Replication enhancer elements within the open reading frame of tick-borne encephalitis virus and their evolution within the Flavivirus genus.

We provide experimental evidence of a replication enhancer element (REE) within the capsid gene of tick-borne encephalitis virus (TBEV, genus Flavivirus). Thermodynamic and phylogenetic analyses predicted that the REE folds as a long stable stem-loop (designated SL6), conserved among all tick-borne flaviviruses (TBFV). Homologous sequences and potential base pairing were found in the corresponding regions of mosquito-borne flaviviruses, but not in more genetically distant flaviviruses. To investigate the role of SL6, nucleotide substitutions were introduced which changed a conserved hexanucleotide motif, the conformation of the terminal loop and the base-paired dsRNA stacking. Substitutions were made within a TBEV reverse genetic system and recovered mutants were compared for plaque morphology, single-step replication kinetics and cytopathic effect. The greatest phenotypic changes were observed in mutants with a destabilized stem. Point mutations in the conserved hexanucleotide motif of the terminal loop caused moderate virus attenuation. However, all mutants eventually reached the titre of wild-type virus late post-infection. Thus, although not essential for growth in tissue culture, the SL6 REE acts to up-regulate virus replication. We hypothesize that this modulatory role may be important for TBEV survival in nature, where the virus circulates by non-viraemic transmission between infected and non-infected ticks, during co-feeding on local rodents.

Origin and evolution of flavivirus 5'UTRs and panhandles: trans-terminal duplications?

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.

Origin and evolution of 3'UTR of flaviviruses: long direct repeats as a basis for the formation of secondary structures and their significance for virus transmission.

The 3' untranslated regions (3'UTRs) of flaviviruses are reviewed and analyzed in relation to short sequences conserved as direct repeats (DRs). Previously, alignments of the 3'UTRs have been constructed for three of the four recognized flavivirus groups, namely mosquito-borne, tick-borne, and nonclassified flaviviruses (MBFV, TBFV, and NCFV, respectively). This revealed (1) six long repeat sequences (LRSs) in the 3'UTR and open-reading frame (ORF) of the TBFV, (2) duplication of the 3'UTR of the NCFV by intramolecular recombination, and (3) the possibility of a common origin for all DRs within the MBFV. We have now extended this analysis and review it in the context of all previous published analyses. This has been achieved by constructing a robust alignment between all flaviviruses using the published DRs and secondary RNA structures as "anchors" to reveal additional homologies along the 3'UTR. This approach identified nucleotide regions within the MBFV, NKV (no-known vector viruses), and NCFV 3'UTRs that are homologous to different LRSs in the TBFV 3'UTR and ORF. The analysis revealed that some of the DRs and secondary RNA structures described individually within each flavivirus group share common evolutionary origins. The 3'UTR of flaviviruses, and possibly the ORF, therefore probably evolved through multiple duplication of an RNA domain, homologous to the LRS previously identified only in the TBFV. The short DRs in all virus groups appear to represent the evolutionary remnants of these domains rather than resulting from new duplications. The relevance of these flavivirus DRs to evolution, diversity, 3'UTR enhancer function, and virus transmission is reviewed.

Direct repeats in the flavivirus 3' untranslated region; a strategy for survival in the environment?

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.

The 3' untranslated region of tick-borne flaviviruses originated by the duplication of long repeat sequences within the open reading frame.

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.

Direct repeats in the 3' untranslated regions of mosquito-borne flaviviruses: possible implications for virus transmission.

Direct repeats (DRs) of 20-45 nucleotide conserved sequences (CS) and repeated CS (RCS), separated by non-conserved sequences up to 100 nucleotides long, were previously described in the 3' untranslated region (3'UTR) of the three major mosquito-borne flavivirus (MBFV) subgroups, represented by Japanese encephalitis virus, Yellow fever virus and Dengue virus. Each subgroup exhibits a specific pattern of DRs, the biological significance of which has not yet been adequately addressed. The DRs were originally identified using conventional alignment programs based on the assumption that genetic variation is driven primarily by nucleotide substitutions. Since there are no recognized alignment programs that can adequately accommodate very divergent sequences, a method has been devised to construct and analyse a substantially improved 3'UTR alignment between these highly divergent viruses, based on the concept that deletions and/or insertions, in addition to substitutions, are important drivers of 3'UTR evolution. This 'robust alignment' approach demonstrated more extensive homologies in the 3'UTR than had been recognized previously and revealed the presence of similar DRs, either intact or as sequence 'remnants', in all the MBFV subgroups. The relevance of these observations is discussed in relation to (i) the function of DRs as elements of replication enhancement, (ii) the evolution of RNA secondary structures and (iii) the significance of DRs and secondary structures in MBFV transmissibility between vertebrate and invertebrate hosts.

