Mosquitoes transmit malaria, dengue and Zika viruses, yellow fever and many other vector-borne diseases and cause millions of infections and even deaths every year . Arbovirus infections usually cause acute pathological symptoms in the human body, which are usually relatively mild in the vector [25, 26]. Since Grace  first established an insect cell line from moths, the S2 cell line from Drosophila melanogaster, the Sua5B cell line from Anopheles gambiae, the C6/36 cell line from Aedes albopictus and many other insect cell lines have been used as important tools for studying insects [28, 29, 30]. Because cell lines are homologous,sensitive and repeatable, subtle changes in response to different types of pathogens and stimuli can be detected. The embryo-derived Aag2 cell line used in this study has been proven to be immunologically active, and because mosquitoes serve as an important vector for mosquito-borne viruses, this cell lines has been used for immune-related experiments . Gao et al. found a small peptide belonging to the defensin family in the Aag2 cell line that was preferentially induced in heat-killed, gram-positive bacteria-treated growing cells, and the subsequent sequencing of the peptide identified two previously described subtypes of defensin A (A1 and A4) and a new subtype (A5) . Sim et al. found that the transcriptional induction of antimicrobial peptides (AMPs) by the Toll and IMD pathways was impaired in the Aag2 cell line after DENV infection, which indicated that DENV can actively suppress the immune responses of infected cells . In addition, Varjak et al. also used the Aag2 cell line to studying the characteristics of Zika virus (ZIKV)-induced RNA interference (RNAi) that regulate the immune responses to virus infection . Based on these studies, we aimed to discover other target genes related to pathogen infection.
In this study, to avoid the effects of RNA degradation and accumulation on physiological functions and to ensure the quality of the cDNA library, we selected the samples collected at 4 dpi that exhibited high intracellular and extracellular virus titers and high virus copy numbers after DENV-2 infection for transcriptome sequencing. This analysis identified 1,199 DEGs (185 upregulated and 1,014 downregulated), which initially indicated that these genes might be related to DENV-2 infection. The results from the enrichment analyses demonstrated that the DEGs mainly participate in the pathways related to material metabolism, synthesis, transport, DNA replication, and signaling. The PPI analysis identified the interactions within the proteins expressed in Aag2 cells during DENV-2 infection (Fig. 5). And further analysis of this results showed that the DEGs were also mainly enriched in DNA replication, longevity regulating pathway-multiple species, metabolic pathways, and purine and pyrimidine metabolism. The effects of DENV-2 infection on the physiological functions of Aag2 cells were further elucidated, but the specific functions need to be further explored and verified.
Analysis of metabolic pathways
Cells change their metabolic pathways to ensure their survival after infection. In this study, the metabolic pathways that were enriched by the upregulated genes were pyrimidine metabolism, retinol metabolism, drug metabolism-other enzymes, starch and sucrose metabolism, tryptophan metabolism, and porphyrin and chlorophyll metabolism. Among these pathways, the most significant enrichment was found in pyrimidine metabolism. Pyrimidine is a structural component of a series of key molecules and is involved in the synthesis of DNAs, RNAs, lipids and carbohydrates, and pyrimidine metabolism includes the synthesis, degradation, recovery, transformation and transportation of these molecules. Previous studies have shown that pyrimidine metabolism participates in programmed cell death (PCD) through a dynamic balance between recovery and degradation . And PCD, as an intrinsic response to viral infection, has been shown to inhibit viral replication [34, 35]. In addition, changes in host cell pyrimidine metabolism after infection also affect viral replication, and previous studies have analyzed the levels of metabolites in cells infected with human cytomegalovirus (HCMV) and herpes simplex virus type-1 (HSV-1), which are viruses that utilize different pyrimidine metabolism-related strategies to promote virus replication . A metabolomics study of intraerythrocytes infected with the malaria parasite Plasmodium falciparum also revealed the effects of changes in the pyrimidine levels on pathogen infection . Pathogens have evolved new metabolic strategies during evolution that provide optimal levels of catabolic metabolites for their own replication . The results of this study also confirm that pyrimidine metabolism is activated in Aag2 cells after infection with DENV-2. We can further study the mechanisms related to mosquito-mediated pyrimidine metabolic pathways. We may be able to use pyrimidine inhibitors or antimetabolites to inhibit and block DENV-2 replication in Aag2 cells.
