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.

Identification of Umbre Orthobunyavirus as a Novel Zoonotic Virus Responsible for Lethal Encephalitis in 2 French Patients with Hypogammaglobulinemia.

BACKGROUND: Human encephalitis represents a medical challenge from a diagnostic and therapeutic point of view. We investigated the cause of 2 fatal cases of encephalitis of unknown origin in immunocompromised patients. METHODS: Untargeted metatranscriptomics was applied on the brain tissue of 2 patients to search for pathogens (viruses, bacteria, fungi, or protozoans) without a prior hypothesis. RESULTS: Umbre arbovirus, an orthobunyavirus never previously identified in humans, was found in 2 patients. In situ hybridization and reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR) showed that Umbre virus infected neurons and replicated at high titers. The virus was not detected in cerebrospinal fluid by RT-qPCR. Viral sequences related to Koongol virus, another orthobunyavirus close to Umbre virus, were found in Culex pipiens mosquitoes captured in the south of France where the patients had spent some time before the onset of symptoms, demonstrating the presence of the same clade of arboviruses in Europe and their potential public health impact. A serological survey conducted in the same area did not identify individuals positive for Umbre virus. The absence of seropositivity in the population may not reflect the actual risk of disease transmission in immunocompromised individuals. CONCLUSIONS: Umbre arbovirus can cause encephalitis in immunocompromised humans and is present in Europe.

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

Genome 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.

Human Usutu Virus Infection with Atypical Neurologic Presentation, Montpellier, France, 2016.

Infection with Usutu virus (USUV) has been recently associated with neurologic disorders, such as encephalitis or meningoencephalitis, in humans. These findings indicate that USUV is a potential health threat. We report an acute human infection with USUV in France putatively associated with a clinical diagnosis of idiopathic facial paralysis.

The Strange Lifestyle of Multipartite Viruses.

Multipartite viruses have one of the most puzzling genetic organizations found in living organisms. These viruses have several genome segments, each containing only a part of the genetic information, and each individually encapsidated into a separate virus particle. While countless studies on molecular and cellular mechanisms of the infection cycle of multipartite viruses are available, just as for other virus types, very seldom is their lifestyle questioned at the viral system level. Moreover, the rare available "system" studies are purely theoretical, and their predictions on the putative benefit/cost balance of this peculiar genetic organization have not received experimental support. In light of ongoing progresses in general virology, we here challenge the current hypotheses explaining the evolutionary success of multipartite viruses and emphasize their shortcomings. We also discuss alternative ideas and research avenues to be explored in the future in order to solve the long-standing mystery of how viral systems composed of interdependent but physically separated information units can actually be functional.

[Usutu virus: let's be vigilant]. / Le virus Usutu : soyons vigilants.

Usutu virus (USUV) is an emerging arbovirus, close to the West Nile virus (WNV), which was first isolated in South Africa in 1959. This flavivirus has spread to a large part of the European continent, causing bird deaths, particularly in 2018. Although human infection seems to be mostly asymptomatic, several cases of neurological complications (encephalitis, meningoencephalitis) have been described. The description in Montpellier of an atypical case of Bell's palsy suggests that the clinical spectrum of USUV-related infections may be more extensive than expected and highlights our limited knowledge of the pathophysiology of this emerging virus.

Demographics of natural oral infection of mosquitos by Venezuelan equine encephalitis virus.

The within-host diversity of virus populations can be drastically limited during between-host transmission, with primary infection of hosts representing a major constraint to diversity maintenance. However, there is an extreme paucity of quantitative data on the demographic changes experienced by virus populations during primary infection. Here, the multiplicity of cellular infection (MOI) and population bottlenecks were quantified during primary mosquito infection by Venezuelan equine encephalitis virus, an arbovirus causing neurological disease in humans and equids.

The Multiplicity of Cellular Infection Changes Depending on the Route of Cell Infection in a Plant Virus.

