[Show abstract][Hide abstract] ABSTRACT: Japanese encephalitis (JE) is arguably one of the most serious viral encephalitis diseases worldwide. China has a long history of high prevalence of Japanese encephalitis, with thousands of cases reported annually and incidence rates often exceeding 15/100,000. In global terms, the scale of outbreaks and high incidence of these pandemics has almost been unique, placing a heavy burden on the Chinese health authorities. However, the introduction of vaccines, developed in China, combined with an intensive vaccination program initiated during the 1970s, as well as other public health interventions, has dramatically decreased the incidence from 20.92/100,000 in 1971, to 0.12/100,000 in 2011. Moreover, in less readily accessible areas of China, changes to agricultural practices designed to reduce chances of mosquito bites as well as mosquito population densities have also been proven effective in reducing local JE incidence. This unprecedented public health achievement has saved many lives and provided valuable experience that could be directly applicable to the control of vector-borne diseases around the world. Here, we review and discuss strategies for promotion and expansion of vaccination programs to reduce the incidence of JE even further, for the benefit of health authorities throughout Asia and, potentially, worldwide.
[Show abstract][Hide abstract] ABSTRACT: The discovery and development of methods for isolation, characterisation and taxonomy of viruses represents an important milestone in the study, treatment and control of virus diseases during the 20th century. Indeed, by the late-1950s, it was becoming common belief that most human and veterinary pathogenic viruses had been discovered. However, at that time, knowledge of the impact of improved commercial transportation, urbanisation and deforestation, on disease emergence, was in its infancy. From the late 1960s onwards viruses, such as hepatitis virus (A, B and C) hantavirus, HIV, Marburg virus, Ebola virus and many others began to emerge and it became apparent that the world was changing, at least in terms of virus epidemiology, largely due to the influence of anthropological activities. Subsequently, with the improvement of molecular biotechnologies, for amplification of viral RNA, genome sequencing and proteomic analysis the arsenal of available tools for virus discovery and genetic characterization opened up new and exciting possibilities for virological discovery. Many recently identified but "unclassified" viruses are now being allocated to existing genera or families based on whole genome sequencing, bioinformatic andphylogenetic analysis. New species, genera and families are also being created following the guidelines of the International Committee for the Taxonomy of Viruses. Many of these newly discovered viruses are vectored by arthropods (arboviruses) and possess an RNA genome. This brief review will focus largely on the discovery of new arthropod-borne viruses.
[Show abstract][Hide abstract] ABSTRACT: RNA secondary structures in the 3'untranslated regions (3'UTR) of the viruses of the family Flaviviridae, previously identified as essential (promoters) or beneficial (enhancers) for replication, have been analysed. Duplicated enhancer elements are revealed as a global feature in the evolution of the 3'UTR of distantly related viruses within the genera Flavivirus and Pestivirus. For the flaviviruses, duplicated structures occur in the 3'UTR of all four distantly related ecological virus subgroups (tick-borne, mosquito-borne, no known vector and insect-specific flaviviruses (ISFV). RNA structural differences distinguish tick-borne flaviviruses with discrete pathogenetic characteristics. For Aedes- and Culex-associated ISFV, secondary RNA structures with different conformations display numerous short ssRNA direct repeats, exposed as loops and bulges. Long quadruplicate regions comprise almost the entire 3'UTR of Culex-associated ISFV. Extended duplicated sequence and associated RNA structures were also discovered in the 3'UTR of pestiviruses. In both the Flavivirus and Pestivirus genera, duplicated RNA structures were localized to the enhancer regions of the 3'UTR suggesting an adaptive role predominantly in wild-type viruses. We propose sequence reiteration might act as a scaffold for dimerization of proteins involved in assembly of viral replicase complexes. Numerous nucleotide repeats exposed as loops/bulges might also interfere with host immune responses acting as a molecular sponge to sequester key host proteins or microRNAs.