Studies related to retinol metabolism have revealed that retinol functions in the maintenance and promotion of immunity. In the absence of retinol, the expression of retinoic acid receptors in immune cells correspondingly decreases, and these decreases affect physiological abnormalities and pathological changes caused by immune function. Drug metabolism-other enzymes refers to the process related to changes in the chemical structure of the drug catalyzed by various drug-metabolizing enzymes, and the mechanism involved in the immune response process needs to be further clarified. These pathways are activated in Aag2 cells after infection with DENV-2 and might play an important role in the regulation of physiological functions in infected cells.
Purine metabolism and tyrosine metabolism were identified as the metabolic pathways in which the downregulated genes were enriched. Purines and pyrimidines are indispensable to all life and have many important functions , and purine metabolism involves the in vivo synthesis and decomposition of purine derivatives, such as the nucleic acid bases adenine and guanine. Purine compounds play an important role in the growth, proliferation and survival of all cells, and the results from the enrichment analysis indicate that the metabolic pathway, as the most enriched pathway, plays an important role in the interaction between Aag2 cells and DENV-2. The findings suggest that further studies can be based on the metabolic pathway, pay particular attention to relevant genes involved in the pyrimidine metabolic pathway, and further investigate the changes in physiological functions fur to host cell-virus infections, and the above-mentioned relevant genes can be used as targets for inhibiting or even promoting their expression and thereby inhibiting or even blocking virus replication.
Analysis of signaling pathways
According to previous studies, PCD is an important part of the host defense system against pathogen invasion. Many viruses generate inhibitors of PCD at an early stage to disrupt these host responses during infection and thereby ensure their own replication and thus the production of latent and persistent infections, and these inhibitors play a key role in the maintenance of the immune system and host defense during virus infection. Therefore, a complex host-pathogen interaction is produced when viruses and cells compete for control of these pathways . During evolution, viruses have developed the ability to regulate a variety of host cell signaling pathways. Most signaling pathways identified in this study are involved in the regulation of viral infections by participating in PCD.
Many of the DEGs identified in this study are enriched in related signaling pathways, and the upregulated DEGs were enriched in the FoxO signaling pathway, which is mainly involved in the regulation of cell life processes such as apoptosis, the cell cycle, and longevity. Previous studies have demonstrated that Japanese encephalitis virus (JEV) infection can reduce the expression of apoptosis-related FoxO signaling pathway genes and that the knockdown and overexpression of the FoxO gene promotes and inhibits apoptosis in JEV-infected Neuro-2a cells, respectively . PI3K/Akt, a central regulatory mechanism of FoxO, also participates in the inhibition of apoptosis through negative regulation of the FoxO signaling pathway and thereby regulates the process of virus replication [41, 42].
In addition, the longevity regulating pathway-multiple species, which was found to be significantly enriched in both up- and downregulated genes, is also involved in the apoptotic process. In Drosophila melanogaster, dietary restriction (DR) inactivates the PI3K/Akt/TOR signaling cascade by reducing signaling through the insulin/insulin-like growth factor signaling and target of rapamycin (IIS/TOR) pathway and thereby activates the FoxO signaling pathway, which is involved in apoptosis and survival processes .
Pathways enriched in downregulated genes, such as the MAPK signaling pathway and Hippo signaling pathway-fly, also participate in this process. The Hippo signaling pathway regulates the proliferation, survival, and morphology of eukaryotic cells, and studies have shown that this pathway is involved in the process of ZIKV infection . The MAPK signaling pathway also participates in the virus-host interaction process by regulating various physiological processes, such as cell growth, differentiation, apoptosis and death [45, 46].