UNLABELLED: The multiplicity of cellular infection (MOI) is the number of virus genomes of a given virus species that infect individual cells. This parameter chiefly impacts the severity of within-host population bottlenecks as well as the intensity of genetic exchange, competition, and complementation among viral genotypes. Only a few formal estimations of the MOI currently are available, and most theoretical reports have considered this parameter as constant within the infected host. Nevertheless, the colonization of a multicellular host is a complex process during which the MOI may dramatically change in different organs and at different stages of the infection. We have used both qualitative and quantitative approaches to analyze the MOI during the colonization of turnip plants by Turnip mosaic virus. Remarkably, different MOIs were observed at two phases of the systemic infection of a leaf. The MOI was very low in primary infections from virus circulating within the vasculature, generally leading to primary foci founded by a single genome. Each lineage then moved from cell to cell at a very high MOI. Despite this elevated MOI during cell-to-cell progression, coinfection of cells by lineages originating in different primary foci is severely limited by the rapid onset of a mechanism inhibiting secondary infection. Thus, our results unveil an intriguing colonization pattern where individual viral genomes initiate distinct lineages within a leaf. Kin genomes then massively coinfect cells, but coinfection by two distinct lineages is strictly limited. IMPORTANCE: The MOI is the size of the viral population colonizing cells and defines major phenomena in virus evolution, like the intensity of genetic exchange and the size of within-host population bottlenecks. However, few studies have quantified the MOI, and most consider this parameter as constant during infection. Our results reveal that the MOI can depend largely on the route of cell infection in a systemically infected leaf. The MOI is usually one genome per cell when cells are infected from virus particles moving long distances in the vasculature, whereas it is much higher during subsequent cell-to-cell movement in mesophyll. However, a fast-acting superinfection exclusion prevents cell coinfection by merging populations originating from different primary foci within a leaf. This complex colonization pattern results in a situation where within-cell interactions are occurring almost exclusively among kin and explains the common but uncharacterized phenomenon of genotype spatial segregation in infected plants.

Circulative Nonpropagative Aphid Transmission of Nanoviruses: an Oversimplified View.

UNLABELLED: Plant virus species of the family Nanoviridae have segmented genomes with the highest known number of segments encapsidated individually. They thus likely represent the most extreme case of the so-called multipartite, or multicomponent, viruses. All species of the family are believed to be transmitted in a circulative nonpropagative manner by aphid vectors, meaning that the virus simply crosses cellular barriers within the aphid body, from the gut to the salivary glands, without replicating or even expressing any of its genes. However, this assumption is largely based on analogy with the transmission of other plant viruses, such as geminiviruses or luteoviruses, and the details of the molecular and cellular interactions between aphids and nanoviruses are poorly investigated. When comparing the relative frequencies of the eight genome segments in populations of the species Faba bean necrotic stunt virus (FBNSV) (genus Nanovirus) within host plants and within aphid vectors fed on these plants, we unexpectedly found evidence of reproducible changes in the frequencies of some specific segments. We further show that these changes occur within the gut during early stages of the virus cycle in the aphid and not later, when the virus is translocated into the salivary glands. This peculiar observation, which was similarly confirmed in three aphid vector species, Acyrthosiphon pisum, Aphis craccivora, and Myzus persicae, calls for revisiting of the mechanisms of nanovirus transmission. It reveals an unexpected intimate interaction that may not fit the canonical circulative nonpropagative transmission. IMPORTANCE: A specific mode of interaction between viruses and arthropod vectors has been extensively described in plant viruses in the three families Luteoviridae, Geminiviridae, and Nanoviridae, but never in arboviruses of animals. This so-called circulative nonpropagative transmission contrasts with the classical biological transmission of animal arboviruses in that the corresponding viruses are thought to cross the vector cellular barriers, from the gut lumen to the hemolymph and to the salivary glands, without expressing any of their genes and without replicating. By monitoring the genetic composition of viral populations during the life cycle of Faba bean necrotic stunt virus (FBNSV) (genus Nanovirus), we demonstrate reproducible genetic changes during the transit of the virus within the body of the aphid vector. These changes do not fit the view that viruses simply traverse the bodies of their arthropod vectors and suggest more intimate interactions, calling into question the current understanding of circulative nonpropagative transmission.

Circulating virus load determines the size of bottlenecks in viral populations progressing within a host.

For any organism, population size, and fluctuations thereof, are of primary importance in determining the forces driving its evolution. This is particularly true for viruses--rapidly evolving entities that form populations with transient and explosive expansions alternating with phases of migration, resulting in strong population bottlenecks and associated founder effects that increase genetic drift. A typical illustration of this pattern is the progression of viral disease within a eukaryotic host, where such demographic fluctuations are a key factor in the emergence of new variants with altered virulence. Viruses initiate replication in one or only a few infection foci, then move through the vasculature to seed secondary infection sites and so invade distant organs and tissues. Founder effects during this within-host colonization might depend on the concentration of infectious units accumulating and circulating in the vasculature, as this represents the infection dose reaching new organs or "territories". Surprisingly, whether or not the easily measurable circulating (plasma) virus load directly drives the size of population bottlenecks during host colonization has not been documented in animal viruses, while in plants the virus load within the sap has never been estimated. Here, we address this important question by monitoring both the virus concentration flowing in host plant sap, and the number of viral genomes founding the population in each successive new leaf. Our results clearly indicate that the concentration of circulating viruses directly determines the size of bottlenecks, which hence controls founder effects and effective population size during disease progression within a host.

