BRESARD, Gautier et al. (2013) International Worm Meeting "Specificity of interaction between Caenorhabditis and their natural viruses."

Felix, Marie-Anne, BRESARD, Gautier

[International Worm Meeting, 2013]

Species involved in host-pathogen relationships exert selective pressures on each other. This co-evolution situation results in an arms race between host and pathogen, which may lead to specialisation of their interactions. We recently found three related horizontally-transmitted RNA viruses that naturally infect C. elegans or C. briggsae, called Orsay, Santeuil and Le Blanc viruses (Felix et al. 2011, Franz et al. 2012). Here we study their specificity for C. elegans vs. C. briggsae, and at the intraspecific level in C. briggsae. We first used viral filtrates to infect a set of C. elegans and C. briggsae isolates, and measured by RT-PCR the virus ability to replicate. We find that the Orsay virus can infect C. elegans but not C. briggsae, whereas Santeuil and Le Blanc viruses infect C. briggsae, but not C. elegans. Thus, each virus shows specificity toward one of these two Caenorhabditis species. Given that C. briggsae can be infected by two viruses, we then measured viral replication after infection of C. briggsae isolates by either Santeuil or Le Blanc viruses, using RT-qPCR. We observed 1) wide variation among C. briggsae isolates; 2) correlation between the sensitivities to each virus; 3) an exception to the correlation. Schematically, C. briggsae isolates can be separated into two groups: sensitive isolates, in which the viruses replicate efficiently; and resistant ones, in which the viruses either disappear or are barely maintained. Strikingly, all sensitive strains belong to the temperate C. briggsae clade, raising the possibility that sensitivity is derived within this clade. The exception to the correlation in sensitivity is HK104, a temperate-clade isolate from Japan. HK104 is sensitive to the Santeuil virus, but resistant to Le Blanc. This result opens the possibility to study specificity of host-pathogen interactions through genetic analysis.

Frezal, Lise et al. (2015) International Worm Meeting "Caenorhabditis briggsae and its two natural viruses, coevolution in a 'menage a trois'."

BRESARD, Gautier, Felix, Marie-Anne, Frezal, Lise

[International Worm Meeting, 2015]

We study the natural coevolution between Caenorhabditis briggsae and its two recently described RNA viruses called Santeuil and Le Blanc (1, 2). The main advantage of this system is to combine the access to wild host and virus populations with powerful molecular tools and experimental evolution designs. We characterized the incidence of the two C. briggsae viruses in France and found that they are found in sympatry. By monitoring the viral RNAs in wild-caught C. briggsae isolates using Fluorescent In Situ Hybridization, we demonstrated that the Le Blanc and Santeuil viruses could coexist in one host population, one animal and one intestinal cell. Molecular variation of the wild-caught viruses was assessed by sequencing their two RNA molecules. While both viruses' diversities are geographically structured, we detected balancing selection on the RNA-dependent RNA polymerase (RdRp) locus in one local Santeuil population. Despite the frequent incidence of coinfection in the wild, we found no evidence for genetic exchange (recombination or RNA reassortment) between the Santeuil and Le Blanc viruses. However, we found clear evidence for RNA reassortment between different Santeuil virus variants. Finally, we investigated natural variation in C. briggsae resistance to each virus. We tested a set of wild isolates -representative of C. briggsae worldwide diversity- for their sensitivity to the Santeuil and Le Blanc viruses. While temperate C. briggsae genotypes are generally susceptible to both viruses, the tested tropical C. briggsae genotypes are resistant to both viruses. Most interestingly, two Japanese C. briggsae genotypes show specific resistance to the Le Blanc virus. To understand the genetic basis of the general and virus-specific resistances of C. briggsae, we carried out a QTL-mapping approach using recombinant inbred lines between AF16 and HK104 (3) and identified a main QTL region on chromosome IV responsible for the variation in resistance to Santeuil virus infection.(1) Felix, Ashe, Piffaretti et al. 2011 PloS Biology. (2) Franz et al. 2012 Journal of Virology. (3) Ross et al. 2011 PLoS Genetics..

Alkan , Cigdem et al. (2017) International Worm Meeting "Interactions between Caenorhabditis nematodes and viruses infecting them naturally."

