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 infuence host physiology through yet unknown phenomena of potential interest to the felds 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 infuence 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 fve 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 fuctuated 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 infuence on mosquito physiology.This work was supported by the 7th Framework Programme of the European Commission (grant FP7-613996 Vmerge), the Montpellier University of Excellence Program (MUSE, ArboSud project), and the Direction Générale de l’Alimentation from the French Ministry of Agriculture and Food (DGAl grant agreement: SPA17 n0079-E). The contents of this publication are the sole responsibility of the authors and do not necessarily refect the views of the European Commission.info:eu-repo/semantics/publishedVersio

Different viral genes modulate virulence in model mammal hosts and Culex pipiens vector competence in Mediterranean basin lineage 1 West Nile virus strains

West Nile virus (WNV) is a single-stranded positive-sense RNA virus (+ssRNA) belonging to the genus Orthoflavivirus. Its enzootic cycle involves mosquito vectors, mainly Culex, and wild birds as reservoir hosts, while mammals, such as humans and equids, are incidental dead-end hosts. It was first discovered in 1934 in Uganda, and since 1999 has been responsible for frequent outbreaks in humans, horses and wild birds, mostly in America and in Europe. Virus spread, as well as outbreak severity, can be influenced by many ecological factors, such as reservoir host availability, biodiversity, movements and competence, mosquito abundance, distribution and vector competence, by environmental factors such as temperature, land use and precipitation, as well as by virus genetic factors influencing virulence or transmission. Former studies have investigated WNV factors of virulence, but few have compared viral genetic determinants of pathogenicity in different host species, and even fewer have considered the genetic drivers of virus invasiveness and excretion in Culex vector. In this study, we characterized WNV genetic factors implicated in the difference in virulence observed in two lineage 1 WNV strains from the Mediterranean Basin, the first isolated during a significant outbreak reported in Israel in 1998, and the second from a milder outbreak in Italy in 2008. We used an innovative and powerful reverse genetic tool, e.g., ISA (infectious subgenomic amplicons) to generate chimeras between Israel 1998 and Italy 2008 strains, focusing on non-structural (NS) proteins and the 3′UTR non-coding region. We analyzed the replication of these chimeras and their progenitors in mammals, in BALB/cByJ mice, and vector competence in Culex (Cx.) pipiens mosquitoes. Results obtained in BALB/cByJ mice suggest a role of the NS2B/NS3/NS4B/NS5 genomic region in viral attenuation in mammals, while NS4B/NS5/3′UTR regions are important in Cx. pipiens infection and possibly in vector competence

A tale of caution:How endogenous viral elements affect virus discovery in transcriptomic data

Large-scale metagenomic and -transcriptomic studies have revolutionized our understanding of viral diversity and abundance. In contrast, endogenous viral elements (EVEs), remnants of viral sequences integrated into host genomes, have received limited attention in the context of virus discovery, especially in RNA-Seq data. EVEs resemble their original viruses, a challenge that makes distinguishing between active infections and integrated remnants difficult, affecting virus classification and biases downstream analyses. Here, we systematically assess the effects of EVEs on a prototypical virus discovery pipeline, evaluate their impact on data integrity and classification accuracy, and provide some recommendations for better practices. We examined EVEs and exogenous viral sequences linked to Orthomyxoviridae, a diverse family of negative-sense segmented RNA viruses, in 13 genomic and 538 transcriptomic datasets of Culicinae mosquitoes. Our analysis revealed a substantial number of viral sequences in transcriptomic datasets. However, a significant portion appeared not to be exogenous viruses but transcripts derived from EVEs. Distinguishing between transcribed EVEs and exogenous virus sequences was especially difficult in samples with low viral abundance. For example, three transcribed EVEs showed full-length segments, devoid of frameshift and nonsense mutations, exhibiting sufficient mean read depths that qualify them as exogenous virus hits. Mapping reads on a host genome containing EVEs before assembly somewhat alleviated the EVE burden, but it led to a drastic reduction of viral hits and reduced quality of assemblies, especially in regions of the viral genome relatively similar to EVEs. Our study highlights that our knowledge of the genetic diversity of viruses can be altered by the underestimated presence of EVEs in transcriptomic datasets, leading to false positives and altered or missing sequence information. Thus, recognizing and addressing the influence of EVEs in virus discovery pipelines will be key in enhancing our ability to capture the full spectrum of viral diversity. </p

Multi-locus sequence analysis reveals great genetic diversity among Mycoplasma capricolum subsp. capripneumoniae strains in Asia

International audienceMulti-Locus Sequence Analysis (MLSA) of Mycoplasma capricolum subsp. capripneumoniae ( Mccp ) strains from Asia revealed unforeseen diversity and a central position for genotyping groups representing strains from Central/East Asia, suggesting a possible origin of contagious caprine pleuropneumonia in this continent. A better assessment of the emergence, diversity and distribution of Mccp in Asia and Africa calls for renewed efforts to dramatically enlarge the sample of strains. Availability and affordability in the field, added to superior typeability (directly from poor samples) and high stability, discriminatory power and concordance with epidemiological and phylogenetic analyses, make MLSA an excellent tool for such investigations

Identification of Eilat virus and prevalence of infection among <em>Culex pipiens L</em>. populations, Morocco, 2016

International audienceEilat 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

The Genome Segments of Bluetongue Virus Differ in Copy Number in a Host-Specific Manner

International audienceGenome segmentation is mainly thought to facilitate reassortment. Here, we show that segmentation can also allow differences in segment abundance in populations of bluetongue virus (BTV). BTV has a genome consisting in 10 segments, and its cycle primarily involves periodic alternation between ruminants and Culicoides biting midges. We have developed a reverse transcription-quantitative PCR (RT-qPCR) approach to quantify each segment in wild BTV populations sampled in both ruminants and midges during an epizootic. Segment frequencies deviated from equimolarity in all hosts. Interestingly, segment frequencies were reproducible and distinct between ruminants and biting midges. Beyond a putative regulatory role in virus expression, this phenomenon could lead to different evolution rates between segments. IMPORTANCE The variation in viral gene frequencies remains a largely unexplored aspect of within-host genetics. This phenomenon is often considered to be specific to multipartite viruses. Multipartite viruses have segmented genomes, but in contrast to segmented viruses, their segments are each encapsidated alone in a virion. A main hypothesis explaining the evolution of multipartism is that, compared to segmented viruses, it facilitates the regulation of segment abundancy, and the genes the segments carry, within a host. These differences in gene frequencies could allow for expression regulation. Here, we show that wild populations of a segmented virus, bluetongue virus (BTV), also present unequal segment frequencies. BTV cycles between ruminants and Culicoides biting midges. As expected from a role in expression regulation, segment frequencies tended to show specific values that differed between ruminants and midges. Our results expand previous knowledge on gene frequency variation and call for studies on its role and conservation beyond multipartite viruses

A library preparation optimized for metagenomics of RNA viruses

International audienceOur 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

Literature analysis of the host range (Host) and geographic range (Country) of known generalist viruses found in this study.

The "Weighted_Avg_contigs_p_id" columns provide the average value of the identity percentages associated with the contigs. The column "Blastn" indicates other virus names given to a virus species in the NCBI database (e.g., different virus strains or naming errors during submission). (XLSX)</p