An ideal model system to study antiviral immunity and host-pathogen co-evolution would combine a genetically tractable small animal with a virus capable of naturally infecting the host organism. The use of C. elegans as a model to define host-viral interactions has been limited by the lack of viruses known to infect nematodes. From wild isolates of C. elegans and C. briggsae with unusual morphological phenotypes in intestinal cells, we identified two novel RNA viruses distantly related to known nodaviruses, one infecting specifically C. elegans (Orsay virus), the other C. briggsae (Santeuil virus). Bleaching of embryos cured infected cultures demonstrating that the viruses are neither stably integrated in the host genome nor transmitted vertically. 0.2 µm filtrates of the infected cultures could infect cured animals. Infected animals continuously maintained viral infection for 6 mo (∼50 generations), demonstrating that natural cycles of horizontal virus transmission were faithfully recapitulated in laboratory culture. In addition to infecting the natural C. elegans isolate, Orsay virus readily infected laboratory C. elegans mutants defective in RNAi and yielded higher levels of viral RNA and infection symptoms as compared to infection of the corresponding wild-type N2 strain. These results demonstrated a clear role for RNAi in the defense against this virus. Furthermore, different wild C. elegans isolates displayed differential susceptibility to infection by Orsay virus, thereby affording genetic approaches to defining antiviral loci. This discovery establishes a bona fide viral infection system to explore the natural ecology of nematodes, host-pathogen co-evolution, the evolution of small RNA responses, and innate antiviral mechanisms. The nematode C. elegans is a robust model organism that is broadly used in biology. It also has great potential for the study of host-microbe interactions, as it is possible to systematically knockout almost every gene in high-throughput fashion to examine the potential role of each gene in infection. While C. elegans has been successfully applied to the study of bacterial infections, only limited studies of antiviral responses have been possible since no virus capable of infecting any Caenorhabditis nematode in laboratory culture has previously been described. Here we report the discovery of natural viruses infecting wild isolates of C. elegans and its relative C. briggsae. These novel viruses are most closely related to the ssRNA nodaviruses, but have larger genomes than other described nodaviruses and clearly represent a new taxon of virus. We were able to use these viruses to infect a variety of laboratory nematode strains. We show that mutant worms defective in the RNA interference pathway, an antiviral system known to operate in a number of organisms, accumulate more viral RNA than wild type strains. The discovery of these viruses will enable further studies of host-virus interactions in C. elegans and the identification of other host mechanisms that counter viral infection.

Background: Astroviruses infect a variety of mammals and birds and are causative agents of diarrhea in humans and other animal hosts. We have previously described the identification of several sequence fragments with limited sequence identity to known astroviruses in a stool specimen obtained from a child with acute diarrhea, suggesting that a novel virus was present. Results: In this study, the complete genome of this novel virus isolate was sequenced and analyzed. The overall genome organization of this virus paralleled that of known astroviruses, with 3 open reading frames identified. Phylogenetic analysis of the ORFs indicated that this virus is highly divergent from all previously described animal and human astroviruses. Molecular features that are highly conserved in human serotypes 1–8, such as a 3'NTR stem-loop structure and conserved nucleotide motifs present in the 5'NTR and ORF1b/2 junction, were either absent or only partially conserved in this novel virus. Conclusion: Based on the analyses described herein, we propose that this newly discovered virus represents a novel species in the family Astroviridae. It has tentatively been named Astrovirus MLB1.

Bacteriophage modulation of microbial populations impacts critical processes in ocean, soil, and animal ecosystems. However, the role of bacteriophages with RNA genomes (RNA bacteriophages) in these processes is poorly understood, in part because of the limited number of known RNA bacteriophage species. Here, we identify partial genome sequences of 122 RNA bacteriophage phylotypes that are highly divergent from each other and from previously described RNA bacteriophages. These novel RNA bacteriophage sequences were present in samples collected from a range of ecological niches worldwide, including invertebrates and extreme microbial sediment, demonstrating that they are more widely distributed than previously recognized. Genomic analyses of these novel bacteriophages yielded multiple novel genome organizations. Furthermore, one RNA bacteriophage was detected in the transcriptome of a pure culture of Streptomyces avermitilis, suggesting for the first time that the known tropism of RNA bacteriophages may include gram-positive bacteria. Finally, reverse transcription PCR (RT-PCR)-based screening for two specific RNA bacteriophages in stool samples from a longitudinal cohort of macaques suggested that they are generally acutely present rather than persistent. Bacteriophages (viruses that infect bacteria) can alter biological processes in numerous ecosystems. While there are numerous studies describing the role of bacteriophages with DNA genomes in these processes, the role of bacteriophages with RNA genomes (RNA bacteriophages) is poorly understood. This gap in knowledge is in part because of the limited diversity of known RNA bacteriophages. Here, we begin to address the question by identifying 122 novel RNA bacteriophage partial genome sequences present in metagenomic datasets that are highly divergent from each other and previously described RNA bacteriophages. Additionally, many of these sequences contained novel properties, including novel genes, segmentation, and host range, expanding the frontiers of RNA bacteriophage genomics, evolution, and tropism. These novel RNA bacteriophage sequences were globally distributed from numerous ecological niches, including animal-associated and environmental habitats. These findings will facilitate our understanding of the role of the RNA bacteriophage in microbial communities. Furthermore, there are likely many more unrecognized RNA bacteriophages that remain to be discovered.

Quick and accurate identification of microbial pathogens is essential for both diagnosis and response to emerging infectious diseases. The advent of next-generation sequencing technology offers an unprecedented platform for rapid sequencing-based identification of novel viruses. We have developed a customized bioinformatics data analysis pipeline, VirusHunter, for the analysis of Roche/454 and other long read Next generation sequencing platform data. To illustrate the utility of VirusHunter, we performed Roche/454 GS FLX titanium sequencing on two unclassified virus isolates from the World Reference Center for Emerging Viruses and Arboviruses (WRCEVA). VirusHunter identified sequences derived from a novel bunyavirus and a novel reovirus in the two samples respectively. Further sequence analysis demonstrated that the viruses were novel members of the Phlebovirus and Orbivirus genera. Both Phlebovirus and Orbivirus genera include many economic important viruses or serious human pathogens.