Ruaud, Albane, Alkan , Cigdem, BRESARD, Gautier, Felix, Marie-Anne

[International Worm Meeting, 2017]

The discovery of RNA viruses that naturally infect C. elegans and C. briggsae serves as an ideal model system to study antiviral immunity and host-pathogen co-evolution. The Orsay virus only infects C. elegans whereas Santeuil and Le Blanc viruses only infect C. briggsae. Intraspecifically, within both species we found a wide variation in viral sensitivity, as well as a positive correlation among wild isolates in sensitivity to both viruses in C. briggsae. An exception to this correlation is the C. briggsae strain HK104, which is specifically resistant to Le Blanc virus but sensitive to Santeuil virus. Taking advantage of this natural variation in the host, we use a genetic approach from the host side and use Recombinant Inbred Lines (RILs) to first map the recombinant genomic regions participating to the resistance/sensitivity in a general and/or specific manner. The RILs were phenotyped for the sensitivity to the relevant viruses using Fluorescent In Situ Hybridization (FISH). The genotype (SNP markers from pool sequencing) and phenotype (resistance/sensitivity from FISH) data were used to perform QTL analysis. Several Near Isogenic Lines (NILs) were created by introgressing the candidate regions. C. briggsae AF16 is resistant to both Santeuil and Le Blanc viruses while C. briggsae HK104 is specifically sensitive to the Santeuil virus. Using AF16xHK104 Advanced Intercrossed RILs (AIRILs) (Ross et al. 2011), two QTLs were detected on chromosomes III and IV for Santeuil virus sensitivity. The NILs in the AF16 background confirm both candidate regions. C. briggsae JU1498 is sensitive to both Santeuil and Le Blanc viruses. Using JU1498xHK104 RILs, a QTL on chromosome II was detected and is being introgressed. Once candidate polymorphisms associated with the virus sensitivity/resistance are identified, we will test them by RNAi knockdown, transformation rescue and/or CRISPR-mediated gene replacement.

Frezal, Lise et al. (2013) International Worm Meeting "Microevolution and coexistence of Santeuil and Le Blanc viruses in Caenorhabditis briggsae. ."

Frezal, Lise, Felix, Marie-Anne

[International Worm Meeting, 2013]

We recently found three viruses, Orsay, Santeuil and Le Blanc, which naturally infect Caenorhabditis nematodes (1,2). These ss(+)RNA viruses cause intestinal cell symptoms and are horizontally transmitted. Whereas C. elegans can so far only be infected by the Orsay virus, European C. briggsae genotypes are susceptible to both Santeuil and Le Blanc viruses, and both viruses have been found in the same locations. This vulnerability of C. briggsae to two viruses enables studies of in vivo viral competition and of the mechanisms driving their short-term evolution, as well as the impact of their competition on worm fitness. RNA viruses may evolve rapidly through both high mutation rates and recombination events. The impact of recombination widely varies from one viral species to another but in all cases, for recombination to occur, different virus types have to infect the same host cell. The first step is thus to assess whether different virus species can co-infect the same worm population, the same animal and the same cell. By using quantitative RT-PCR, we demonstrate that the Le Blanc and Santeuil viruses can coexist in a worm population, even when originally introduced at widely different concentrations. The two viruses are jointly maintained over 10 worm generations. We presently investigate the co-infection at the whole organism and single cell levels by tracking the viral RNAs in co-infected worms using Fluorescent In Situ Hybridization. 1- Felix, Ashe, Piffaretti et al. 2011 PloS biology. 2- Franz et al. 2012 Journal of virology.

Chen, Kevin et al. (2015) International Worm Meeting "Exploring novel antiviral immunity in C. elegans."

Wang, David, Jiang, Hongbing, Wu, Guang, Franz, Carl, Renshaw, Hilary, Chen, Kevin

[International Worm Meeting, 2015]

Model organisms have played a critical role in our understanding of innate immunity. The recent discovery of Orsay virus, the 1st virus capable of infecting C. elegans, and the discoveries of Santeuil and Le Blanc viruses which infect C. briggsae, provide a unique opportunity to define virus host interactions in these model hosts. In order to identify candidate antiviral genes, we have performed a time course transcriptional profiling with RNA-seq. In C. elegans, we identified 151 genes that were differentially expressed upon Orsay virus infection. In this set, only 36 have annotation; 22 genes contain domains involved in ubiquitin-mediated proteolysis. By further defining the transcriptional response of the orthologous genes in C. briggsae to Santeuil and Le Blanc virus infection, we identified 39 conserved genes induced in both hosts by the three viruses. Strikingly, 17 of the 39 conserved response genes are paralogs of a single gene family that is exemplified by C17H1.3. This gene family has a human ortholog, but no known function has been associated to these orthologous genes. The conserved induction of these genes in response to infection by multiple viruses strongly suggests they may play a role in antiviral defense. Efforts to define such function by targeted gene deletion and overexpression are underway. .

