviruses by disturbing virus assembly and favoring the production of noninfectious but highly immunogenic subviral particles opens up a promising new avenue for the development of live flavivirus vaccines.     

我国新分离森林脑炎病毒E基因特征分析

为了解分离自黑龙江省大兴安岭林区全沟硬蜱中的DXAL-5、12、13、16、18,21共6株森林脑炎(TBE)病毒E蛋白基因特征并确定病毒基因型,应用RT-PCR技术对6株病毒E蛋白基因进行体外扩增、克隆、测序.结果发现,6株病毒E蛋白基因的核苷酸序列长均为1 488 bp,推导的氨基酸序列长均为496 aa.与TBE参考毒株E蛋白基因进行比较,这6株病毒与远东亚型同源性最高,其次是西伯利亚亚型,与欧洲亚型同源性最差;在决定亚型特征的氨基酸位点多数属于TBE病毒远东亚型.E蛋白基因推导的氨基酸种系发生树分析表明,6株病毒均在远东亚型分枝内.因此就E蛋白基因而言,DXAL-5、12、13、16、18、21株均属于TBE病毒的远东亚型.新分离毒株与Senzhang株同源性较高,种系发生关系也比较接近,推测疫苗株对新分离毒株仍具有很好的保护作用.但是在E蛋白的A、B和C抗原决定区内,6株病毒均有不同程度的氨基酸改变,这些突变有可能影响E蛋白的功能.     

Attenuation of tick-borne encephalitis virus by structure-based site-specific mutagenesis of a putative flavivirus receptor binding site

The impact of a specific region of the envelope protein E of tick-borne encephalitis (TBE) virus on the biology of this virus was investigated by a site-directed mutagenesis approach. The four amino acid residues that were analyzed in detail (E308 to E311) are located on the upper-lateral surface of domain III according to the X-ray structure of the TBE virus protein E and are part of an area that is considered to be a potential receptor binding determinant of flaviviruses. Mutants containing single amino acid substitutions, as well as combinations of mutations, were constructed and analyzed for their virulence in mice, growth properties in cultured cells, and genetic stability. The most significant attenuation in mice was achieved by mutagenesis of threonine 310. Combining this mutation with deletion mutations in the 3'-noncoding region yielded mutants that were highly attenuated. The biological effects of mutation Thr 310 to Lys, however, could be reversed to a large degree by a mutation at a neighboring position (Lys 311 to Glu) that arose spontaneously during infection of a mouse. Mutagenesis of the other positions provided evidence for the functional importance of residue 308 (Asp) and its charge interaction with residue 311 (Lys), whereas residue 309 could be altered or even deleted without any notable consequences. Deletion of residue 309 was accompanied by a spontaneous second-site mutation (Phe to Tyr) at position 332, which in the three-dimensional structure of protein E is spatially close to residue 309. The information obtained in this study is relevant for the development of specific attenuated flavivirus strains that may serve as future live vaccines.     

Tick-borne encephalitis virus in northern Italy: molecular analysis, relationships with density and seasonal dynamics of Ixodes ricinus

Ixodes ricinus ticks were collected from dragging vegetation and from shot roe deer in the province of Trento and Belluno in northern Italy. Ticks were pooled for analyses and from 1060 pools of ticks collected in the province of Belluno and 12390 tick samples collected in Trentino, four proved positive by immunofluorescence microscopy using a tick-borne encephalitis (TBE)-specific antiserum. The identity of the virus isolates was determined by RT-PCR cycle sequencing and they were all found to be closely similar (> 98% nucleotide identity) to typical western European TBE complex viruses as found in Austria. The isolates from Trentino differed from the Neudorfl strain of western European TBE virus at eight nucleotide positions but as these nucleotide substitutions were all synonymous, there were no amino acid changes. These results imply that the virus isolates in Trentino have changed slightly from the typical European strains isolated in nearby Austria. The abundance of questing ticks and ticks feeding on roe deer was greater in TBE positive hunting districts than in hunting districts where TBE complex viruses were only probable or believed to be absent. In TBE positive and probable districts synchrony in the seasonal dynamics of larvae and nymphs of L. ricinus was observed. This study provides evidence to suggest that roe deer may have an important role to play in the maintenance of tick density and in the persistence of TBE virus.     

