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Castiglioni, V. G.

Publications and source records attributed to Castiglioni, V. G..

15 recordsLinked to original sources

Orsay virus infection is associated with reduced locomotion and alters cue-dependent food choice in Caenorhabditis elegans

Behavioral responses are an important component of host defense; however, most evidence in Caenorhabditis elegans comes from bacterial pathogens. Here, we tested whether infection with Orsay virus (OrV) alters host dispersal and food choice and whether these responses involve the RNA-processing factor EOL-1. When measuring distance travelled over a certain period of time in control and infected wild-type animals, we observed that OrV infection was associated with lower locomotion. Moreover, when presenting infected and control animals with food choices, we observed that infected animals preferred OrV infected food sources only when the alternative was a pure food source, preferring a non-inoculated food source when both control and viral food sources contained a C. elegans filtrate. Moreover, loss of EOL-1 altered baseline locomotion and attenuated the infection-associated reduction detected in the earlier motility assay, while also modifying the food choice response, suggesting that eol-1 contributes to the motility and behavioral changes induced by OrV infection. Collectively, these results support an overall association between OrV infection and reduced host dispersal, whereas its effect on food choice depends on the sensory context. These findings extend infection-associated behavioral responses to a natural viral pathogen and identify eol-1 as a modulator of their expression.

microbiology↗

The hidden diversity: Replication-strand asymmetry shapes short-term nucleotide and structural variation in an RNA virus

Orsay virus (OrV), a positive-sense RNA virus infecting Caenorhabditis elegans, establishes latent infections with minimal impact on host fitness. Using strand-specific RNA-seq across twelve time points spanning larval development, we characterized OrV replication dynamics and short-term evolutionary patterns. Viral accumulation followed four distinct phases and exhibited strong strand asymmetry, consistent with a predominance of the stamping-machine replication mechanism. Diversity analyses revealed that negative-strand genomes harbor more mutations than positive strands, with hotspots concentrated in the replicase C-terminal tail and structural protein regions. Many negative-strand variants failed to propagate to positive strands, suggesting within-cell constraints or purifying selection at the RNA level. We also report the first evidence of non-standard viral genomes in OrV, dominated by deletions in RNA2, including a recurrent large deletion that may function as a subgenomic RNA encoding a shorter version of the {delta} protein. These findings highlight replication asymmetry, strand-specific mutational filtering, and pervasive non-standard genomes as key features shaping OrV evolution during a primary infection.

evolutionary biology↗

Native virion structure reveals stochastic genome assortment underlying dose-dependent Orsay virus infection

Orsay virus (OrV), infecting Caenorhabditis elegans, is a valuable model for studying host-virus interactions, yet its bipartite RNA genome packaging and assembly mechanisms remain unclear. To investigate these mechanisms, we combine infection assays, single-molecule imaging, and cryo-electron microscopy. We find that the two RNA segments accumulate asymmetrically, and that productive infection depends on coinfection by multiple particles. We therefore asked whether unequal segment availability generates heterogeneous virions. Cryo-EM analysis of OrV, including symmetric and asymmetric reconstructions to 2.5 [A] resolution, unveils uniform internal density, excluding large populations of empty or semi-empty particles. We show the genome forms a semi-ordered network contacting the electropositive capsid interior through sequence-independent interactions with the N-terminus and penton region. We also resolve the capsid-8 linker, identifying the penton as a likely site coordinating capsid assembly. Together, we propose a model in which stochastic genome assortment emerges during assembly modulated by segment-specific replication dynamics.

microbiology↗

Simulated microgravity alters short-term evolutionary trajectories of Orsay virus in Caenorhabdidits elegans