The abovementioned signal pathways interact with each other and form a huge complex signal transmission network through activation or negative regulation. This provides us with new ideas that can interfere with the PCD process by regulating Aag2 cell-related signaling pathways and achieve the purpose of inhibiting or even blocking DENV-2 replication. And we suspect that the PCD process involved in these signaling pathways may be related to the nonlytic persistent infection phenotype without morphological and CPE changes in DENV-2-infected Aag2 cells, which needs further study.
Analysis of immune pathways
Previous research on the mosquito immune response has provided evidence demonstrating that its immune response is similar to the mammalian adaptive immune response, but the innate immune response also exerts a marked effect on the outcome of infection [47, 48]. The immunoregulatory mechanisms in mosquitoes include the RNAi, Toll, and JAK-STAT pathways. The RNAi pathway is triggered by double-stranded RNA (dsRNA) after virus infection, and the activation of this pathway leads to the production of small interfering RNAs (siRNAs), which aid the degradation of viral mRNAs that show homology to the dsRNA trigger sequence and thereby inhibit viral replication . Viruses, fungi, and gram-positive bacteria can activate the Toll pathway, which regulates the immune response mainly by controlling the expression of multiple AMPs . The JAK-STAT pathway, which is another antiviral mechanism that is independent of the Toll and RNAi pathways, exerts its antiviral activity through one or several STAT-regulated effectors. The silencing of the receptors Domeless (Dome) and Janus kinase (Hop) increases the susceptibility of mosquitoes to viruses, and the silencing of the negative regulator PIAS increases their resistance to viruses . In addition, many other mechanisms are involved in the antiviral and escape mechanisms in mosquito, and these include many miRNAs, the type 1 IFN response, the complement system response, cellular intrinsic autophagy and apoptosis mechanisms, and the PI3K/Akt pathway [48, 52].
The downregulated DEGs identified in this study, were only enriched in the Toll and Imd signaling pathways and not in the JAK-STAT signaling pathway and the RNAi pathway, which suggests that the Toll and Imd signaling pathways might differ from the JAK-STAT signaling pathway and the RNAi pathway. This result is similar to the results obtained after the infection of Aedes albopictus cells with Bluetongue virus [53, 54]. Moreover, the Toll and Imd signaling pathways were inhibited in DENV-2-infected Aag2 cells, and these findings were consistent with those from previous studies that showed that viruses such as DENV and CHIKV inhibit the Toll signaling pathway and thereby inhibit antiviral activity, which might reflect a “DENV downregulation trend” [55, 56, 57].
Analysis of other pathways
The downregulated DEGs were significantly enriched in the circadian rhythm pathway-fly. The circadian rhythm is important not only for behavioral research but also for basic physiological processes such as immunity. Although the molecular mechanisms of these interactions are not fully understood, the effects of disrupting circadian rhythms on infections and diseases have been well documented . Studies in this field have mainly been performed using the model organism Drosophila melanogaster. Studies conducted by Schneider et al. have shown that bacterial infections disrupt the circadian rhythm. After the exposure of Drosophila melanogaster to lethal doses of pathogenic bacteria, the time of death of flies with mutations in the proteins related to the circadian rhythm is significantly earlier than that of wild-type flies, which reveals a functional relationship between circadian rhythm and innate immunity . Lee et al. used Drosophila melanogaster infected with the human opportunistic pathogen Pseudomonas aeruginosa as a model to study the effects of physiological regulation on immunity . The results show that medical intervention strategies incorporating chronobiological considerations can enhance the innate immune response and improve the effect of combating pathogenic infections . These behavioral studies and the results obtained in this study provide new strategies and ideas for the immune regulation of vectors.
