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Our knowledge on the diversity of eukaryotic viruses has recently undergone a massive expansion. This diversity could influence host physiology through yet unknown phenomena of potential interest to the fields of health and food production. However, the assembly processes in eukaryotic viromes of terrestrial animals remain elusive. This situation hinders hypothesis-driven tests of virome influence on host physiology. Here, we explore virome assembly at different spatial scales in the eukaryotic virome of the mosquitoCulex pipiens. This mosquito is a vector of human pathogens worldwide. Several new aspects of virome assembly were unveiled through a sampling involving five countries in Africa and Europe and large sample sizes. A group of viruses was conserved inC. pipienspopulations in all countries. This core group represented a relatively large and diverse fraction of the virome. However, core viruses were not shared by all host individuals in a given country, and their infection rates fluctuated between countries and years. Moreover, the distribution of co-infections in individual mosquitoes suggested random co-occurrence of certain core viruses. We also observed differences in the virome depending on geography, with viromes tending to cluster depending on the continent. Thus, our results unveil that taxon conservation in a eukaryotic virome changes with spatial scale. Thus, predictions on virome assembly seem possible at a large geographical scale inC. pipiens.IMPORTANCEThe study of the eukaryotic virome of mosquitoes is an emerging research field. Beyond its fundamental interest, this field could lead to the development of control tools against the transmission of mosquito-borne human pathogens. However, we yet know little on the assembly patterns in the eukaryotic viromes of mosquitoes, as well as of terrestrial animals in general. This situation hampers the design of hypothesis-driven studies on the influence of the virome on pathogen transmission. Here, we have analyzed virome assembly in the mosquito vectorCulex pipienswithin and between countries in Africa and Europe. Our results show that integrating contrasted spatial scales allows to identify deterministic patterns in virome assembly. Such patterns can guide future studies of virome influence on mosquito physiology.

International audience; Our understanding of the viral communities associated to animals has not yet reached the level attained on the bacteriome. This situation is due to, among others, technical challenges in adapting metagenomics using high-throughput sequencing to the study of RNA viromes in animals. Although important developments have been achieved in most steps of viral metagenomics, there is yet a key step that has received little attention: the library preparation. This situation differs from bacteriome studies in which developments in library preparation have largely contributed to the democratisation of metagenomics. Here, we present a library preparation optimized for metagenomics of RNA viruses from insect vectors of viral diseases. The library design allows a simple PCR-based preparation, such as those routinely used in bacterial metabarcoding, that is adapted to shotgun sequencing as required in viral metagenomics. We first optimized our library preparation using mock viral communities and then validated a full metagenomic approach incorporating our preparation in two pilot studies with field-caught insect vectors; one including a comparison with a published metagenomic protocol. Our approach provided a fold increase in virus-like sequences compared to other studies, and nearly-full genomes from new virus species. Moreover, our results suggested conserved trends in virome composition within a population of a mosquito species. Finally, the sensitivity of our approach was compared to a commercial diagnostic PCR for the detection of an arbovirus in field-caught insect vectors. Our approach could facilitate studies on viral communities from animals and the democratization of metagenomics in community ecology of viruses.

International audience; Eilat virus (EILV) is described as one of the few alphaviruses restricted to insects. We report the record of a nearly-complete sequence of an alphavirus genome showing 95% identity with EILV during a metagenomic analysis performed on 1488 unblood-fed females and 1076 larvae of the mosquito Culex pipiens captured in Rabat (Morocco). Genetic distance and phylogenetic analyses placed the EILV-Morocco as a variant of EILV. The observed infection rates in both larvae and adults suggested an active circulation of the virus in Rabat and its maintenance in the environment either through vertical transmission or through horizontal infection of larvae in breeding sites. This is the first report of EILV out of Israel and in Culex pipiens populations.