Characterization of Cross-Species Transmission of Drosophila melanogaster Nora Virus.

Drosophila melanogaster Nora virus (DmNV) is a novel picorna-like virus first characterized in 2006. Since then, Nora virus has been detected in several non-Drosophila species, including insects in the Orders Hymenoptera, Lepidoptera, Coleoptera, and Orthoptera. The objective of this study was to determine if DmNV could infect individuals of other species of invertebrates besides D. melanogaster. The presence of DmNV in native invertebrates and commercially available stocks was determined. Laboratory-reared D. yakuba, D. mercatorum, Gryllodes sigillatus, Tenebrio molitor, Galleria mellonella, and Musca domestica were intentionally infected with DmNV. In addition, native invertebrates were collected and D. melanogaster stocks were purchased and screened for DmNV presence using reverse transcription-polymerase chain reaction (RT-PCR) before being intentionally infected for study. All Drosophila species and other invertebrates, except M. domestica, that were intentionally infected with DmNV ended up scoring positive for the virus via RT-PCR. DmNV infection was also detected in three native invertebrates (Spilosoma virginica, Diplopoda, and Odontotaenius disjunctus) and all commercially available stocks tested. These findings suggest that DmNV readily infects individuals of other species of invertebrates, while also appearing to be an endemic virus in both wild and laboratory D. melanogaster populations. The detection of DmNV in commercially available stocks presents a cautionary message for scientists using these stocks in studies of virology and immunology.

In vitro assembly and evaluation of Nora virus VLPs.

Nora virus is a RNA picorna-like virus that produces a persistent infection in Drosophila melanogaster. The genome is approximately 12,300 bases and is divided into four open reading frames (ORFs). Structurally, there are four important viral proteins: VP3, VP4A, VP4B, and VP4C. Three proteins (VP4A, VP4B, and VP4C) that form the virion's capsid are encoded by ORF 4, which produces a polyprotein that is post-translationally cleaved. The fourth protein (VP3) is encoded by ORF 3 and it is hypothesized to play a role in virion stability. The genes for these proteins were individually cloned into Escherichia coli, expressed, and the proteins were purified. Virus-like particles (VLPs) were assembled in vitro by mixing the proteins together in different combinations and measured via electron microscopy. Assemblies that contained VP4A and/or VP3 created VLPs with similar sizes to purified empty Nora virus capsids, potentially indicating that VP4A and/or VP3 are vital for Nora virus capsid structure, assembly, and/or stability. Not only does this study provide insight into the role of Nora virus proteins, but it may also lead to a deeper understanding of how Nora virus or other picorna-like viruses undergo assembly.  Keywords: RNA viruses; Nora virus; picorna-like virus; virus-like particles; capsid assembly.

Effect of Nora virus infection on native gut bacterial communities of Drosophila melanogaster.

Gastrointestinal microflora is a key component in the maintenance of health and longevity across many species. In humans and mice, nonpathogenic viruses present in the gastrointestinal tract enhance the effects of the native bacterial microbiota. However, it is unclear whether nonpathogenic gastrointestinal viruses, such as Nora virus that infects Drosophila melanogaster, lead to similar observations. Longevity analysis of Nora virus infected (NV+) and uninfected (NV-) D. melanogaster in relationship to presence (B+) or absence (B-) of the native gut bacteria using four different treatment groups, NV+/B+, NV+/B-, NV-/B+, and NV-/B-, was conducted. Data from the longevity results were tested via Kaplan-Meier analysis and demonstrated that Nora virus can be detrimental to the longevity of the organism, whereas bacterial presence is beneficial. These data led to the hypothesis that gastrointestinal bacterial composition varies from NV+ to NV- flies. To test this, NV+ and NV- virgin female flies were collected and aged for 4 days. Surface sterilization followed by dissections of the fat body and the gastrointestinal tract, divided into crop (foregut), midgut, and hindgut, were performed. Ribosomal 16S DNA samples were sequenced to determine the bacterial communities that comprise the microflora in the gastrointestinal tract of NV+ and NV- D. melanogaster. When analyzing operational taxonomic units (OTUs), the data demonstrate that the NV+ samples consist of more OTUs than NV- samples. The NV+ samples were both more rich and diverse in OTUs compared to NV-. When comparing whole body samples to specific organs and organ sections, the whole fly was more diverse in OTUs, whereas the crop was the most rich. These novel data are pertinent in describing where Nora virus infection may be occurring within the gastrointestinal tract, as well as continuing discussion between the relationship of persistent viral and bacterial interaction.

Nora Virus VP4b and ORF1 Circulate in Hemolymph of Infected D. melanogaster with Coordinate Expression of Vago and Vir-1.

