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Marseille, France

Tuplin A.,University of Warwick | Evans D.J.,University of Warwick | Buckley A.,Cranfield University | Jones I.M.,University of Reading | And 2 more authors.
Nucleic Acids Research

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. © 2011 The Author(s). Source

Barnard R.T.,University of Queensland | Hall R.A.,University of Queensland | Gould E.A.,Unite des Virus Emergents
Expert Review of Molecular Diagnostics

Extrapolation from recent disease history suggests that changes in the global environment, including virus, vector and human behavior, will continue to influence the spectrum of viruses to which humans are exposed. In this article, these environmental changes will be enumerated, and their potential impact on target-focused, nucleic acid-based diagnostic tests will be considered, followed by a presentation of some emerging technological responses. © 2011 Expert Reviews Ltd. Source

Schnettler E.,University of Glasgow | Schnettler E.,Roslin Institute | Tykalova H.,University of South Bohemia | Watson M.,Roslin Institute | And 23 more authors.
Nucleic Acids Research

Arboviruses are transmitted by distantly related arthropod vectors such as mosquitoes (class Insecta) and ticks (class Arachnida). RNA interference (RNAi) is the major antiviral mechanism in arthropods against arboviruses. Unlike in mosquitoes, tick antiviral RNAi is not understood, although this information is important to compare arbovirus/host interactions in different classes of arbovirus vectos. Using an Ixodes scapularis-derived cell line, key Argonaute proteins involved in RNAi and the response against tick-borne Langat virus (Flaviviridae) replication were identified and phylogenetic relationships characterized. Analysis of small RNAs in infected cells showed the production of virusderived small interfering RNAs (viRNAs), which are key molecules of the antiviral RNAi response. Importantly, viRNAs were longer (22 nucleotides) than those from other arbovirus vectors and mapped at highest frequency to the termini of the viral genome, as opposed to mosquito-borne flaviviruses. Moreover, tick-borne flaviviruses expressed subgenomic flavivirus RNAs that interfere with tick RNAi. Our results characterize the antiviral RNAi response in tick cells including phylogenetic analysis of genes encoding antiviral proteins, and viral interference with this pathway. This shows important differences in antiviral RNAi between the two major classes of arbovirus vectors, and our data broadens our understanding of arthropod antiviral RNAi. © The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research. Source

Gould E.A.,Unite des Virus Emergents | Gould E.A.,UK Center for Ecology and Hydrology
Molecular Ecology

Following the announcement of the first case of rabbit haemorrhagic disease (RHD) in a pet rabbit, housed indoors in Canada for more than 1 year, I submitted an evidence-based explanation to ProMed explaining how RHD might have caused the death of 'one' of the three pet rabbits. I suggested with supporting evidence, that it may have been persistently infected with rabbit haemorrhagic disease virus (RHDV) which may have reactivated to cause the fatal disease. However, in this issue, Peacock et al. have proposed an alternative 'hypothesis' for the appearance of RHD in the pet rabbit. They hypothesise that a non-identified insect or fomite might have become contaminated by a Chinese strain of RHDV somewhere in the US. This insect/fomite then flew or was windborne, from the US to Canada where it entered the house containing three pet rabbits and infected one of them. RHD is non-endemic and is rarely reported in the US, where it has only been observed in domestic European rabbits, held in rabbitries. My proposal was based on the details provided by ProMed, the veterinary report from Canada, where RHDV has never previously been identified and the epidemiological, ecological and evolutionary history of RHDV which includes serological and phylogenetic evidence that ancestral RHDV lineages circulated before 1984. The flying insect hypothesis of Peacock et al. is based on circumstantial evidence and, I believe, has a lower probability of being correct than my evidence-based long-term infection proposal. © 2012 Blackwell Publishing Ltd. Source

Gritsun D.J.,University of Reading | Jones I.M.,University of Reading | Gould E.A.,Unite des Virus Emergents | Gritsun T.S.,University of Reading

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. © 2014 Gritsun et al. Source

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