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.

Host influence on the eukaryotic virome of sympatric mosquitoes and abundance of diverse viruses with a broad host range.

Mosquitoes harbor a large diversity of eukaryotic viruses. Those viromes probably influence mosquito physiology and the transmission of human pathogens. Nevertheless, their ecology remains largely unstudied. Here, we address two key questions in virome ecology. First, we assessed the influence of mosquito species on virome taxonomic diversity and relative abundance. Contrary to most previous studies, the potential effect of the habitat was explicitly included. Thousands of individuals of Culex poicilipes and Culex tritaeniorhynchus, two vectors of viral diseases, were concomitantly sampled in three habitats over two years. A total of 95 viral taxa from 25 families were identified with meta-transcriptomics, with 75% of taxa shared by both mosquitoes. Viromes significantly differed by mosquito species but not by habitat. Differences were largely due to changes in relative abundance of shared taxa. Then, we studied the diversity of viruses with a broad host range. We searched for viral taxa shared by the two Culex species and Aedes vexans, another disease vector, present in one of the habitats. Twenty-six out of the 163 viral taxa were found in the three mosquitoes. These taxa encompassed 14 families. A database analysis supported broad host ranges for many of those viruses, as well as a widespread geographical distribution. Thus, the viromes of mosquitoes from the same genera mainly differed in the relative abundance of shared taxa, whereas differences in viral diversity dominated between mosquito genera. Whether this new model of virome diversity and structure applies to other mosquito communities remains to be determined.

Mosquito (Diptera: Culicidae) populations in contrasting areas of the western regions of Burkina Faso: species diversity, abundance and their implications for pathogen transmission.

BACKGROUND: Mosquitoes (Diptera: Culicidae) can have a significant negative impact on human health. The urbanization of natural environments and their conversion for agricultural use, as well as human population growth, may affect mosquito populations and increase the risk of emerging or re-emerging mosquito-borne diseases. We report on the variety and number of adult mosquitoes found in four environments with varying degrees of human impact (rural, urban, rice fields, and forest) located in a savannah zone of West Africa. METHODS: Mosquitoes were collected from two regions (Hauts-Bassins and Sud-Ouest) of Burkina Faso during five periods between August 2019 and June 2021. Sampling sites were grouped according to environment. Mosquitoes were collected using BG-Sentinel traps and double net traps, and Prokopack Aspirators. Statistical analyses were performed using R software version 4.1.2. Logistic regression, using generalised mixed linear models, was used to test the effect of environment on mosquito abundance and diversity. Alpha diversity analysis was also performed, using the vegan package. RESULTS: A total of 10,625 adult mosquitoes were collected, belonging to 33 species and five genera: Culex, Aedes, Anopheles, Mansonia, and Ficalbia. The most dominant species were Culex quinquefasciatus, Anopheles gambiae sensu lato and Aedes aegypti. Alpha diversity was similar in the two regions. Habitat had a significant effect on mosquito species richness, the Shannon index and the Simpson index. The rural environment had the highest species richness (n = 28) followed by the forest environment (n = 24). The highest number of mosquitoes (4977/10,625) was collected in the urban environment. CONCLUSIONS: The species composition of the mosquito populations depended on the type of environment, with fewer species in environments with a high human impact such as urban areas and rice fields. Due to the diversity and abundance of the mosquito vectors, the human populations of all of the environments examined are considered to be at potential risk of mosquito-borne diseases.

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.

Dynamics of the multiplicity of cellular infection in a plant virus.