The 3' untranslated regions of Kamiti River virus and Cell fusing agent virus originated by self-duplication.

Previously, it was shown that the 3' untranslated region (3'UTR) of Kamiti River virus (KRV) is nearly twice as long as the 3'UTR of other flaviviruses (1208 nucleotides compared with 730 nucleotides for the longest 3'UTR of any virus in the Tick-borne encephalitis virus species). Additionally, KRV and the closely related Cell fusing agent virus (CFAV) were shown to contain two short, almost perfect repeat sequences of 67 nucleotides. However, the construction of a robust comparative nucleotide alignment has now revealed that the double-length 3'UTR and the direct repeats resulted from the virtually complete duplication of a primordial KRV 3'UTR. We also propose that the CFAV 3'UTR was derived from a KRV-like precursor sequence with a large deletion that nevertheless preserved the two direct repeat sequences. These data provide new insights into the evolution of the flavivirus 3'UTR.

The 3' untranslated region of tick-borne flaviviruses originated by the duplication of long repeat sequences within the open reading frame.

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 virus diseases of human interest in Europe.

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.

Tick-borne encephalitis.

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.

Characterization of a siberian virus isolated from a patient with progressive chronic tick-borne encephalitis.

A strain of Tick-borne encephalitis virus designated Zausaev (Za) was isolated in Siberia from a patient who died of a progressive (2-year) form of tick-borne encephalitis 10 years after being bitten by a tick. The complete genomic sequence of this virus was determined, and an attempt was made to correlate the sequence with the biological characteristics of the virus. Phylogenetic analysis demonstrated that this virus belongs to the Siberian subtype of Tick-borne encephalitis virus. Comparison of Za virus with two related viruses, a Far Eastern isolate, Sofjin, and a Siberian isolate, Vasilchenko, revealed differences among the three viruses in pathogenicity for Syrian hamsters, cytopathogenicity for PS cells, plaque morphology, and the electrophoretic profiles of virus-specific nonstructural proteins. Comparative amino acid alignments revealed 10 individual amino acid substitutions in the Za virus polyprotein sequence that were different from those of other tick-borne flaviviruses. Notably, the dimeric form of the Za virus NS1 protein migrated in polyacrylamide gels as a heterogeneous group of molecules with a significantly higher electrophoretic mobility than those of the Sofjin and Vasilchenko viruses. Two amino acid substitutions, T(277)-->V and E(279)-->G, within the NS1 dimerization domain are probably responsible for the altered oligomerization of Za virus NS1. These studies suggest that the patient from whom Za virus was isolated died due to increased pathogenicity of the latent virus following spontaneous mutagenesis.

Tick-borne flaviviruses.

Tick-borne encephalitis (TBE), one of the most dangerous neuroinfections in Europe and Asia, is caused by tick-borne encephalitis virus (TBEV) and currently involves approximately 11,000 human cases annually, mostly in Russia. This chapter describes the main problems associated with the epidemiology, ecology, pathogenesis, and control of this disease. We have attempted to review the factors that influence the incidence and distribution of TBE, and to discuss possible reasons for the different clinical manifestations including most commonly observed asymptomatic infections, fever forms, acute encephalitis, and the less frequently registered biphasic milk fever and chronic encephalitis. Epidemiologic data concerning the other tick-borne flaviviruses, namely Louping ill virus, Langat virus, and Powassan virus that also produce encephalitis on a smaller scale, are also presented. Here we describe the history and current epidemiological role of Omsk hemorrhagic fever virus and Kyasanur forest disease virus, two viruses that are genetically closely related to TBEV, but produce hemorrhagic fever instead of encephalitis, and provide possible explanations for these differences. The other viruses in the tick-borne flavivirus group are also included despite the fact that they do not play an essential epidemiologic role in humans. This chapter contains a brief history of vaccination against TBE including the trials with live attenuated vaccine and reviews the modern trends in development of vaccine virus strains.

The degree of attenuation of tick-borne encephalitis virus depends on the cumulative effects of point mutations.