PLoS ONE 01/2014; 9(3):e92056. · 3.53 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Biotic factors contributing to the survival of tick-borne viruses in nature are poorly understood. Using tick-borne encephalitis virus (TBEV) and its principal European vector, Ixodes ricinus, we examined the relative roles of salivary gland infection, co-feeding transmission, and moulting in virus survival. Virus titres in the salivary glands increased after blood-feeding in a time- and dose-dependent manner. This was observed in ticks infected by inoculation but not in ticks infected by the natural route of co-feeding. Amplification of infection prevalence occurred via co-feeding. However, when larvae or nymphs subsequently moulted, the infection prevalence dramatically declined although this was not observed when ticks were infected by inoculation. Trans-stadial survival is a hitherto overlooked parameter that may contribute to the low incidence of TBEV infection in field-collected I. ricinus ticks.
Ticks and Tick-borne Diseases 01/2014; · 2.35 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: West Nile virus (WNV), a mosquito-borne flavivirus in the Japanese encephalitis antigenic group, has caused sporadic outbreaks in humans, horses and birds throughout many of the warmer regions of Europe for at least 20 years. Occasional cases of West Nile encephalitis have also been associated with infected blood transfusions and organ donations. Currently, WNV appears to be expanding its geographical range in Europe and causing increasing numbers of epidemics/outbreaks associated with human morbidity and mortality. This brief review reports on the current epidemic situation regarding WNV in Europe, highlighting the clinical, diagnostic and preventive measures available for controlling this apparently emerging human pathogen.
Clinical Microbiology and Infection 03/2013; · 4.58 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The polymerase chain reaction (PCR) has become an essential method for the detection of viruses in tissue specimens. However, it is well known that the presence of PCR inhibitors in tissue samples may cause false-negative results. Hence the identification of PCR inhibitors and evaluation and optimization of nucleic acid extraction and preservation methods is of prime concern in virus discovery programs dealing with animal tissues. Accordingly, to monitor and remove inhibitors we have performed comparative analyses of two commonly used tissue storage methods and 5 RNA purification techniques using a variety of animal tissues, containing quantified levels of added MS2 bacteriophages as the indicator of inhibition. The results showed (i) no significant difference between the two methods of sample preservation, viz direct storage at -80C or 4C in RNAlater, (ii) lung rodent tissues contained lower levels of inhibitor than liver, kidney and spleen, (iii) RNA extraction using the EZ1+PK RNA kit was the most effective procedure for removal of RT-PCR inhibitors.
Journal of virological methods 03/2013; · 2.13 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Duck egg-drop syndrome virus (DEDSV) is a newly emerging pathogenic flavivirus causing avian diseases in China. The infection occurs in laying ducks characterized by a severe drop in egg production with a fatality rate of 5-15 %. The virus was found to be most closely related to Tembusu virus (TMUV), an isolate from mosquitoes in South-east Asia. Here, we have sequenced and characterized the full-length genomes of seven DEDSV strains, including the 5'- and 3'-non-coding regions (NCRs). We also report for the first time the ORF sequences of TMUV and Sitiawan virus (STWV), another closely related flavivirus isolated from diseased chickens. We analysed the phylogenetic and antigenic relationships of DEDSV in relation to the Asian viruses TMUV and STWV, and other representative flaviviruses. Our results confirm the close relationship between DEDSV and TMUV/STWV and we discuss their probable evolutionary origins. We have also characterized the cleavage sites, potential glycosylation sites and unique motifs/modules of these viruses. Additionally, conserved sequences in both 5'- and 3'-NCRs were identified and the predicted secondary structures of the terminal sequences were studied. Antigenic cross-reactivity comparisons of DEDSV with related pathogenic flaviviruses identified a surprisingly close relationship with dengue virus (DENV) and raised the question of whether or not DEDSV may have a potential infectious threat to man. Importantly, DEDSV can be efficiently recognized by a broadly cross-reactive flavivirus mAb, 2A10G6, derived against DENV. The significance of these studies is discussed in the context of the emergence, evolution, epidemiology, antigenicity and pathogenicity of the newly emergent DEDSV.
Journal of General Virology 07/2012; 93(Pt 10):2158-70. · 3.13 Impact Factor
[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 firstname.lastname@example.org.
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. · 8.81 Impact Factor
[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: 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: 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.