[show abstract][hide abstract] ABSTRACT: Tick-borne flaviviruses (TBF) are widely dispersed across Africa, Europe, Asia, Oceania, and North America, and some present a significant threat to human health. Seminal studies on tick-borne encephalitis viruses (TBEV), based on partial envelope gene sequences, predicted a westward clinal pattern of evolution and dispersal across northern Eurasia, terminating in the British Isles. We tested this hypothesis using all available full-length open reading frame (ORF) TBF sequences. Phylogenetic analysis was consistent with current reports. However, linear and nonlinear regression analysis of genetic versus geographic distance combined with BEAST analysis identified two separate clines, suggesting that TBEV spread both east and west from a central point. In addition, BEAST analysis suggested that TBF emerged and dispersed more than 16,000 years ago, significantly earlier than previously predicted. Thus, climatic and ecological changes may have played a greater role in TBF dispersal than humans.
Journal of Virology 06/2012; 86(16):8663-71. · 5.08 Impact Factor
[show abstract][hide abstract] ABSTRACT: The European Virus Archive (EVA) was conceived as a direct response to the need for a coordinated and readily accessible collection of viruses that could be made available to academia, public health organisations and industry, initially within Europe, but ultimately throughout the world. Although scientists worldwide have accumulated virus collections since the early twentieth century, the quality of the collections and the viruses collected may vary according to the personal interests and agenda of the scientists. Moreover, when laboratories are re-organised or closed, collections are no longer maintained and gradually cease to exist. The tragedy of 9/11 and other disruptive activities have also meant that some previously available biological reagents are no longer openly exchanged between countries. In 2008, funding under the FP7-EU infrastructure programme enabled the initiation of the EVA. Within three years, it has developed from a consortium of nine European laboratories to encompass associated partners in Africa, Russia, China, Turkey, Germany and Italy. There is every reason to believe that EVA will continue to expand and ultimately exist as a globally networked, quality-controlled non-profit archive for the benefit of science. Organizations or individuals who would like to be considered as contributors are invited to contact the EVA coordinator, Jean-Louis Romette, at email@example.com.
Antiviral research 05/2012; 95(2):167-71. · 3.61 Impact Factor
[show abstract][hide abstract] ABSTRACT: 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.
Nucleic Acids Research 05/2011; 39(16):7034-48. · 8.28 Impact Factor
[show abstract][hide abstract] ABSTRACT: Dengue epidemics in Cuba have repeatedly demonstrated a month-to-month increase in clinical severity during secondary infections. The dengue 2 outbreak that occurred in Santiago de Cuba in 1997 was accompanied by the most severe intraepidemic increase in disease severity reported to date. It was initially proposed that the appearance of neutralization escape mutants during the course of the epidemic might explain this phenomenon. Recent studies have revealed that during the course of this epidemic, nucleotide substitutions appeared only in nonstructural (NS) genes, most of which were silent, except for one change in the NS1 gene. To study whether or not variation in the NS1 gene might be associated with increased disease severity during the epidemic, this gene was partially sequenced from 15 isolates obtained at different times during the 1997 epidemic. Early epidemic isolates differed from those obtained later by replacement only of threonine with serine at position 164 in the NS1 protein, an amino acid rarely found in any genotype of dengue 2 virus. All viruses isolated from patients located in Health Districts, where dengue 2 transmissions occurred late in the epidemic, contained Serine at position 164, indicating that this change was fixed within a few months. Here we argue that this single mutation contributes to viral survival or replication efficiency, resulting in enhanced infection in the presence of enhancing antibodies, a phenomenon that we term increased virus "fitness" in contrast to "virulence," an intrinsic property of the virus.
[show abstract][hide abstract] ABSTRACT: Serum and liver samples collected monthly, during 2005, from healthy wild rabbits at a site in Pitroddie, Scotland, were analysed by ELISA and RT-PCR sequencing. Sera collected in January and February had high antibody titres against RHDV. However, during the rabbit breeding season average antibody titres were lower but increased again as the year progressed. Between March and August, RHDV-specific RNA was detected in healthy rabbits spanning a wide range of age and antibody titres. Importantly, two virus lineages were identified; a novel widely divergent strain, recovered between March and August, and a strain related to UK epidemic strains, was recovered between May and July from juvenile rabbits. We propose that a non-virulent widely divergent strain of RHDV circulated asymptomatically amongst the wild rabbits potentially inducing immunity against the introduced epidemic strain that predominantly causes high fatality rates in young immunologically naïve rabbits.
