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 virus-derived 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.

Centre for Immunity Infection and Evolution University of Edinburgh EH9 3JT UK

Faculty of Science University of South Bohemia and Biology Centre Institute of Parasitology Czech Academy of Sciences 37005 České Budějovice Czech Republic

Innate Immunity and Pathogenesis Unit Laboratory of Virology Rocky Mountain Laboratories Division of Intramural Research National Institute of Allergy and Infectious Diseases National Institutes of Health Hamilton MT 59840 USA

Institute of Evolutionary Biology and Centre for Infection Immunity and Evolution University of Edinburgh EH9 3JT UK

Laboratory of Virology Wageningen University 6708 PB Wageningen The Netherlands

Markey Centre for Structural Biology Department of Biological Sciences Purdue University West Lafayette IN 47907 USA

MRC University of Glasgow Centre for Virus Research Glasgow G11 5JR UK

MRC University of Glasgow Centre for Virus Research Glasgow G11 5JR UK The Roslin Institute and Royal School of Veterinary Studies University of Edinburgh Easter Bush Midlothian EH25 9RG UK

The Roslin Institute and Royal School of Veterinary Studies University of Edinburgh Easter Bush Midlothian EH25 9RG UK

Unité des Virus Emergents Faculté de Médicine Timone 13385 Marseille Cedex 05 France Centre for Hydrology and Ecology Maclean Building Oxon OX10 8BB UK

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