Franz, Carl J. et al. (2013) International Worm Meeting "Tissue Tropism and Infection Characterization of Novel Viruses in Caenorhabditis Nematodes."

Franz, Carl J., Frezal, Lise, Jiang, Yanfang, Wang, David, Felix, Marie-Anne, Renshaw, Hilary

[International Worm Meeting, 2013]

Orsay, Santeuil and Le Blanc viruses were recently discovered, enabling for the first time the study of virus-host interactions using a natural pathogen in the well-established model organism Caenorhabditis elegans and its relative Caenorhabditis briggsae. All three viruses share less than 50% amino acid identity and are most closely related to nodaviruses, which are positive sense RNA viruses with bipartite genomes. Comparison of their complete genomes demonstrated unique coding and noncoding features absent in known nodaviruses. Le Blanc virus, similar to Santeuil virus, was capable of infecting wild C. briggsae isolates but not the AF16 C. briggsae laboratory reference strain nor any tested C. elegans strains. We characterized the tissue tropism of infection in Caenorhabditis nematodes by all three viruses. Using immunofluorescence assays targeting viral proteins, as well as in situ hybridization, we demonstrated that viral proteins and RNAs localized primarily to intestinal cells in larval stage Caenorhabditis nematodes. The viral proteins could be detected in one to six of the 20 intestinal cells present in Caenorhabditis nematodes. In Orsay virus-infected C. elegans, viral proteins could be detected as early as six hours post infection. Furthermore, the RNA-dependent RNA polymerase and capsid proteins of Orsay virus exhibited different subcellular localization patterns from each other. Collectively, these observations broaden our understanding of viral infection in Caenorhabditis nematodes.

Belicard, Tony et al. (2011) International Worm Meeting "Evolution in viral sensitivity to the Orsay virus in natural C. elegans isolates."

Belicard, Tony, Felix, Marie-Anne

[International Worm Meeting, 2011]

We isolated the first natural viruses infecting Caenorhabditis nematodes: the Orsay virus in C. elegans isolate JU1580 and the Santeuil virus in C. briggsae JU1264 (Felix & al., 2011). We more recently found a third virus in C. briggsae JU1498 (Le Blanc virus). These viruses cause disorders in intestinal cells of their host and are horizontally transmitted. Their genomes are composed of two single-stranded positive RNA segments carrying 3 ORFs. One of them, the ORF d, has no homology with any known ORF (Felix & al., 2011). We aim to identify its role during infection. We thus cloned it and are currently expressing it in a JU1580 background in order to know whether it affects the anti-viral response of the worm. In order to evaluate natural variation in sensitivity to these viruses, we scored the susceptibility of natural isolates and standard laboratory strains of C. elegans and C. briggsae. The results reveal i) a species specificity of infection by each virus and ii) intraspecific variation in sensitivity within both species for their respective viruses. First, we found a species specificity of each virus for a specific Caenorhabditis host species. Indeed, the Santeuil and Le Blanc viruses do not infect JU1580, while the Orsay virus does not infect JU1264 and JU1498 Second, we evaluated the geographic and genetic distribution of Orsay virus susceptibility in a worldwide set of 25 C. elegans isolates representing wild genetic diversity. We measured the viral load by RT-qPCR. Preliminary results suggest that only a subset of isolates from the Old world are sensitive to the virus and none of the "New World". This diversity seems to be partially linked with their ability to perform a small RNA response that acts in anti-viral defense (Felix & al., 2011; poster by Nuez & Felix). We plan to determine the genetic architecture and identify the molecular basis for this intraspecific variation in Orsay virus susceptibility. One approach is to cross closely related sensitive and resistant strains to obtain Recombinant Inbred Lines. We will test the susceptibility to the virus in these lines in order to find loci involved in the last evolutionary event causing resistance/sensitivity to the virus. By identifying these loci, we will be able to describe the last step in the "arms race" between C. elegans and its natural virus.