BackgroundEnvironmental conditions shape the evolutionary trajectories of RNA viruses, yet little is known about how complex physical stressors such as microgravity influence host-virus interactions and viral evolution. Here, we investigated the short-term evolutionary consequences of simulated microgravity on the Caenorhabditis elegans - Orsay virus (OrV) system. MethodsOrV was subjected to six serial passages in hosts acclimated to low-shear modeled microgravity, with parallel evolution under standard-gravity. Evolutionary outcomes were evaluated using virulence, transmission, and replication traits, all measured under standard-gravity conditions. ResultsViral load fluctuated across passages in both environments, with lower mean accumulation in microgravity-evolved lineages. After evolution, we detected no significant changes in virulence. Transmission increased in standard-gravity lineages but not in microgravity-evolved ones, while viral replication decreased in all lineages, with a stronger decline in those evolved under microgravity. However, the magnitude of phenotypic changes was generally modest. DiscussionThese results indicate that evolution under microgravity can alter viral phenotypic trajectories over short timescales. However, because all traits were assayed under standard-gravity conditions, we cannot directly assess local adaptation to microgravity, and the observed differences may reflect environment-specific trade-offs rather than reduced fitness per se. Furthermore, the limited number of passages and the modest magnitude of phenotypic change suggest that evolutionary responses may still be in an early stage. ConclusionOverall, our findings provide initial evidence that simulated microgravity can influence the evolutionary dynamics of an RNA virus, while highlighting the need for reciprocal fitness assays and longer-term experiments to fully characterize adaptation to altered gravitational environments.

evolutionary biology↗

Species-specific barriers constrict Orsay virus host range across the Caenorhabditis genus

Predicting the host range and spillover potential of RNA viruses requires understanding how ecological, immunological, and evolutionary factors jointly shape viral life cycles across related hosts. Here we integrate population-level viral load dynamics, single-animal heterogeneity, tissue-level progression, transmission competence, and evolutionary sustainability to map the eco evolutionary barriers that Orsay virus encounters across six Caenorhabditis species. We show that host species identity determines the timing and completeness of the viral life cycle, producing species specific combinations of susceptibility, replication kinetics, RNA2 to RNA1 stoichiometric balance, virion egress, and onward transmission. These phenotypes correspond to distinct host competence phenotypes, ranging from permissive (Caenorhabditis elegans) to restrictive or evolutionarily dead end host species. Alternative host species disrupt viral life cycle synchrony through delayed replication, truncated cycles, or failure to produce lumen localized virus, thereby reducing transmission and preventing viral adaptation upon serial passage. Our results demonstrate how temporal mismatches between viral replication and host physiology create a series of eco evolutionary barriers to emergence, offering a mechanistic framework for predicting viral host range.

evolutionary biology↗

Cosmic silence and viral noise: transcriptomic crosstalk in Caenorhabditis elegans under simulated space conditions

Spaceflight environments pose unique physiological challenges due to altered gravity and radia-tion exposure. To investigate how these abiotic stressors interact with viral infection, we analyzed the transcriptomic response of Caenorhabditis elegans acclimated to simulated microgravity ({micro}G) and below-background muon radiation flux (BBR), upon infection with Orsay virus (OrV). Using RNA-seq, we characterized gene expression profiles across single and combined stress condi-tions. Both {micro}G and BBR elicited distinct stress responses, including modulation of oxidative stress, lipid metabolism, and immune pathways. OrV infection alone induced robust transcrip-tional changes, but its impact was significantly attenuated when combined with either abiotic stress, suggesting antagonistic interactions. Notably, proviral genes such as drl-1, fat-7 and hipr-1 were downregulated under BBR and {micro}G, potentially impairing viral replication. Gene ontology analyses revealed enrichment in immune effectors, RNA metabolism, and proteostasis-related pathways, particularly under BBR. Viral load and RNA2/RNA1 ratios were reduced in both stress conditions, indicating a shift in viral replication dynamics. Moreover, genomic diversity and de-fective viral genome formation were differentially affected, with increased genetic diversity and structural variation under stress. These findings suggest that acclimation to off-Earth conditions primes the host for a dampened response to an acute viral infection, potentially through resource reallocation and transcriptional attenuation. This study provides transcriptomic insight into viral infection under space-relevant conditions, highlighting complex stress interactions and their im-plications for host-pathogen dynamics in extraterrestrial environments.

microbiology↗

The interplay between Caenorhabditis elegans larval development and Orsay virus infection