[show abstract][hide abstract] ABSTRACT: The alphaviruses were amongst the first arboviruses to be isolated, characterized and assigned a taxonomic status. They are globally very widespread, infecting a large variety of terrestrial animals, insects and even fish, and circulate both in the sylvatic and urban/peri-urban environment, causing considerable human morbidity and mortality. Nevertheless, despite their obvious importance as pathogens, there are currently no effective antiviral drugs with which to treat humans or animals infected by any of these viruses. The EU-supported project-VIZIER (Comparative Structural Genomics of Viral Enzymes Involved in Replication, FP6 Project: 2004-511960) was instigated with an ultimate view of contributing to the development of antiviral therapies for RNA viruses, including the alphaviruses [Coutard, B., Gorbalenya, A.E., Snijder, E.J., Leontovich, A.M., Poupon, A., De Lamballerie, X., Charrel, R., Gould, E.A., Gunther, S., Norder, H., Klempa, B., Bourhy, H., Rohayemj, J., L'hermite, E., Nordlund, P., Stuart, D.I., Owens, R.J., Grimes, J.M., Tuckerm, P.A., Bolognesi, M., Mattevi, A., Coll, M., Jones, T.A., Aqvist, J., Unger, T., Hilgenfeld, R., Bricogne, G., Neyts, J., La Colla, P., Puerstinger, G., Gonzalez, J.P., Leroy, E., Cambillau, C., Romette, J.L., Canard, B., 2008. The VIZIER project: preparedness against pathogenic RNA viruses. Antiviral Res. 78, 37-46]. This review highlights some of the major features of alphaviruses that have been investigated during recent years. After describing their classification, epidemiology and evolutionary history and the expanding geographic distribution of Chikungunya virus, we review progress in understanding the structure and function of alphavirus replicative enzymes achieved under the VIZIER programme and the development of new disease control strategies.
Antiviral research 08/2009; 87(2):111-24. · 3.61 Impact Factor
[show abstract][hide abstract] ABSTRACT: Rabbit haemorrhagic disease (RHD) was first recognised in 1984 following the introduction of apparently healthy rabbits into China from Germany. The aetiological agent Rabbit haemorrhagic disease virus (RHDV) has subsequently killed hundreds of millions of domestic and wild rabbits particularly in Europe, China and Australia. Previously, using phylogenetic analysis we have attempted to understand the underlying factors that determine why this virus emerged, and why it has such an unpredictable epidemiology. Here we report the use of tree congruency supported by bootscanning analysis to detect recombination amongst both closely related, and widely divergent strains of RHDV. We show that recombination occurs commonly and in several different regions of the RHDV genome. Moreover, the first identified strain of RHDV, i.e. from China in 1984, showed evidence of recombination in the capsid gene, with a virus or viruses containing lineages in German strains. These observations imply that recombination may play a significant role in the evolution, epidemiology and diversity of RHDV.
[show abstract][hide abstract] ABSTRACT: Life-threatening RNA viruses emerge regularly, and often in an unpredictable manner. Yet, the very few drugs available against known RNA viruses have sometimes required decades of research for development. Can we generate preparedness for outbreaks of the, as yet, unknown viruses? The VIZIER (VIral enZymes InvolvEd in Replication) (http://www.vizier-europe.org/) project has been set-up to develop the scientific foundations for countering this challenge to society. VIZIER studies the most conserved viral enzymes (that of the replication machinery, or replicases) that constitute attractive targets for drug-design. The aim of VIZIER is to determine as many replicase crystal structures as possible from a carefully selected list of viruses in order to comprehensively cover the diversity of the RNA virus universe, and generate critical knowledge that could be efficiently utilized to jump-start research on any emerging RNA virus. VIZIER is a multidisciplinary project involving (i) bioinformatics to define functional domains, (ii) viral genomics to increase the number of characterized viral genomes and prepare defined targets, (iii) proteomics to express, purify, and characterize targets, (iv) structural biology to solve their crystal structures, and (v) pre-lead discovery to propose active scaffolds of antiviral molecules.
