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Flavivirus replication is mediated by interactions between complementary ssRNA sequences of the 5′- and 3′-termini that form dsRNA cyclisation stems or panhandles, varying in length, sequence and specific location in the mosquito-borne, tick-borne, non-vectored and non-classified flaviviruses. In this manuscript we manually aligned the flavivirus 5′UTRs and adjacent capsid genes and revealed significantly more homology than has hitherto been identified. Analysis of the alignments revealed that the panhandles represent evolutionary remnants of a long cyclisation domain that probably emerged through duplication of one of the UTR termini.

Comparative alignment of the 3′untranslated regions (3′UTRs) of tick-borne flaviviruses has previously revealed short direct repeat sequences about 25–70 nucleotides long [Gritsun, T.S., Venugopal, K., Zanotto, P.M., Mikhailov, M.V., Sall, A.A., Holmes, E.C., Polkinghorne, I., Frolova, T.V., Pogodina, V.V., Lashkevich, V.A., Gould, E.A., 1997. Complete sequence of two tick-borne flaviviruses isolated from Siberia and the UK: analysis and significance of the 5′ and 3′-UTRs. Virus Res. 49 (1) 27–39; Wallner, G., Mandl, C.W., Kunz, C., Heinz, F.X., 1995. The flavivirus 3′-noncoding region: extensive size heterogeneity independent of evolutionary relationships among strains of tick-borne encephalitis virus. Virology, 213 (1) 169–178]. We now show that these short sequences appear to have originated from longer repeat sequences (LRSs) that are present both in the 3′UTR and the open reading frame of the genome. We propose that the 3′UTR, and possibly the open reading frame, evolved through multiple duplications, deletions and mutations of a primordial sequence element.

Tick-borne encephalitis (TBE) is one of the most dangerous human infections occurring in Europe and many parts of Asia. The etiological agent Tick-borne encephalitis virus (TBEV), is a member of the virus genus Flavivirus, of the family Flaviviridae. TBEV is believed to cause at least 11,000 human cases of encephalitis in Russia and about 3000 cases in the rest of Europe annually. Related viruses within the same group, Louping ill virus (LIV), Langat virus (LGTV) and Powassan virus (POWV), also cause human encephalitis but rarely on an epidemic scale. Three other viruses within the same group, Omsk hemorrhagic fever virus (OHFV), Kyasanur Forest disease virus (KFDV) and Alkhurma virus (ALKV), are closely related to the TBEV complex viruses and tend to cause fatal hemorrhagic fevers rather than encephalitis. This review describes the clinical manifestations associated with TBEV infections, the main molecular-biological properties of these viruses, and the different factors that define the incidence and severity of disease. The role of ticks and their local hosts in the emergence of new virus variants with different pathogenic characteristics is also discussed. This review also contains a brief history of vaccination against TBE including trials with live attenuated vaccine and modern tendencies in developing of vaccine virus strains.

To elucidate the processes controlling the emergence and spread of dengue-2 virus (DEN-2) we examined the evolution of viral isolates sampled from both local (Viet Nam) and global populations. Our phylogenetic analysis, incorporating envelope (E) glycoprotein sequences from 147 isolates of DEN-2, provided a more complete picture of viral diversity, with a newly defined "Cosmopolitan" genotype having a near global distribution and two other genotypes restricted to Asia. By analyzing rates of synonymous and nonsynonymous substitution we determined that genotypes have experienced different selection pressures, with some evidence of positive selection in the Cosmopolitan genotype and one of the two Asian genotypes, but that the transition from sylvatic to human transmission was not accompanied by adaptive evolution of the E gene. Although there was no association between selection pressures acting on the E gene and proposed virulence differences among genotypes, some putatively selected amino acid sites have previously been implicated in changing viral pathogenicity, most notably E-390, and may also affect transmittability. These findings have implications for the future spread of DEN-2. © 2002 Elsevier Science (USA).

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.

We propose a novel method for direct cloning of foreign genes into baculoviruses which avoids the use of bacterial transfer vectors. The foreign gene to be inserted is derived by PCR using appropriate primers each of which contains an additional 50 nt of baculovirus sequence for homologous recombination between the PCR-derived DNA and the baculovirus DNA, thus accomplishing insertion of the foreign gene into the baculovirus. The direct cloning of green fluorescent protein and beta-glucuronidase in different baculovirus loci is described. The method is simple and avoids the use of cumbersome techniques associated with enzymatic treatment and DNA purification.

Previously, direct repeats (DRs) of 20-70 nucleotides were identified in the 3' untranslated regions (3'UTR) of flavivirus sequences. To address their functional significance, we have manually generated a pan-flavivirus 3'UTR alignment and correlated it with the corresponding predicted RNA secondary structures. This approach revealed that intra-group-conserved DRs evolved from six long repeated sequences (LRSs) which, as approximately 200-nucleotide domains were preserved only in the genomes of the slowly evolving tick-borne flaviviruses. We propose that short DRs represent the evolutionary remnants of LRSs rather than distinct molecular duplications. The relevance of DRs to virus replication enhancer function, and thus survival, is discussed.

Epidemics of tick-borne encephalitis involving thousands of humans occur annually in the forested regions of Europe and Asia. Despite the importance of this disease, the underlying basis for the development of encephalitis remains undefined. Here, we prove the key role of CD8+ T-cells in the immunopathology of tick-borne encephalitis, as demonstrated by prolonged survival of SCID or CD8−/− mice, following infection, when compared with immunocompetent mice or mice with adoptively transferred CD8+ T-cells. The results imply that tick-borne encephalitis is an immunopathological disease and that the inflammatory reaction significantly contributes to the fatal outcome of the infection.

An attenuated strain (263) of the tick-borne encephalitis virus, isolated from field ticks, was either serially subcultured, 5 times in mice, or at 40 °C in PS cells, producing 2 independent strains, 263-m5 and 263-TR with identical genomes; both strains exhibited increased plaque size, neuroinvasiveness and temperature-resistance. Sequencing revealed two unique amino acid substitutions, one mapping close to the catalytic site of the viral protease. These observations imply that virus adaptation from ticks to mammals occurs by selection of pre-existing virulent variants from the quasispecies population rather than by the emergence of new random mutations. The significance of these observations is discussed.

Full-length genomic sequences from six DENV-2 isolates sampled at different times during a dengue outbreak that occurred in Cuba in 1997 were determined. Phylogenetic analysis indicated that these isolates fall into the “American/Asian” genotype. Genome analysis revealed strong conservation of the structural proteins and the non-coding regions (5′ NCR and 3′ NCR). Nucleotide substitutions were observed in non-structural genes and most notably in the NS5 gene. There was a clear pattern of virus evolution during the epidemic; the earliest isolates sampled differed from those sampled later by amino acid replacements in the NS1 and NS5 proteins, although there was no evidence that these represented escape mutants. Further studies are therefore required to define the functional role of amino acid replacements observed and their possible relation to disease severity.