Analysis of immune-related genes during Nora virus infection of <em>Drosophila melanogaster</em> using next generation sequencing

Abstract

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.

Heterogeneity in the response of different subtypes ofDrosophila melanogasterenteroendocrine cells to viral infections

“…In our case, we failed to detect any considerable up-regulation of immune genes in the cellular response to either TV or DMelNV. Interestingly, the time course analysis of the response to DMelNV found that expression of immune related genes increased overtime (Lopez et al, 2018). As the time of infection is an important factor to regulation of the host transcriptome, and the scRNAseq data used here is from 5-7 days old flies, we may have failed to detect a more substantial change to the host transcriptome.…”

“…The fact that disruption of essential genes in the interactome might diminish the infection process of pathogens, in accordance with the centrality-lethality rule (Jeong et al, 2001;Ahmed et al, 2018), corroborates with the first hypothesis; whereas the fact that some cells exhibited a large amount of DMelNV RNA and the expression of some ribosomal protein genes is negatively correlated to DMelNV replication level corroborates with the latter hypothesis. Interestingly, previous microarray and bulk RNA-seq studies were unable to find a similar under-expression of ribosomal genes in DMelNV-infected flies (Cordes et al, 2013;Lopez et al, 2018). In fact, some ribosomal proteins were over-expressed in the bulk RNA-seq data (supplementary file S1).…”

“…As the time of infection is an important factor to regulation of the host transcriptome, and the scRNAseq data used here is from 5-7 days old flies, we may have failed to detect a more substantial change to the host transcriptome. The up-regulation of immune genes in the bystander response to IAV (Steuerman et al, 2018) and SARS-CoV-2 (Ravindra et al, 2020) and in the bulk RNAseq analysis of DMelNV-infected flies (Lopez et al, 2018) indicates that activation of immunology-related genes is triggered as a systemic response to viral infections.…”

“…A list of DEGs from DMelNV-infected female flies was obtained from Lopez et al (2018). This data contains DEGs detected at two, 10, 20, and 30 days post-eclosion.…”

“…DMelNV infection led to a downregulation of essential hubs and bottlenecks of interactions on the D. melanogaster interactome, which are mostly composed by ribosomal proteins. By analyzing a publicly available bulk RNAseq data from DMelNV-infected flies (Lopez et al, 2018), we found that in this systemic response to DMelNV, only bottleneck genes were down-regulated, and no significant perturbation of hubs was found. Similarly, cellular response to TV infection was characterized by a down-regulation of bottleneck genes only.…”