Antiviral Research 05/2008; 78(1):37-46. · 3.93 Impact Factor
[show abstract][hide abstract] ABSTRACT: 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.
[show abstract][hide abstract] ABSTRACT: 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.
[show abstract][hide abstract] ABSTRACT: With the exception of virus strains Ashington and RCV, other recognised strains of Rabbit haemorrhagic disease virus (RHDV) share relatively close genetic homology. Using serology and phylogenetic analysis, we have identified a third disparate virus lineage in healthy rabbits on Lambay Island off the east coast of Eire, where disease due to RHDV has never been observed. ELISA tests revealed high titre RHDV-specific antibodies in 81% of the sera from 11 healthy rabbits captured on this island, indicating that the virus is actively circulating amongst these rabbits. Nevertheless, infectious virus has not been isolated from rabbits living on this island. The detection of antibodies and the disparate Lambay virus lineage in an apparently healthy and isolated wild rabbit population provides the most convincing evidence yet that at least some strains of RHDV can circulate harmlessly for long periods of time in wild rabbits possibly by producing persistent or latent infections.
[show abstract][hide abstract] ABSTRACT: 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.
Advances in Virus Research 02/2007; 69:203-48. · 2.84 Impact Factor
[show abstract][hide abstract] ABSTRACT: We have determined the nucleotide sequence of DNA extracted from pustules, saliva, and blood of camels presenting with contagious ecthyma, in Bahrain and also from a sample (SACamel) of infected tissue from a camel that had presented with contagious ecthyma in 1998 in Saudi Arabia (1). Sequence homologies and phylogenetic analysis showed that this extracted DNA was more closely related to Pseudocowpox virus (PCPV) than Orf virus (ORFV), which infects sheep, goats, and other animal species. The phylogeny also demonstrated that PCPV in Arabian camels was phylogenetically distinct from, and circulates independently of, ruminant-associated PCPV from Europe.
[show abstract][hide abstract] ABSTRACT: 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.
Journal of General Virology 12/2006; 87(Pt 11):3297-305. · 3.13 Impact Factor
[show abstract][hide abstract] ABSTRACT: 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.
[show abstract][hide abstract] ABSTRACT: 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.
Journal of General Virology 10/2006; 87(Pt 9):2615-9. · 3.13 Impact Factor
[show abstract][hide abstract] ABSTRACT: During the past 50 years two readily distinguishable rabbit-specific diseases caused by Myxoma virus (MYXV) and Rabbit haemorrhagic disease virus (RHDV) respectively, have decimated wild rabbit populations worldwide. Combined with the use of these viruses as biocontrol agents, the consequences for farming, commercial rabbit breeding and rare habitat conservation dependent on rabbit grazing, have been both positive and negative. Moreover, rare predators that rely on rabbits as a food resource, and even hunters, have suffered the consequences of rabbit populations being affected by one or other of these viruses.Rabbit haemorrhagic disease virus was first identified after thousands of domestic rabbits died suddenly in China in 1984. Similar epidemics subsequently occurred in other regions of Asia, the Middle East, Europe and North America, suggesting that the virus had dispersed widely following its emergence in China. However, the discovery that RHDV had circulated apparently harmlessly for many years before the first recognised epidemic in China prompted us to investigate the evolution, emergence and dispersal of this virus in relation to its impact on conservation of wildlife species. Accordingly, we have sequenced new isolates of RHDV and combined these data with a global selection of available RHDV sequences. Using phylogenetic analysis we demonstrate that the Chinese epidemic virus diverged from European viruses that circulated many years prior to 1984. We also demonstrate that the lineages of the pathogenic viruses that emerged in the UK in the early 1990s, are distinct from and pre-date those of the 1984 Chinese virus. In other words, European strains of RHDV emerged from apparently harmless strains to cause epidemic outbreaks, independently of the Chinese 1984 epidemic virus. These studies demonstrate how understanding viral epidemiology can improve the development of strategies to conserve rabbits, rare predator species and the habitat.