Coevolution of Caenorhabditis nematodes and viral pathogens – CAENOVIRUS

Aim I: Characterization of the infection of the model organism C. elegans by the newly discovered Orsay virus and development of infection markers. One specific goal here is to find adequate quantitative markers of the course of infection in different tissues, because the present scoring of intestinal cell symptoms is painstaking and difficult to scale up.

Aim II: Reconstitution of the virus through DNA transgenesis in the worm and role of viral ORF delta. We aim to develop a transgenic virus approach allowing for manipulation of the viral genome for structure-function studies. One particular objective is to define the role of the viral ORF delta.

Aim III: Extent and genetic analysis of intraspecific genetic variation in sensitivity of natural Caenorhabditis isolates to Orsay and Santeuil viruses. We aim to study the distribution of Orsay virus susceptibility among C. elegans wild isolates. One specific aim is to select specific isolates for genetic and genomic studies for the identification of molecular variants underlying immune response evolution.

Aim IV: Characterization of the specificity of virus and Caenorhabditis species and experimental co-evolution of host and pathogen. We aim to understand the molecular basis of C. elegans vs. C. briggsae host specificity of Orsay vs. Santeuil viruses. The goal of the experimental evolution is to test specific hypotheses about rate of variation and diversification during co-evolution. In this case where the host mounts an immune response through small RNAs matching the viral genome, which may be passed on trans-generationally, a particularly exciting avenue is to test in a controlled manner the influence of the epigenetic immune state of the host on virus evolution and diversification.

I. We can visualize the viruses by single-molecule FISH, which turns out to be an efficient method to quantify infection.
II. In collaboration with the Wang lab, the reconstitution of infectious Orsay virus through transgenesis in the worm was successful, which opens up many possibilities, especially for us in the study of host-pathogen specificity determinants.
III. We found that the main determinant of variation in sensitivity to the Orsay virus in C. elegans natural populations is a deletion polymorphism in the drh-1 gene, homolog of the RIG-I family of viral sensors in vertebrates. The small RNA response of the drh-1 mutant and the natural isolate with the deletion has been studied in the Miska lab. drh-1 appears necessary for 1° siRNA formation.
IV. The Orsay virus is specific to C. elegans, the Santeuil and Le Blanc viruses to C. briggsae. Among the C. briggsae isolates that we tested for sensitivity to Santeuil and Le Blanc viruses, some are sensitive to both, some to neither, and quite unexpectedely, one is very sensitive to Santeuil and not at all to Le Blanc. This provides us with a great system to study the evolution of host-pathogen specificity, within one host species. We are starting to make Recombinant Inbred Lines to find the genetic basis for this variation in sensitivity and specificity. We will also use the transgenesis approach to pinpoint key features of specificity on the virus side.

We are continuing the project as scheduled.

Franz, C., Zhao, G., Félix, M.-A., Wang, D. (2012). Complete genome sequence of Le Blanc virus, a third Caenorhabditis nematode infecting virus. J. Virology 86, 11940.

Natural pathogens exert strong selective pressures on their host. Host defense in turn provides a changing environment for the pathogen. This reciprocal evolutionary interaction may drive an evolutionary "arms race" between pathogen and host, as evidenced by the fast evolution of relevant genes.
The nematode Caenorhabditis elegans is a major animal model for genetic and molecular studies. Laboratory studies on immunity of C. elegans so far mostly used human pathogens. However, because natural pathogens leave an imprint on recent evolution of their host, they are obviously more relevant to study C. elegans immunity and co-evolution between host and pathogen. We have isolated natural populations of Caenorhabditis that are infected by pathogens, and recently identified the first natural viruses infecting C. elegans and C. briggsae, called Orsay and Santeuil viruses, respectively (Félix et al., PLoS Biology 2011). These RNA(+) viruses are related to nodaviruses and horizontally transmitted. Their small genome encodes a putative RNA-dependent RNA polymerase on the RNA1 segment, and a putative capsid protein and an ORF d of unknown function on the RNA2 segment. Infection slows down progeny production by the nematodes, but can be maintained indefinitely in culture.
Infection by these viruses elicits the formation of small RNAs corresponding to the viral genome. While the reference C. elegans isolate N2 is relatively resistant to Orsay virus infection, mutants deficient in the RNAi response are rendered sensitive to this virus. These results demonstrate a role for RNAi in C. elegans antiviral defense.
Different wild C. elegans isolates display differential susceptibility to infection by the Orsay virus, thereby affording genetic approaches to defining naturally variant loci acting in antiviral defense. Natural variation among C. elegans isolates in part affects somatic RNAi efficiency, but also other aspects of C. elegans-virus interactions.
These results establish the first natural and experimental viral infection system in C. elegans, suitable for probing all facets of the host antiviral response and co-evolutionary interactions between both partners. The present project proposes to build on these preliminary results and undertake the following studies about C. elegans anti-viral immunity and viral host specificity:
Task 1: Characterization of the infection of the model organism C. elegans by the newly discovered Orsay virus and development of infection markers.
Task 2: Reconstitution of the virus through DNA transgenesis in the worm for structure-function studies; role of the viral ORF delta, especially in the counter-defense of host immune responses.
Task 3: Characterization of intraspecific genetic variation in sensitivity of natural Caenorhabditis isolates to Orsay and Santeuil viruses and of its molecular basis.
Task 4: Range and mechanistic basis of the host specificity of Orsay and Santeuil viruses for C. elegans versus C. briggsae.
Task 5: Experimental evolution and co-evolution of host and pathogen, including genetic and epigenetic host evolution.

This project would lay the foundations for the study of host specificity and co-evolution in a new model system involving an animal and a pathogen (virus), which would be the simplest and fastest available. Such a model system allowing to combine laboratory and field studies of host-virus co-evolution on an animal that is already by itself a laboratory top model in genetics is particularly exciting.