Caenorhabditis elegans development can be altered by its interactions with pathogens, such as bacteria1,2 or fungi3. However, the impact of viral infections on the nematodes development remains largely unexplored, and conversely, whether molting periods affect viral replication is unknown. Studying infections with Orsay virus (OrV), the only known natural C. elegans virus4, we investigated the role of molting on virus replication dynamics and the impact of OrV infection on C. elegans larval development. We found that OrV replicates during C. elegans molting periods. Indeed, nematodes inoculated near molt initiation exhibited higher OrV viral loads and the virus infected more intestinal cells. Moreover, nematodes inoculated right after hatching accelerated the start of the first molt and lengthened the second larval stage, effectively resynchronizing normal developmental timing. Overall, our work enhances understanding of OrV dynamics in C. elegans and elucidates the interplay between viral infection and nematode development. HIGHLIGHTSO_LIrsay virus replication peaks during molting periods, with larvae acquiring virus later in the first larval stage producing higher viral loads and more widespread intestinal infection than larvae acquiring it right after hatching. C_LIO_LIinfection alters the expression of key developmental regulators, particularly those with oscillatory profiles synchronized with molting cycles (such as let-7, lin-42 and nhr-23). C_LIO_LInematodes display temporal plasticity in development: accelerating the first molt while extending the second larval stage, ultimately resynchronizing their developmental timeline. C_LIO_LIis a bidirectional interaction between Orsay virus infection and C. elegans development: molting appears to transiently weaken antiviral defenses, while host development adapts to infection through temporal plasticity. C_LI

microbiology↗

Chronic infection of Caenorhabditis elegans by Orsay virus induces age-dependent immunity and superinfection exclusion

Orsay virus (OrV) is a natural pathogen of C. elegans, which mounts an antiviral response upon infection and enables the investigation of the mechanisms governing infection and immunity. Here, we focus on two of these features, namely the effect of life-long infections and superinfection dynamics. By following the course of an infection throughout the lifespan of a synchronous wild-type population, we describe several viral load peaks followed by sharp decreases in viral load, suggesting that the infection is chronic and that animals manage to suppress viral replication successfully throughout most of their lives. Moreover, we show that animals that have been previously exposed to the virus are able to control viral replication upon a subsequent inoculation, indicative of superinfection exclusion. Primary infections produced transcriptomic and small RNA alterations, whose extent was highly dependent on the developmental stage of the worm at the time of infection and the time until sampling. Superinfection, in turn, had little impact on the overall transcriptome, but showed a misregulation of piRNAs, rRNAs, and tsRNAs. Superinfection exclusion was robust throughout larval development and adulthood, providing protection against various OrV isolates. However, the protective effect of the initial infection diminished over time, suggesting different mechanisms of action and ultimately favoring the primary infecting virus. This phenomenon was dependent on a functional RNA interference pathway.

genomics↗

Microgravity and low muon radiation affect viral pathogenesis and physiology in Caenorhabditis elegans

Environmental conditions fundamentally shape host-pathogen interactions, yet how multiple extreme abiotic stressors combine to influence infection outcomes remains poorly understood. Organisms evolved under specific gravitational and radiation regimes; deviations from these conditions --whether in extreme terrestrial environments or beyond Earth-- may alter immune function and pathogen replication. In this study, we investigated the effects of reduced gravity and lowered muon flux on Orsay virus infection in the nematode Caenorhabditis elegans. We employed a fully factorial experimental design, examining how each factor, alone and in combination, influences physiological traits and viral load. While below-background radiation radically affected viral accumulation dynamics, reduced gravity had a minor effect. Both factors significantly impacted reproduction and morphology, with some effects magnified by viral infection. These results reveal how even partial modifications of Earth-like gravity and radiation levels can alter pathogen-host interactions. By integrating experimental observations with mathematical modeling, we show that these environmental stressors primarily affect prezygotic reproductive processes and modulate viral replication through distinct and sometimes antagonistic mechanisms. Although this work does not encompass the full complexity of space environments, where cosmic radiation includes high-energy protons and heavy ions, it provides insight into how adjustable models of reduced gravity and radiation can advance our understanding of biological adaptation beyond standard terrestrial conditions. IMPORTANCEUnderstanding how extreme environmental conditions affect host-pathogen interactions is critical both for safeguarding biological systems during spaceflight and for exploring fundamental principles of stress biology. This study demonstrates that reduced gravity and diminished muon radiation flux can significantly alter viral infection dynamics and host physiology in Caenorhabditis elegans. By integrating experimental data with mathematical modeling, we reveal that these abiotic stresses impact prezygotic reproductive processes and modulate viral replication in distinct and sometimes antagonistic ways. Our findings suggest that even partial deviations from Earth-like conditions can reshape infection outcomes and developmental trajectories, highlighting the need for deeper mechanistic insights into biological adaptation beyond terrestrial norms. These results have implications for space biosciences, evolutionary virology, radiation hormesis theory, and the design of countermeasures to preserve organismal health in extreme or non-terrestrial habitats.

