Transcriptome analysis of Aedes aegypti Aag2 cells in response to dengue virus-2 infection

Abstract

Background: Dengue virus (DENV) is a flavivirus transmitted by mosquitoes that is prevalent in tropical and subtropical countries and has four serotypes (DENV1-4). Aedes aegypti, as the main transmission vector of DENV, exhibits strong infectivity and transmission. With the aim of obtaining a better understanding of the Ae. aegypti-DENV interaction, the transcriptome changes in DENV-2-infected Aag2 cells were studied to describe the immune responses of mosquitoes using the Ae. aegypti Aag2 cell line as a model. Methods: RNAseq technology was used to sequence the transcripts of the Ae. aegypti Aag2 cell line before and after infection with DENV-2. A bioinformatics analysis was then performed to assess the biological functions of the differentially expressed genes, and the sequencing data were verified by quantitative reverse transcription-polymerase chain reaction (qRT-PCR). Results: The transcriptome analysis generated 8866 unigenes that were found in both groups, 225 unigenes that were only found in the infection group, and 683 unigenes that only existed in the control group. A total of 1199 differentially expressed genes, including 1014 upregulated and 185 downregulated genes, were identified. The bioinformatics analysis showed that the differentially expressed genes were mainly involved in the longevity regulating pathway, circadian rhythm, DNA replication, and peroxisome, purine, pyrimidine, and drug metabolism. The qRT-PCR verification results showed the same trend, which confirmed that the expression of the differentially expressed genes had changed, and that the transcriptome sequencing data were reliable. Conclusions: This study investigated the changes in the transcriptome levels in the DENV-2-infected Ae. aegypti Aag2 cell line, which provides a faster and effective method for discovering genes related to Ae. aegypti pathogen susceptibility. The findings provide basic data and directions for further research on the complex mechanism underlying host-pathogen interactions.

Transcriptome Analysis of Responses to Dengue Virus 2 Infection in Aedes albopictus (Skuse) C6/36 Cells

“…Similar to the results of our previous research in Aag2 cells [29], cells maintain a dynamic balance by changing their metabolic pathways during the long-term evolution process to ensure their survival after infection [37]. Among these processes, pyrimidine metabolism, which we also observed to be enriched in this study, has been proven to affect virus replication and pathogen infection [38,39].…”

“…The results indicated that the virus copy number gradually increased, reaching a peak at 6 to 7 dpi and then plateauing. Cytopathic effect (CPE) or morphological changes were observed in DENV2-infected C6/36 cells but not mock-infected cells at 4 dpi, with the infect cells gradually showing obvious phenotypes such as cavitation, collapse, and fusion, which is different from the results in Aag2 cells found in our previous research, which showed no changes in morphology or CPE following DENV2 infection (Figure 1b) [29]. DENV2-and mock-infected cells were harvested at 4 dpi for transcriptome sequencing analysis.…”

“…The subsequent bioinformatics analysis methods used are the same, and the screening criteria for differentially expressed genes used are the same (padj < 0.05 and |Log2 (FC)| > 1). A total of 16 DEGs overlapped between Aag2 cells (which we studied before [ 29 ]) and C6/36 cells ( Figure 5 a). GO enrichment analysis revealed 7 overlapping terms enriched for upregulated DEGs ( Figure 5 b), which mainly included phosphate-containing compound and phosphorus metabolic process, biosynthetic processes such as nucleobase-containing compound, aromatic compound, heterocycle, organic cyclic compound, and DNA binding.…”

“…As we explained in previous studies [ 29 ], the molecular mechanism of this pathway is not fully understood, but many studies have fully demonstrated the impact of circadian rhythm disruption on infections and diseases. In addition, the upregulated and downregulated DEGs were simultaneously enriched in the Toll and Imd signaling pathways, but the JAK-STAT signaling pathway was not identified, which is similar to the results of our previous analysis of the transcriptome of DENV2-infected Aag2 cells [ 29 ]. These findings strongly suggest that the Toll and Imd signaling pathways may participate in the regulation of DENV2–vector interactions.…”

Bioinformatic and cell-based tools for pooled CRISPR knockout screening in mosquitos

“…5 ), could have several immediate applications in mosquito research. Several studies have generated genesets from proteomic or differential expression analyses (e.g., host proteins that interact with viral proteins 58 , or genes up- or down-regulated in response to pathogen infection 26 , 34 , 37 , 59 , 60 ). Focused CRISPR screening based on these genesets can provide functional validation of these data.…”