Stanley I Shyn et al. (2001) International C. elegans Meeting "Calcium imaging of vulval muscle activity in egg-laying behavior"

William R Schafer, Stanley I Shyn

[International C. elegans Meeting, 2001]

Serotonin (5HT) is a neurotransmitter which often functions as a modulator of tissue excitability and behavioral states. In C. elegans , one behavioral effect it produces is the stimulation of egg-laying. 1 One mechanism by which it appears to do this is by shifting the worm's vulval muscles from a quiescent or inactive mode to a more active one during which eggs are laid in a cluster. Through a combination of genetic and pharmacologic approaches, we have identified mutants in several genes to be 5HT-resistant for this behavior: egl-19 (L-type voltage gated Ca 2+ channel alpha-1 subunit), tpa-1 (PKC homolog), acy-1 (adenylate cyclase), & gpa-14 (novel G-protein). 2,3 While implicated in 5HT-signalling, these molecules may also constitute effectors from other neurotransmitter systems known to interact with 5HT in egg-laying, including neuropeptide signalling: Neuropeptides derived from the flp-1 gene, for example, are known to potentiate 5HT-response. 4,5 To gain more insight into these issues, we have been utilizing Cameleon, a genetically encodable and ratiometric Ca 2+ -sensor 6,7 in both intact & cut-worm preparations to study how vulval muscle physiology changes in the presence or absence of 5HT & and how it responds to agents like forskolin or FLP peptides in N2s and in various mutant backgrounds. Preliminary results indicate that, in the absence of 5HT, vulval muscles in N2 worms typically show sporadic but often clustered Ca 2+ transients. Upon application of exogenous 5HT (5 mg/ml), this pattern gives way to a more rhythmic train of small transient events (~0.5 Hz). This data, along with initial mutant characterization, will be presented at the June meeting. 1 Horvitz HR et al , Science 216 :1012-4 2 Waggoner LE et al , Neuron 21 :203-214 3 Shyn S & Schafer W, 2000 WCWM abstract 228 4 Schinkmann K & Li C, J Comp Neurol 316 :251-260 5 Waggoner LE et al , Genetics 154 :1181-1192 6 Miyawaki A et al , Nature 388 :882-887 7 Kerr R et al , Neuron 26 :583-594

Schroeder, Frank et al. (2021) International Worm Meeting "Carboxyesterases and intestinal granules in the biosynthesis of novel families of nematode small molecule signals"

Wrobel, Chester, Schroeder, Frank, Sternberg, Paul, Cohen, Sarah

[International Worm Meeting, 2021]

Signaling molecules derived from attachment of diverse primary metabolic building blocks to ascarosides play a central role in the life history of C. elegans and other nematodes; however, the extent to which various inter- and intra-organismal signaling pathways are controlled by small molecules is unclear. Using comparative metabolomics, we show that a pathway mediating formation of intestinal lysosome-related organelles (LROs) is required for biosynthesis of a large library of small molecules, including most modular ascarosides as well as previously undescribed modular glucosides (1). Similar to modular ascarosides, the modular glucosides are derived from highly selective assembly of moieties from nucleoside, amino acid, neurotransmitter, and lipid metabolism. We further show that a family of carboxylesterases (CEST's) that localize to intestinal organelles are required for the assembly of both modular ascarosides and glucosides, suggesting that modular glucosides, like the ascarosides, serve signaling functions. For example, biosynthesis of some modular glucosides is starkly upregulated in long-lived daf-2 mutants, suggesting that insulin signaling is modulated by small molecules produced in a lysosome-dependent manner. Parallel studies in C. briggsae and other nematode species indicate that assembly of modular metabolites via CEST enzymes is widely conserved in nematodes. Further exploration of LRO function and cest homologs in C. elegans and other animals may reveal additional new compound families and signaling paradigms. (1) H. H. Le, C. J. J. Wrobel, S. M. Cohen, J. Yu, H. Park, M. J. Helf, B. J. Curtis, P. R. Rodrigues, P. W. Sternberg, F. C. Schroeder. Modular metabolite assembly in C. elegans depends on carboxylesterases and formation of lysosome-related organelles. eLife, 9, e61886, 2020.

Bhagwati P Gupta et al. (2002) West Coast Worm Meeting "Genetic analysis of the vulval mutants in C. briggsae"

Shahla Gharib, Bhagwati P Gupta, Paul W Sternberg, Jennifer X Li

[West Coast Worm Meeting, 2002]

To understand the evolution of developmental mechanisms, we are doing a comparative analysis of vulval patterning in C. elegans and C. briggsae. C. briggsae is closely related to C. elegans and has identical looking vulval morphology. However, recent studies have indicated subtle differences in the underlying mechanisms of development. The recent completion of C. briggsae genome sequence by the C. elegans Sequencing Consortium is extremely valuable in identifying the conserved genes between C. elegans and C. briggsae.