An infectious clone (pGGVs) of the tick-borne encephalitis complex virus Vasilchenko (Vs) was constructed previously. Virus recovered from pGGVs produced slightly smaller plaques than the Vs parental virus. Sequence analysis demonstrated five nucleotide differences between the original Vs virus and pGGVs; four of these mutations resulted in amino acid substitutions, while the fifth mutation was located in the 3' untranslated region (3'UTR). Two mutations were located in conserved regions and three mutations were located in variable regions of the virus genome. Reverse substitutions from the conserved regions of the genome, R(496)-->H in the envelope (E) gene and C(10884)-->T in the 3'UTR, were introduced both separately and together into the infectious clone and their biological effect on virus phenotype was evaluated. The engineered viruses with R(496) in the E protein produced plaques of smaller size than viruses with H(496) at this position. This mutation also affected the growth and neuroinvasiveness of the virus. In contrast, the consequence of a T(10884)-->C substitution within the 3'UTR was noticeable only in cytotoxicity and neuroinvasiveness tests. However, all virus mutants engineered by modification of the infectious clone, including one with two wild-type mutations, H(496) and T(10884), showed reduced neuroinvasiveness in comparison with the Vs parental virus. Therefore, although the H(496)-->R and T(10884)-->C substitutions clearly reduce virus virulence, the other mutations within the variable regions of the capsid (I(45)-->F) and the NS5 (T(2688)-->A and M(3385)-->I) genes also contribute to the process of attenuation. In terms of developing flavivirus vaccines, the impact of accumulating apparently minor mutations should be assessed in detail.

Diagnostic immunoassays for tick-borne encephalitis virus based on recombinant baculovirus protein expression.

The baculovirus expression system that utilizes Autographa californica nuclear polyhedrosis virus was used to express the highly antigenic envelope protein E of a tick-borne encephalitis (TBE) complex virus, as well as a C-terminally truncated form of protein E (Etr). The recombinant proteins were produced with a histidine-tag at their carboxy-terminus. Protein purification by nickel agarose chromatography resulted in high concentrations of pure Etr protein, but only poor yields of E protein. Therefore, Etr was used to develop a sensitive and specific enzyme-linked immunosorbent assay (ELISA), as well as an immunoblot assay to detect TBE virus-specific antibodies in sera from immunized human blood donors. Sera from non-vaccinated blood donors were used as controls. The data show that the recombinant TBE virus-specific Etr protein exhibits the antigenic epitopes and conformation necessary for specific antigen-antibody recognition. Thus, the baculovirus expression system provides a cheap and easy method to generate recombinant viral antigens for TBE virus-specific serodiagnosis.

Biological consequences of deletions within the 3'-untranslated region of flaviviruses may be due to rearrangements of RNA secondary structure.

It was previously reported that deletions introduced into the 3'-untranslated region (3'-UTR) of dengue type 4 (DEN 4) virus (Men, R., Bray, M., Clark, D., Chanock, R.M., Lai, C.J., 1996. DEN 4 virus mutants containing deletions in the 3'-noncoding region of the RNA genome: analysis of growth restriction in cell culture and altered viremia pattern and immunogenicity in Rhesus monkeys. J. Virol. 70, 3930-3937), tick-borne encephalitis (TBE) virus (Mandl, C.W., Holzmann, H., Meixner, T., Rauscher, S., Stadler, P.F., Allison, S.L. , Heinz, F.X., 1998. Spontaneous and engineered deletions in the 3'-noncoding region of TBE virus: construction of highly attenuated mutants of a flavivirus. J. Virol. 72, 2132-2140) and subgenomic replicons of Kunjin virus (Khromykh, A.A., Westaway, E.G., 1997. Subgenomic replicons of the flavivirus Kunjin: construction and applications. J. Virol. 71, 1497-1505) altered the infectivity of the mutants and reduced the efficiency of RNA replication. Here, these deletions were superimposed onto the models of secondary structure we constructed previously and the folding of the modified 3'-UTR sequences was simulated. The analysis showed that most of the deletions disrupted or reshaped conserved elements of secondary structure and that the biological effects of these deletions are likely to represent structural rearrangements in the 3'-UTR, rather than the loss of sequence motifs. The analysis also suggested that the overall structural integrity of the flaviviral 3'-UTR is essential for optimal performance of its promotor function, although two distinct parts can be defined: the most 3'-terminal structures and sequences which may be critical for the initiation of minus-strand RNA synthesis, and more proximal structures and sequences that possibly function as enhancers of viral RNA replication. The functional significance of certain structural elements and their possible effect on the efficiency of viral replication in different cells are also discussed.

Development and analysis of a tick-borne encephalitis virus infectious clone using a novel and rapid strategy.

In less than 1 month we have constructed an infectious clone of attenuated tick-borne encephalitis virus (strain Vasilchenko) from 100 microl of unpurified virus suspension using long high fidelity PCR and a modified bacterial cloning system. Optimization of the 3' antisense primer concentration was essential to achieve PCR synthesis of an 11 kb cDNA copy of RNA from infectious virus. A novel system utilising two antisense primers, a 14-mer for reverse transcription and a 35-mer for long PCR, produced high yields of genomic length cDNA. Use of low copy number Able K cells and an incubation temperature of 28 degrees C increased the genetic stability of cloned cDNA. Clones containing 11 kb cDNA inserts produced colonies of reduced size, thus providing a positive selection system for full length clones. Sequencing of the infectious clone emphasised the improved fidelity of the method compared with conventional PCR and cloning methods. A simple and rapid strategy for genetic manipulation of the infectious clone is also described. These developments represent a significant advance in recombinant technology and should be applicable to positive stranded RNA viruses which cannot easily be purified or genetically manipulated.

