The epidemiology of tick-borne encephalitis virus was investigated by comparative sequence analysis of virus strains isolated in endemic areas of Europe and Asia. Phylogenetic relationships were determined from the nucleotide and amino acid sequences of the major envelope (E) protein of 16 newly sequenced strains and nine previously published sequences. Three genetic lineages could be clearly distinguished, corresponding to a European, a Far Eastern and a Siberian subtype. Amino acids characteristic for each of the subtypes (' signature ' amino acids) were identified and their location in the atomic structure of protein E was determined. The degree of variation between strains within subtypes was low and exhibited a maximum of only 2n2 % at the amino acid level. A maximum difference of 5n6 % was found between the three subtypes, which is in the range of variation reported for other flaviviruses.

Infectious cDNA clones of two strains of tick-borne encephalitis (TBE) virus, i.e. European subtype prototypic strain Neudoerfl and the closely related but more virulent strain Hypr, were constructed. The recombinant constructs consisted of cDNAs stably inserted into the bacterial plasmid pBR322 under the control of T7 promoter elements. The genome of TBE virus strain Neudoerfl was successfully cloned, both as a full-length cDNA and as two partial cDNAs. In the case of strain Hypr, the genome is represented by two cDNA clones corresponding to the 5h-and 3h-

Benign paroxysmal positional vertigo is a common cause of disabling vertigo with a high rate of recurrence. Although connections between vitamin D deficiency and osteoporosis, as well as between osteoporosis and benign paroxysmal positional vertigo have been suggested respectively in the literature, we are not aware of any publication linking vitamin D and benign paroxysmal positional vertigo. As a hypothesis, we suggest that there is a relation between insufficient vitamin D level and benign paroxysmal positional vertigo. In order to test this hypothesis, in a small retrospective pilot study, 25-hydroxyvitamin D levels in serum of patients with benign paroxysmal positional vertigo and frequency of recurrence after correction of serum level were assessed retrospectively. Patients with idiopathic positional vertigo had a low average serum level of 25-hydroxyvitamin D (23 ng/mL) similar to that of the general Austrian population, which has a high prevalence of hypovitaminosis D. In 4 cases with chronically recurrent severe vertigo episodes, average levels of serum 25-hydroxyvitamin D were even significantly lower than in the other vertigo patients, who had their first episode. Vertigo attacks did not recur after supplementation with vitamin D. We raise the possibility that patients with benign paroxysmal positional vertigo who have low vitamin D levels may benefit from supplementation and suggest further epidemiological investigations to determine the effect of correcting vitamin D deficiency on the recurrence of vertigo. Given the many known benefits of vitamin D, the authors recommend the measurement of vitamin D in patients with benign paroxysmal positional vertigo and supplementation if necessary.

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 Nd⌬ME 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 ؋ 10 7 /ml in the supernatant of these cells. Another replicon (Nd⌬CME) 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 Nd⌬ME-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.Subgenomic replicons of positive-stranded RNA viruses contain all of the genetic elements needed to amplify themselves in susceptible host cells but lack some or all of the genes coding for structural proteins. Consequently, these RNAs are replicated in cells but are not packaged into viral particles. Replicons have proven to be valuable tools for studying replication independently of virion assembly and maturation (4,20,30). Moreover, they have great potential as molecular tools for gene expression and as vectors for therapeutic and prophylactic purposes (15,19,40). The delivery of replicon RNAs to host cells can be achieved in three different ways: (i) transfection with in vitro-transcribed replicon RNA, (ii) transfection with plasmid DNA encoding replicon sequences under the control of a cellular RNA polymerase II promoter, and (iii) infection with virus-like particles (VLPs) generated by encapsidation of replicon RNA with viral structural proteins provided in trans. VLPs are capable of initiating a single round of infection, providing an efficient and easy way to deliver the replicon vector into specific host cells, and are therefore particularly useful tools in gene therapy or for immunization purposes (19).Flaviviruses, members of the genus Flavivirus (family Flaviviridae), are positive-stranded RNA viruses and include important human pathogens such as yellow fever virus, the four serotypes of dengue virus, Japanese encephaliti...

Escape mutants of tick-borne encephalitis (TBE) virus were selected using neutralizing monoclonal antibodies (MAbs) that react with three different and previously unrecognized epitopes in the envelope protein E of TBE virus. Two of these variants (V-IC3 and V-IE3) exhibited a significantly reduced reactivity with their selecting MAbs, as determined by ELISA, whereas with one variant (V-IO3), reactivity was completely unchanged. Comparative sequence analyses demonstrated that each of the variants differed from the wild-type virus by a single amino acid substitution located at exposed positions within domains I, II and III of protein E. In the mouse model, all three mutants were still neuro-virulent but exhibited a significantly reduced neuro-invasiveness after subcutaneous inoculation. Virus replication, however, was sufficient to induce a specific antibody response. The observed alterations in virulence properties were not associated with reduced growth rates in vertebrate cell cultures, but one variant (V-IE3) exhibited a small plaque phenotype. The mutation of variant V-IO3 resulted in a temperature-sensitive phenotype and a significant elevation of the pH-threshold of the conformational change necessary for fusion activity.