microbiology↗

Experimental evolution of an RNA virus in Caenorhabditis elegans

The discovery of Orsay virus (OrV), the first virus infecting wild populations of Caenorhabditis elegans, has boosted studies of viral immunity pathways in this nematode. Considering the many advantages that C. elegans offers for fundamental research in host-pathogen interactions, this pathosystem has high potential to become a model system for experimental virus evolution studies. However, the evolutionary constraints operating in this pathosystem have barely been explored. Here we describe for the first time an evolution experiment of two different OrV strains in C. elegans. After 10 serial passages of evolution, we report slight changes in infectivity and non-synonymous mutations fixed in the evolved viral populations. In addition, we observed numerous minor variants emerging in the viral population. These minor variants were not randomly distributed along the genome but concentrated in polymorphic genomic regions. Overall, our work established the grounds for future experimental virus evolution studies using Caenorhabditis nematodes. HIGHLIGHTSO_LICaenorhabditis elegans-Orsay virus is a convenient pathosystem to study virus evolution. C_LIO_LIThe approach used to test the viral strains may interfere with the infection phenotypes observed. C_LIO_LIThere may be specific genomic hotspots regions of nucleotide diversity important for the evolution of Orsay virus. C_LIO_LIThe substitution rate observed for Orsay virus was low, suggesting that the two strains studied might be already well adapted to laboratory conditions. C_LI

evolutionary biology↗

Story of an infection: viral dynamics and host responses in the Caenorhabditis elegans-Orsay virus pathosystem

Orsay virus (OrV) is the only known natural virus affecting Caenorhabditis elegans, with minimal impact on the worms fitness due to its robust innate immune response. This study aimed to understand the interactions between C. elegans and OrV by tracking the infections progression during larval development. Four distinct stages of infection were identified based on viral load, with a peak in capsid- encoding RNA2 coinciding with the first signs of viral egression. Transcriptomic analysis revealed temporal changes in gene expression and functions induced by the infection. A specific set of up- regulated genes remained active throughout the infection, and genes correlated and anticorrelated with virus accumulation were identified. Responses to OrV mirrored reactions to other biotic stressors, distinguishing between virus-specific responses and broader immune responses. Additionally, mutants of early response genes and defense-related processes showed altered viral load progression, uncovering new players in the antiviral defense response.

systems biology↗

Orsay virus infection increases Caenorhabditis elegans resistance to heat-shock

The heat shock response plays a role in the immune defense against viruses across various organisms. Studies on model organisms show that inducing this response prior to viral exposure enhances host resistance to infections, while deficient responses make individuals more vulnerable. Moreover, viruses rely on components of the heat shock response for their own stability and viral infections improve thermal tolerance in plants, giving infected individuals an advantage in extreme conditions, which aids the virus in replication and transmission. Here, we examine the interaction between the nematode Caenorhabditis elegans and its natural pathogen the Orsay virus (OrV) under heat stress. We found that OrV infection leads to differential gene expression related to heat stress, and infected populations showed increased resistance to heat shock. This resistance was associated with increased expression of argonautes alg-1 and alg-2, which are crucial for survival after heat shock and for OrV replication. Overall, our study suggests an environmental-dependent mutualistic relationship between the worm and OrV, potentially expanding the worms ecological niche and providing the virus with extra opportunities for replication and adaptation to extreme conditions. IMPORTANCEViruses have traditionally been viewed as self-serving pathogens that harm their hosts to ensure their own survival. However, recent studies are painting a different picture, revealing that viruses are ubiquitous and not always linked to diseases. In the realm of plant pathology, it has been long noted that the outcome of an infection hinges on environmental factors. Here, we show that the interaction between an animal virus and its natural host can be mutualistic under harsh temperature conditions. Our research highlights that this shift towards mutualism hinges on the expression of argonaute proteins.

evolutionary biology↗

Characterization of the composition and functioning of the Crumbs complex in C. elegans