Complete sequence of two tick-borne flaviviruses isolated from Siberia and the UK: analysis and significance of the 5' and 3'-UTRs.

The complete nucleotide sequence of two tick-transmitted flaviviruses, Vasilchenko (Vs) from Siberia and louping ill (LI) from the UK, have been determined. The genomes were respectively, 10928 and 10871 nucleotides (nt) in length. The coding strategy and functional protein sequence motifs of tick-borne flaviviruses are presented in both Vs and LI viruses. The phylogenies based on maximum likelihood, maximum parsimony and distance analysis of the polyproteins, identified Vs virus as a member of the tick-borne encephalitis virus subgroup within the tick-borne serocomplex, genus Flavivirus, family Flaviviridae. Comparative alignment of the 3'-untranslated regions revealed deletions of different lengths essentially at the same position downstream of the stop codon for all tick-borne viruses. Two direct 27 nucleotide repeats at the 3'-end were found only for Vs and LI virus. Immediately following the deletions a region of 332-334 nt with relatively conserved primary structure (67-94% identity) was observed at the 3'-non-coding end of the virus genome. Pairwise comparisons of the nucleotide sequence data revealed similar levels of variation between the coding region, and the 5' and 3'-termini of the genome, implying an equivalent strong selective control for translated and untranslated regions. Indeed the predicted folding of the 5' and 3'-untranslated regions revealed patterns of stem and loop structures conserved for all tick-borne flaviviruses suggesting a purifying selection for preservation of essential RNA secondary structures which could be involved in translational control and replication. The possible implications of these findings are discussed.

A new, rapid and simple procedure for direct cloning of PCR products into baculoviruses.

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.

Infectious transcripts of tick-borne encephalitis virus, generated in days by RT-PCR.

Construction of infectious clones of flaviviruses can be problematic owing to instability, toxicity, and recombination events occurring while cloning cDNA in the bacterial vectors. To overcome these difficulties we have devised a rapid and simple method for producing an infectious genetically engineered tick-borne encephalitis virus in less than 10 days using viral RNA from an unpurified virus suspension. The experimental protocol utilized the high fidelity reverse transcription-polymerase chain reaction to produce two long (5.7 and 5.2 kb) overlapping cDNA segments. To produce full-length cDNA the two overlapping segments were either ligated or fused by polymerase chain reaction. The cDNA was then transcribed and the derived full-length RNA was injected intracerebrally into young mice which reproduced the infectious virus within 8-20 days. To differentiate the engineered virus from parent virus, a Sunl restriction site was introduced by substituting nucleotides at positions 5688 and 5691 of the viral genome. This restriction site was present in the engineered virus recovered from infected mice. Antigenic and electrophoretic analysis of the proteins recovered from the engineered virus confirmed that it was indistinguishable from parent virus. In addition to its applicability as a rapid method of producing infectious engineered virus, this protocol offers the opportunity to introduce changes by site-directed mutagenesis without needing to clone the viral DNA. The method should be applicable to most viruses possessing an infectious RNA molecule and reduces the time required to produce a genetically engineered virus from years to days. When appropriate, the choice of mice for transfection of RNA has the advantage of being extremely simple, very sensitive, and producing high titers of stable virus.

Analysis of flavivirus envelope proteins reveals variable domains that reflect their antigenicity and may determine their pathogenesis.

Studies on the molecular basis of flavivirus neutralisation, attenuation and tropism indicate that amino acid substitutions, in different parts of the envelope gene, may be responsible for the altered phenotypes. However, the association of particular substitutions with individual characteristics has proven difficult. Comparative analysis of all known tick-borne flavivirus envelope proteins through sequence alignment and a sliding window, reveals clusters of amino acid variation distributed throughout the envelope protein coding region. Further comparison with mosquito-borne flaviviruses reveals essentially the same profile of variability throughout the envelope protein sequence although there is a major difference within the postulated B domain of these viruses which may reflect their different evolutionary development. Most phenotypically variant properties, such as serotypic differences, variants characteristic of vaccine strains, altered tropisms and neutralisation escape mutants, map within the variable clusters. Thus, we propose that natural mutagenesis and selection may occur at specific sites that do not destroy the secondary and tertiary E protein structure and that the variable clusters represent the exposed surface amino acids of the envelope protein defining antigenicity, tropicity and pathogenesis.