Highly efficient induction of protective immunity by a vaccinia virus vector defective in late gene expression

Vaccinia viruses defective in the essential gene coding for the enzyme uracil DNA glycosylase (UDG) do not undergo DNA replication and do not express late genes in wild-type cells. A UDG-deficient vaccinia virus vector carrying the tick-borne encephalitis (TBE) virus prM/E gene, termed vD4-prME, was constructed, and its potential as a vaccine vector was evaluated. High-level expression of the prM/E antigens could be demonstrated in infected complementing cells, and moderate levels were found under noncomplementing conditions. The vD4-prME vector was used to vaccinate mice; animals receiving single vaccination doses as low as 10(4) PFU were fully protected against challenge with high doses of virulent TBE virus. Single vaccination doses of 10(3) PFU were sufficient to induce significant neutralizing antibody titers. With the corresponding replicating virus, doses at least 10-fold higher were needed to achieve protection. The data indicate that late gene expression of the vaccine vector is not required for successful vaccination; early vaccinia virus gene expression induces a potent protective immune response. The new vaccinia virus-based defective vectors are therefore promising live vaccines for prophylaxis and cancer immunotherapy.     

The clinico-epidemiological characteristics of Powassan encephalitis in the southern Soviet Far East]

The present communication deals with the analysis of 14 cases of Powassan encephalitis. As shown in this study, the course of this infection may be accompanied by symptoms indicating the presence of cerebral and meningeal lesions (in 7 cases meningoencephalitic forms with one fatal outcome and in 2 cases meningeal forms were registered) or take febrile and inapparent forms (5 cases). Powassan encephalitis was found to give characteristic symptoms of cerebellovestibular lesions, differentiating this disease from tick-borne encephalitis (TBE). The cases of mixed infections caused by TBE virus, Powassan encephalitis virus and tick-born Borrelia were found to be possible.     

Correlation of TBE Incidence with Red Deer and Roe Deer Abundance in Slovenia

Tick-borne encephalitis (TBE) is a virus infection which sometimes causes human disease. The TBE virus is found in ticks and certain vertebrate tick hosts in restricted endemic localities termed TBE foci. The formation of natural foci is a combination of several factors: the vectors, a suitable and numerous enough number of hosts and in a habitat with suitable vegetation and climate. The present study investigated the influence of deer on the incidence of tick-borne encephalitis. We were able to obtain data from deer culls. Using this data, the abundance of deer was estimated and temporal and spatial analysis was performed. The abundance of deer has increased in the past decades, as well as the incidence of tick-borne encephalitis. Temporal analysis confirmed a correlation between red deer abundance and tick-borne encephalitis occurrence. Additionally, spatial analysis established, that in areas with high incidence of tick-borne encephalitis red deer density is higher, compared to areas with no or few human cases of tick-borne encephalitis. However, such correlation could not be confirmed between roe deer density and the incidence of tick-borne encephalitis. This is presumably due to roe deer density being above a certain threshold so that availability of tick reproduction hosts has no apparent effect on ticks'' host finding and consequently may not be possible to correlate with incidence of human TBE.     

Tick-borne encephalitis virus prevalence in Ixodes ricinus ticks collected in high risk habitats of the south-Bohemian region of the Czech Republic

A survey for tick-borne encephalitis virus in Ixodes ricinus ticks was conducted in May 2000 in two districts of the South-Bohemian region of the Czech Republic with a high occurrence of tick-borne encephalitis (TBE) in humans. Homogenized ticks were tested on PS cells, which were examined for any cytopathic effect, plaque assay and indirect fluorescent antibody test (IFT). The IFT has proved to be the most sensitive and most rapid test to demonstrate the virus in ticks. TBE virus was found in 17 of 187 pooled samples, consisting of a total of 2,968 ticks. The mean minimum infection rate was 0.6% for all tick stages combined. Infection rates in nymphs collected indifferent locations varied between 0.2 and 1.3% and between 5.9 and 11.1% in adult ticks. The observed TBE prevalence in ticks was compared with data obtained elsewhere in the Czech Republic. It is concluded that screening of ticks for TBE virus prevalence using IFT is a valuable indicator for the degree of risk to contract TBE in as particular habitat. This revised version was published online in July 2006 with corrections to the Cover Date.