The apical domain of epithelial cells can acquire a diverse array of morphologies and functions, which is critical for the function of epithelial tissues. The Crumbs proteins are evolutionary conserved transmembrane proteins with essential roles in promoting apical domain formation in epithelial cells. The short intracellular tail of Crumbs proteins interacts with a variety of proteins, including the scaffolding protein Pals1 (protein associated with LIN7, Stardust in Drosophila). Pals1 in turn binds to a second scaffolding protein termed PATJ (Pals1-associated tight junction protein), to form the core Crumbs/Pals1/PATJ Crumbs complex. While essential roles in epithelial organization have been shown for Crumbs proteins in Drosophila and mammalian systems, the three Caenorhabditis elegans crumbs genes are dispensable for epithelial polarization and animal development. Moreover, the presence and functioning of orthologs of Pals1 and PATJ has not been investigated. Here, we identify MAGU-2 and MPZ-1 as the C. elegans orthologs of Pals1 and PATJ, respectively. We show that MAGU-2 interacts with all three Crumbs proteins as well as MPZ-1, and localizes to the apical membrane domain in a Crumbs-dependent fashion. Similar to crumbs mutants, a magu-2 null mutant shows no developmental or epithelial polarity defects. Finally, we show that overexpression of the Crumbs proteins EAT-20 or CRB-3 in the C. elegans intestine can lead to apical membrane expansion. Our results shed light into the composition of the C. elegans Crumbs complex and indicate that the role of Crumbs proteins in promoting apical domain formation is conserved.

developmental biology↗

Caenorhabditis elegans LET-413 Scribble is essential in the epidermis for growth, viability, and directional outgrowth of epithelial seam cells

The conserved adapter protein Scribble (Scrib) plays essential roles in a variety of cellular processes, including polarity establishment, proliferation, and directed cell migration. While the mechanisms through which Scrib promotes epithelial polarity are beginning to be unraveled, its roles in other cellular processes including cell migration remain enigmatic. In C. elegans, the Scrib ortholog LET-413 is essential for apical-basal polarization and junction formation in embryonic epithelia. However, whether LET-413 is required for postembryonic development or plays a role in migratory events is not known. Here, we use inducible protein degradation to investigate the functioning of LET-413 in larval epithelia. We find that LET-413 is essential in the epidermal epithelium for growth, viability, and junction maintenance. In addition, we identify a novel role for LET-413 in the polarized outgrowth of the epidermal seam cells. These stem cell-like epithelial cells extend anterior and posterior directed apical protrusions in each larval stage to reconnect to their neighbors. We show that the role of LET-413 in seam cell outgrowth is mediated at least in part by the junctional component DLG-1 discs large, which appears to restrict protrusive activity to the apical domain. Finally, we demonstrate that the Rho-family GTPases CED-10 Rac and CDC-42 can regulate seam cell outgrowth and may also function downstream of LET-413. Our data uncover multiple essential functions for LET-413 in larval development and shed new light on the regulation of polarized outgrowth of the seam cells.

developmental biology↗

Epidermal PAR-6 and PKC-3 are essential for postembryonic development of Caenorhabditis elegans and control non-centrosomal microtubule organization

The cortical polarity regulators PAR-6, PKC-3 and PAR-3 are essential for the polarization of a broad variety of cell types in multicellular animals, from the first asymmetric division of the C. elegans zygote to apical-basal polarization of epithelial cells. In C. elegans, the roles of the PAR proteins in embryonic development have been extensively studied, yet little is known about their functions during larval development. Using auxin-inducible protein depletion, we here show that PAR-6 and PKC-3, but not PAR-3, are essential for postembryonic development. We also demonstrate that PAR-6 and PKC-3 are required in the epidermal epithelium to support animal growth and molting, and the proper timing and pattern of seam cell divisions. Finally, we uncovered a novel role for PAR-6 in controlling the organization of non-centrosomal microtubule arrays in the epidermis. PAR-6 was required for the localization of the microtubule organizer NOCA-1/Ninein, and microtubule defects in a noca-1 mutant are highly similar to those caused by epidermal PAR-6 depletion. As NOCA-1 physically interacts with PAR-6, we propose that PAR-6 promotes non-centrosomal microtubule organization through localization of NOCA-1/Ninein. SummaryUsing inducible protein degradation, we show that PAR-6 and PKC-3/aPKC are essential for postembryonic development of C. elegans and control the organization of non-centrosomal microtubule bundles in the epidermis, likely through recruitment of NOCA-1/Ninein.

cell biology↗