Aurlie Blanc et al. (2005) International Worm Meeting "A French functional genomics platform."

Daniel Wong, Jerome Reboul, Jonathan Ewbank, Aurlie Blanc, Yohann Duverger

[International Worm Meeting, 2005]

For the last two years, we have offered a service of worm sorting based around the Union Biometrica COPAS platform. The COPAS machine is fully equipped with Zymark twister robot, allowing automated analysis of multiple 96 well plates, and the Profiler that generates 500 individual fluorescence measurements per worm. This equipment has been applied to a wide range of biological questions. They include quantifying the level of fluorescent reporter gene expression, large-scale RNAi and genetic screens and combinatorial library drug screening. Examples of each will be presented.This platform is now part of a fully integrated functional genomics facility open to the academic community (see http://www.ciml.univ-mrs.fr/EWBANK_jonathan/RIO.html). Other resources include the ORFeome (developed in Marc Vidals laboratory), and Julie Ahringers RNAi library, together with whole-genome microarrays. For the latter, through a collaboration with the Genome Sequencing Center at Washington, and the transcriptome platform at Nice, we have spotted the Illumina long oligo set onto glass slides and provide microarrays to the French C. elegans community.This French functional genomics platform has been made possible through funding from the National Genopole<sym02> network, Marseille-Nice genopole<sym02>, the CNRS and collaboration with Union Biometrica.

Blanc, Caroline et al. (2021) International Worm Meeting "Meiosis modifications at the origin of asexuality in Mesorhabditis pseudogamous nematodes"

Saclier, Nathanaelle, Galtier, Nicolas, Rey, Carine, Delattre, Marie, Blanc, Caroline

[International Worm Meeting, 2021]

Sexuality is widespread and asexuality is a derived character. Despite the recognized costs associated with sex, asexuals remain rare, which constitutes one of the most intriguing paradox in evolutionary biology. To understand the adaptative value of sex, there is clearly a need for further exploration of the asexual world, and in particular of the transition to asexuality. We recently characterized the nematode genus Mesorhabditis, in which regular sexual species are found, as well as species featuring progressive loss of males and sexuality. These pseudo-sexual species are composed of 90% females and only 10% males. Males and sperm are needed for most eggs to develop by gynogenesis (the sperm is needed to activate the oocytes but its DNA is not used, i.e pseudogamy). In the few cases where the sperm DNA is incorporated, the eggs develop as males, because only the Y-bearing sperm of males are competent to fertilize (Grosmaire &amp; al. Science 2019; Launay &amp; al. BMC Evol Bio 2020). In this intriguing system, females are thus produced asexually whereas males are produced sexually. Mesorhabditis nematodes represent an ideal system to study the evolutionary consequences of transition to asexuality in closely related species and to explore the molecular origins of asexuality. There are many ways to lose sex. Here we asked which modifications to meiosis led to the production of diploid oocytes in asexuals. Using cytological descriptions, we found that meiosis of asexual females is similar to sexual species up to anaphase I. Chromosome pairing and crossing-over occur and bivalent forming chiasmata are present in diakinesis. Next, the first meiotic spindle forms and bivalent chromosomes initiate their segregation. However, chromosome segregation eventually stops and all univalents realign at metaphase of meiosis II. Meiosis II then proceeds normally with the segregation of sister chromatids and formation of the single polar body. Hence, meiosis in these asexuals is characterized by the maintenance of recombination and the assortment of non-sister chromatids in the diploid oocyte. In parallel, we are comparing the level of recombination in the sexual and asexual species of this genus. For that, we have sequenced the genome of a dozen strain for one sexual and one asexual species, to measure linkage desequilibrium. The theoretical consequence of such modified meiosis is genome-wide homozygosity, except maintenance of heterozygosity on chromosome centers. This will be contrasted with the actual measure of heterozygosity.

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.

Franz CJ et al. (2012) J Virol "Complete genome sequence of Le Blanc virus, a third Caenorhabditis nematode-infecting virus."

Felix MA, Zhao G, Franz CJ, Wang D

[J Virol, 2012]

Orsay virus and Santeuil virus, the first known viruses capable of naturally infecting the nematodes Caenorhabditis elegans and Caenorhabditis briggsae, respectively, were recently identified by high-throughput sequencing of wild Caenorhabditis strains. By similar analysis of another wild C. briggsae isolate, we have now discovered and sequenced the complete genome of a third novel virus, Le Blanc virus, that is distantly related to Orsay and Santeuil viruses. All three viruses are positive-sense RNA viruses with bipartite genomes that are most closely related to nodaviruses. Identification of a third virus capable of infecting Caenorhabditis nematodes enables comparative analysis of this clade of viruses and strengthens this model for investigating virus-host interactions.

Franz CJ et al. (2014) Virology "Orsay, Santeuil and Le Blanc viruses primarily infect intestinal cells in Caenorhabditis nematodes."

Felix MA, Frezal L, Renshaw H, Wang D, Jiang Y, Franz CJ

[Virology, 2014]

The discoveries of Orsay, Santeuil and Le Blanc viruses, three viruses infecting either Caenorhabditis elegans or its relative Caenorhabditis briggsae, enable the study of virus-host interactions using natural pathogens of these two well-established model organisms. We characterized the tissue tropism of infection in Caenorhabditis nematodes by these viruses. Using immunofluorescence assays targeting proteins from each of the viruses, and in situ hybridization, we demonstrate viral proteins and RNAs localize to intestinal cells in larval stage Caenorhabditis nematodes. Viral proteins were detected in one to six of the 20 intestinal cells present in Caenorhabditis nematodes. In Orsay virus-infected C. elegans, viral proteins were detected as early as 6h post-infection. The RNA-dependent RNA polymerase and capsid proteins of Orsay virus exhibited different subcellular localization patterns. Collectively, these observations provide the first experimental insights into viral protein expression in any nematode host, and broaden our understanding of viral infection in Caenorhabditis nematodes.

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..

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.

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.

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.