Spatial scale influences the distribution of viral diversity in the eukaryotic virome of the mosquito Culex pipiens.

Our knowledge of 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 of this diversity remain elusive in the eukaryotic viromes of terrestrial animals. This situation hinders hypothesis-driven tests of virome influence on host physiology. Here, we compare taxonomic diversity between different spatial scales in the eukaryotic virome of the mosquito Culex pipiens. This mosquito is a vector of human pathogens worldwide. The experimental design involved sampling in five countries in Africa and Europe around the Mediterranean Sea and large mosquito numbers to ensure a thorough exploration of virus diversity. A group of viruses was found in all countries. This core group represented a relatively large and diverse fraction of the virome. However, certain 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 coinfections in individual mosquitoes suggested random co-occurrence of those core viruses. Our results also suggested differences in viromes depending on geography, with viromes tending to cluster depending on the continent. Thus, our results unveil that the overlap in taxonomic diversity can decrease with spatial scale in the eukaryotic virome of C. pipiens. Furthermore, our results show that integrating contrasted spatial scales allows us to identify assembly patterns in the mosquito virome. Such patterns can guide future studies of virome influence on mosquito physiology.

One Health surveillance of West Nile and Usutu viruses: a repeated cross-sectional study exploring seroprevalence and endemicity in Southern France, 2016 to 2020.

BackgroundWest Nile virus (WNV) and Usutu virus (USUV), two closely related flaviviruses, mainly follow an enzootic cycle involving mosquitoes and birds, but also infect humans and other mammals. Since 2010, their epidemiological situation may have shifted from irregular epidemics to endemicity in several European regions; this requires confirmation, as it could have implications for risk assessment and surveillance strategies.AimTo explore the seroprevalence in animals and humans and potential endemicity of WNV and USUV in Southern France, given a long history of WNV outbreaks and the only severe human USUV case in France in this region.MethodsWe evaluated the prevalence of WNV and USUV in a repeated cross-sectional study by serological and molecular analyses of human, dog, horse, bird and mosquito samples in the Camargue area, including the city of Montpellier, between 2016 and 2020.ResultsWe observed the active transmission of both viruses and higher USUV prevalence in humans, dogs, birds and mosquitoes, while WNV prevalence was higher in horses. In 500 human samples, 15 were positive for USUV and 6 for WNV. Genetic data showed that the same lineages, WNV lineage 1a and USUV lineage Africa 3, were found in mosquitoes in 2015, 2018 and 2020.ConclusionThese findings support existing literature suggesting endemisation in the study region and contribute to a better understanding of USUV and WNV circulation in Southern France. Our study underlines the importance of a One Health approach for the surveillance of these viruses.

A library preparation optimized for metagenomics of RNA viruses.

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.

Emergence of two Usutu virus lineages in Culex pipiens mosquitoes in the Camargue, France, 2015.

Usutu virus (USUV) was detected in 11 Culex pipiens mosquito pools collected in 2015 in Camargue (France) using quantitative real-time RT-PCR assays. Phylogenetic analysis of recovered virus sequences identified USUV lineages Africa 2 and Africa 3, demonstrating the simultaneous occurrence of different strains within the mosquito population. This is the first report on USUV in mosquitoes from France that concurrently accompanied the emergence of Usutu virus in blackbirds and a human case in France during 2015/2016.

No association between the use of Bti for mosquito control and the dynamics of non-target aquatic invertebrates in French coastal and continental wetlands.

The environmental safety of Bacillus thuringiensis subsp. israelensis (Bti) is still controversial, mainly because most of the previous field studies on its undesired effects were spatially limited and did not address the relationship between community similarity and application time and frequency. No general statement can therefore be drawn on the usage conditions of Bti that insure protection of non-target organisms. The present study was conducted in eight sites distributed over the main geographical sectors where mosquito control is implemented in mainland France and Corsica. Changes in non-target aquatic invertebrates were followed at elapsed time after repeated applications of two Bti formulations (VectoBac® WDG or 12AS) up to four consecutive years. We examined the influence of both larvicide treatments and environmental variables on community dynamics and dissimilarity between treated and control areas. As it can be argued that chironomids are the most vulnerable group of non-target invertebrates, we scrutinised potential Bti-related effects on the dynamics of their community. The use of VectoBac® WDG and 12AS in coastal and continental wetlands had no immediate or long-term detectable effect on the taxonomic structure and taxa abundance of non-target aquatic invertebrate communities, including chironomids. This applied to the main habitats where mosquito larvae occur, regardless of their geographic location. Flooding, whose frequency and duration depend on local meteorological and hydrological conditions, was identified as the main environmental driver of invertebrate community dynamics. Our findings add support to the environmental safety of currently available Bti formulations when following recommended application rates and best mosquito control practices.