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.

Keywords: Aag2; Ae. aegypti; DENV2; RNAseq; Transcriptome.

Fig. 1

Heatmap of the correlations between samples (a) and the principal components analysis (b). Mock refers to the uninfected group, and DENV2 is the DENV-2 infected group. Four biological replicates of each group were included in the analyses

Fig. 2

Volcano map of differentially expressed genes. The differentially expressed genes were selected based on the criteria Log₂ (FC) > 1 and Padj < 0.05. The red dots represent the 185 upregulated genes, legend is “Up”. The blue dots represent the 1014 downregulated genes, legend is “Down”. The grey dots represent the unchanged gene, legend is “No”

Fig. 3

Bubble map of the top 30 most enriched GO terms and KEGG pathways. a Top 30 GO terms enriched in the upregulated genes. b Top 30 GO terms enriched in the downregulated genes. c Top 30 KEGG pathways enriched in the upregulated genes. d Top 30 KEGG pathways enriched in the downregulated genes. Y-axis label represents the distinct GO terms or KEGG pathways, and X-axis label represents the Gene Ratio. Gene Ratio refers to the ratio of DEGs annotated in the term or pathway to total number of genes annotated in the term or pathway. The size of the bubble represents the number of enriched DEGs (big size indicates large number of enriched genes). The color represents the P-value of the enrichment (green color indicates high enrichment)

Fig. 4

Protein–protein interaction analysis. The analysis of 550 differentially expressed protein-coding genes revealed 108 interacting proteins with more than two edges, as shown in the maps. The connecting lines represent predicted functional associations. The thickness of the lines shows the strength of the data support

Fig. 5

qRT-PCR verification of 12 differentially expressed genes identified by RNAseq. Mock refers to an untreated sample, and the default gene expression was unchanged, and the FC value was set to 1. The relative expression level (FC) of a gene in DENV-2-infected Aag2 cells was calculated using the 2^-ΔΔCT method and presented as fold changes relative to the expression in uninfected cells

Fig. 6

Protein–protein interaction analysis of significantly differentially expressed genes validated by qRT-PCR. a The PPI analyses of AAEL002721, AAEL013828 and AAEL002583 (the KEGG pathways are distinguished by different colors: red, ubiquitin-mediated proteolysis; blue, Toll and Imd signaling pathways; and green, cysteine and methionine metabolism). b The PPI analyses of AAEL004174. The connecting lines represent the predicted functional associations. The thickness of the lines indicates the strength of the data support