Study of the novel RNA virus, Nora virus, which is a persistent, picorna-like virus that replicates in the gut of Drosophila melanogaster offers insight into human innate immunity and other picorna-like viruses. Nora virus infection leads to a locomotor abnormality and upregulation of two candidate target proteins, Vago and Virus-induced RNA 1 (Vir-1). These proteins are uncharacterized in response to Nora virus. We hypothesize that Nora virus is circulating in the hemolymph of Nora virus-infected D. melanogaster, allowing for migration beyond the primary site of replication in the gut. Analysis by qRT-PCR demonstrated biphasic viral load and corresponding vago and vir-1 transcription levels, suggesting transcription of vago and vir-1 occurs in response to viral infection. However, Vir-1 is also present in virus-free D. melanogaster suggesting basal expression or alternative functions. Presence of Nora virus RNA and the Viral Protein 4b (VP4b), in hemolymph of infected D. melanogaster supports the hypothesized circulation of Nora virus in the hemolymph. The study suggests that impaired locomotor function may be due to transport of Nora virus from the gut to the brain via the hemolymph.

Analysis of immune-related genes during Nora virus infection of Drosophila melanogaster using next generation sequencing.

Drosophila melanogaster depends upon the innate immune system to regulate and combat viral infection. This is a complex, yet widely conserved process that involves a number of immune pathways and gene interactions. In addition, expression of genes involved in immunity are differentially regulated as the organism ages. This is particularly true for viruses that demonstrate chronic infection, as is seen with Nora virus. Nora virus is a persistent non-pathogenic virus that replicates in a horizontal manner in D. melanogaster. The genes involved in the regulation of the immune response to Nora virus infection are largely unknown. In addition, the temporal response of immune response genes as a result of infection has not been examined. In this study, D. melanogaster either infected with Nora virus or left uninfected were aged for 2, 10, 20 and 30 days. The RNA from these samples was analyzed by next generation sequencing (NGS) and the resulting immune-related genes evaluated by utilizing both the PANTHER and DAVID databases, as well as comparison to lists of immune related genes and FlyBase. The data demonstrate that Nora virus infected D. melanogaster exhibit an increase in immune related gene expression over time. In addition, at day 30, the data demonstrate that a persistent immune response may occur leading to an upregulation of specific immune response genes. These results demonstrate the utility of NGS in determining the potential immune system genes involved in Nora virus replication, chronic infection and involvement of antiviral pathways.

Characterization of Nora Virus Structural Proteins via Western Blot Analysis.

Nora virus is a single stranded RNA picorna-like virus with four open reading frames (ORFs). The coding potentials of the ORFs are not fully characterized, but ORF3 and ORF4 are believed to encode the capsid proteins (VP3, VP4a, VP4b, and VP4c) comprising the virion. To determine the polypeptide composition of Nora virus virions, polypeptides from purified virus were compared to polypeptides detected in Nora virus infected Drosophila melanogaster. Nora virus was purified from infected flies and used to challenge mice for the production of antisera. ORF3, ORF4a, ORF4b, and ORF4c were individually cloned and expressed in E. coli; resultant recombinant proteins purified and were used to make monospecific antisera. Antisera were evaluated via Western blot against whole virus particles and Nora virus infected fly lysates. Viral purification yielded two particle types with densities of ~1.31 g/mL (empty particles) and ~1.33 g/mL (complete virions). Comparison of purified virus polypeptide composition to Nora virus infected D. melanogaster lysate showed the number of proteins in infected cell lysates is less than purified virus. Our results suggest the virion is composed of 6 polypeptides, VP3, VP4a, two forms of VP4b, and two forms of VP4c. This polypeptide composition is similar to other small RNA insect viruses.

Genome-Wide Gene Expression in relation to Age in Large Laboratory Cohorts of Drosophila melanogaster.

Aging is a complex process characterized by a steady decline in an organism's ability to perform life-sustaining tasks. In the present study, two cages of approximately 12,000 mated Drosophila melanogaster females were used as a source of RNA from individuals sampled frequently as a function of age. A linear model for microarray data method was used for the microarray analysis to adjust for the box effect; it identified 1,581 candidate aging genes. Cluster analyses using a self-organizing map algorithm on the 1,581 significant genes identified gene expression patterns across different ages. Genes involved in immune system function and regulation, chorion assembly and function, and metabolism were all significantly differentially expressed as a function of age. The temporal pattern of data indicated that gene expression related to aging is affected relatively early in life span. In addition, the temporal variance in gene expression in immune function genes was compared to a random set of genes. There was an increase in the variance of gene expression within each cohort, which was not observed in the set of random genes. This observation is compatible with the hypothesis that D. melanogaster immune function genes lose control of gene expression as flies age.

Advances in age-old questions.

Drosophila melanogaster is an ideal model organism for various types of aging studies. They are easy to maintain, relatively inexpensive, have short life cycles, provide large sample sizes, and can be genetically manipulated via various methods for testing. The 49(th) Annual Drosophila Research Conference, held in San Diego, CA (April 2-6, 2008), had over 30 poster presentations and eight platform talks devoted to physiology and aging, and seven presentations in a longevity and functional senescence workshop. The data presented via these avenues included life span manipulation, physiological related genes, candidate aging genes, gene expression, signaling, and using D. melanogaster as a model for age related disease, to name a few. This report provides highlights of some of the information presented in the poster, platform and workshop presentations.