Recombination, complementation and competition profoundly influence virus evolution and epidemiology. Since viruses are intracellular parasites, the basic parameter determining the potential for such interactions is the multiplicity of cellular infection (cellular MOI), i.e. the number of viral genome units that effectively infect a cell. The cellular MOI values that prevail in host organisms have rarely been investigated, and whether they remain constant or change widely during host invasion is totally unknown. Here, we fill this experimental gap by presenting the first detailed analysis of the dynamics of the cellular MOI during colonization of a host plant by a virus. Our results reveal ample variations between different leaf levels during the course of infection, with values starting close to 2 and increasing up to 13 before decreasing to initial levels in the latest infection stages. By revealing wide dynamic changes throughout a single infection, we here illustrate the existence of complex scenarios where the opportunity for recombination, complementation and competition among viral genomes changes greatly at different infection phases and at different locations within a multi-cellular host.

Screening of Circulation of Usutu and West Nile Viruses: A One Health Approach in Humans, Domestic Animals and Mosquitoes in Burkina Faso, West Africa.

Usutu virus (USUV) and West Nile virus (WNV) are phylogenetically closely related arboviruses. These viruses mainly follow an enzootic cycle involving mosquitoes and birds, but they occasionally infect humans and other mammals, inducing neurotropic disorders. Since the discovery of USUV, only two human cases have been reported in Africa, including one in Burkina Faso in 2004. Since then, no studies have been conducted to measure the extent of the circulation of this virus in Burkina Faso, and no study regarding the circulation of WNV has been conducted. Our study aimed to determine the seroprevalence of USUV and WNV in blood donations and in animals (horses, dogs, chickens and pigeons) and to perform molecular screening in patients with febrile fever and in Culex quinquefasciatus and Aedes aegypti mosquitoes. The prevalence of USUV and WNV was studied by serological (ELISA and microneutralization tests) and molecular analyses (RT-qPCR) of mosquito, dog, domestic bird, horse, and human samples in Burkina Faso between 2019 and 2021. We detected a very active transmission of both viruses in Burkina Faso. WNV and USUV seroprevalence is particularly high in humans (19.16% and 14.17%, respectively) and horses (17.28% and 6.17%). Molecular screening did not detect WNV or USUV in the mosquito or human samples tested. Our study shows an active spread of USUV and WNV in Burkina Faso, especially for WNV. This study highlights the value of developing surveillance programs to better prevent, detect, and alert people to USUV and WNV circulation in both primary and incidental hosts.

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.

Evidence of Exposure to USUV and WNV in Zoo Animals in France.

West Nile virus (WNV) and Usutu virus (USUV) are zoonotic arboviruses. These flaviviruses are mainly maintained in the environment through an enzootic cycle involving mosquitoes and birds. Horses and humans are incidental, dead-end hosts, but can develop severe neurological disorders. Nevertheless, there is little data regarding the involvement of other mammals in the epidemiology of these arboviruses. In this study, we performed a serosurvey to assess exposure to these viruses in captive birds and mammals in a zoo situated in the south of France, an area described for the circulation of these two viruses. A total of 411 samples comprising of 70 species were collected over 16 years from 2003 to 2019. The samples were first tested by a competitive enzyme-linked immunosorbent assay. The positive sera were then tested using virus-specific microneutralization tests against USUV and WNV. USUV seroprevalence in birds was 10 times higher than that of WNV (14.59% versus 1.46%, respectively). Among birds, greater rhea (Rhea Americana) and common peafowl (Pavo cristatus) exhibited the highest USUV seroprevalence. Infections occurred mainly between 2016-2018 corresponding to a period of high circulation of these viruses in Europe. In mammalian species, antibodies against WNV were detected in one dama gazelle (Nanger dama) whereas serological evidence of USUV infection was observed in several Canidae, especially in African wild dogs (Lycaon pictus). Our study helps to better understand the exposure of captive species to WNV and USUV and to identify potential host species to include in surveillance programs in zoos.

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

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.

A multicellular way of life for a multipartite virus.

A founding paradigm in virology is that the spatial unit of the viral replication cycle is an individual cell. Multipartite viruses have a segmented genome where each segment is encapsidated separately. In this situation the viral genome is not recapitulated in a single virus particle but in the viral population. How multipartite viruses manage to efficiently infect individual cells with all segments, thus with the whole genome information, is a long-standing but perhaps deceptive mystery. By localizing and quantifying the genome segments of a nanovirus in host plant tissues we show that they rarely co-occur within individual cells. We further demonstrate that distinct segments accumulate independently in different cells and that the viral system is functional through complementation across cells. Our observation deviates from the classical conceptual framework in virology and opens an alternative possibility (at least for nanoviruses) where the infection can operate at a level above the individual cell level, defining a viral multicellular way of life.