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

Date Published: February 27, 2018

Publisher: AIMS Press

Author(s): Wilfredo Lopez, Alexis M. Page, Darby J. Carlson, Brad L. Ericson, Matyas F. Cserhati, Chittibabu Guda, Kimberly A. Carlson.


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.

Partial Text

Nora virus is a picorna-like virus that infects various Drosophila species and produces a non-pathogenic persistent infection [1],[2]. The single-stranded, positive-sense RNA viral genome is approximately 12 kilobases (kb) and encodes four open readings frames (ORFs) [1], unlike other picorna-like viruses such as Drosophila C virus, which only has two ORFs [3]. ORF1 encodes an RNA interference (RNAi) suppressor and suggests this may contribute to the establishment of persistent infection. In addition, other known innate immune pathways are not involved in regulation of infection [4]. ORF2 encodes a replicative cassette, which consists of a helicase, protease and RNA-dependent RNA polymerase [1]. ORF3 encodes VP3 and is essential for the stability of the viral capsid during environmental stress, such as heat or protease exposure [5],[6]. ORF4 encodes VP4, which is processed into three major proteins: VP4A, VP4B and VP4C [7]. Viral localization, replication, and assembly is hypothesized to occur in the gut and transmitted fecal-orally with no exhibited pathology in gut tissue [8]. Gene expression due to viral infection, as well as control of gene regulation due to viral replication, spread and chronic infection, is not well understood. Interactions of the virus with the Drosophila immune system are essential in understanding these processes. Major mechanisms of innate immunity include the immune deficiency pathway (Imd) [9], Toll-Dorsal pathway (Toll) [10], Janus kinase/signal transducer and activator of transcription pathway (JAK/STAT) [11] and RNA interference (RNAi) [12]. Prior research was conducted with cDNA microarray to determine genetic expression due to infection four days post-eclosion of D. melanogaster. The results of the study were limited in that only 58 genes were able to be analyzed (46 upregulated and 12 downregulated) and only a small number of these were related to the innate immune response. The main limitation of this study was that it only assessed a single early time point that did not represent the overall time course of infection in D. melanogaster[13]. In addition, the technology used in this study is currently outdated and is being “sunsetted” in many institutions. However, technological advances have provided better tools to analyze in-depth gene expression during various time points of infection. Next generation sequencing (NGS) allows for faster, inexpensive, and more accurate, reliable results than prior sequencing methods. In addition, NGS does not require validation of results via quantitative RT-PCR (qRT-PCR) [14]. In this study, we expanded the sampling days to 2, 10, 20 and 30 post-eclosion to capture a more complete picture of gene expression profile changes over time. From this data, we used NGS to determine differentially expressed genes (DEGs) during Nora virus infection at day 2, 10, 20 and 30, which were time points selected from the data of the time course. The objective of this study was to determine the immune related genes that are regulated by Nora virus infection and how this changes over time. The data from this study can be used to further analyze and determine pathways that may be involved in regulation of infection, viral replication and potential pathogenesis.

Next generation sequencing was conducted during Nora virus infection to identify immune related genes for further analysis. Genes involved during Nora virus infection were identified by comparison of NV+ and NV− flies aged to day 2, 10, 20 and 30. All NV+ samples demonstrated a positive RT-PCR product at 790 bp and the NV− samples did not show a product, as was expected (data not shown). Viral load was assessed by qRT-PCR and found to increase from day 2 to day 10, decrease on day 20, and increase again at day 30. This bimodal trend was again seen when the differentially expressed genes were submitted to PANTHER analysis for further classification (Table 1), as well as the manual analysis of the immune related genes for total number of genes per day either up or downregulated (Table 2; Supplemental Table 4). In addition, the total upregulated genes for the manually characterized immune related genes (Table 2) followed the same bimodal distribution as was seen with Nora virus load. This suggests that as Nora virus load changes, the genes regulated by this virus, either directly or indirectly, change to match the level of virus in the fly. The kinetics of Nora virus infection are largely unknown. Similar bimodal infection distribution and upregulation of genes is seen in human viruses including influenza A [23] and HIV [24]. In terms of the innate immune response, influenza A was correlated with interferon (IFN) release and found to show a bimodal distribution [23]. This suggests that the bimodal distribution of Nora virus load may be correlated with an interferon-like molecule or other cytokine that follows the same pattern.




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