In addition, the significant enrichment of downregulated DEGs in peroxisome preliminarily indicates that this pathway is also involved in the interaction between Aag2 cells and DENV-2. Previous studies have shown that peroxisome-mediated metabolism is necessary for the immune response to infection in Drosophila melanogaster . Peroxisomes fight bacterial infections through classic innate immune signals, and studies have shown that the reduction of peroxisome function impairs the turnover of the oxidative burst necessary for fighting infection. Further research on peroxisomes and their role in the immune system is needed .
The downregulated DEGs were also enriched in ABC transporters. As the largest family of active transporters, ABC transporters play a vital role in the cell metabolic balance, immunity and detoxification. Previous research on ABC transporters has mostly focused on pesticide sensitivity and metabolism [63, 64], but the results of this study indicate that these transporters might also participate in the cellular immune process. All three pathways were inhibited in the DENV-2-infected Aag2 cells, which suggests that the genes related to these pathway might be involved in the host-virus interaction process and that enhancing the expression of these genes might inhibit or even block virus transmission.
qRT-PCR verification of gene expression levels
For verification of the sequencing data, 12 genes that were found to show significantly upregulated expression were selected for qRT-PCR verification, and cell samples collected at 4 dpi were used in this analysis. Here, we mainly focused on genes related to immune activation and enhanced infection, and we thus selected some of the upregulated genes for validation. The results from the qRT-PCR verification analysis were consistent with the transcriptome sequencing results, and the expression levels increased after infection, which indicated that the expression of the selected genes in Aag2 cells changed after infection.
In addition, to observe the changes in the expression levels of these 12 genes in cells, a qRT-PCR analysis was performed at 1, 2, and 4 dpi. The results showed that genes exhibit dynamic changes during the interaction between Aag2 and DENV-2, and the expression of most of these genes was inhibited in the early stage and enhanced at the later stage (Table 2). This finding confirms that the genes might participate in the regulation of Aag2 processes in response to the virus and thus provides potential targets for blocking intracellular virus replication. For example, Toll-like receptor 7 (TOLL7) was upregulated at 1, 2, and 4 dpi and exhibited significantly increased expression at 1 and 4 dpi (P < 0.05). Studies using Drosophila melanogaster have revealed that TLRs are involved in the development of the dorsoventral axis, synaptogenesis, and axonal initiation and have demonstrated that these processes participate in the immune response [65, 66]. Mutations in the Toll signaling pathway significantly reduce the survival of Drosophila melanogaster after fungal infection and are activated either by a distinct Imd signaling pathway or by combined activation of both Imd and dorsoventral pathways . In humans, TOLL7 is a transmembrane protein that recognizes viral ssRNAs and specific small-molecule agonists and plays a very important role in the signal transduction pathways involved in innate immunity . Further clarification of the relationship between TOLL7 activity and viral infection in mosquitoes will aid the formulation of new mosquito-borne virus intervention strategies. The T-box transcription factor TBX6 was upregulated at 1, 2, and 4 dpi and significantly upregulated at 4 dpi (P < 0,05). The T-box gene is essential for the development of arthropod limbs. Eight genes in the Drosophila genome encode the T-box, and six of these genes are expressed in limb ontogenesis; in addition, three TBX6-related Dorsocross genes are required for epithelial remodeling during wing development , but their role in virus regulation needs to be further verified.
We then performed a PPI analysis of four DEGs (PEM, M1Pi, TOLL7, and TBX6), as shown in Fig. 6. PEM, M1Pi and TOLL7 are linked by a series of genes, and PEM and M1Pi are linked by cell division cycle 20 (CDC20, AAEL014025) and cell division cycle protein 23 homolog (CDC23, AAEL003273), which are two genes that control mitosis and the cell cycle. TOLL7 interacts with other genes through baculoviral IAP repeat-containing protein 7 (IAP2, AAEL006633), which is involved in regulating the inhibition of apoptosis, and then through mitotic spindle assembly checkpoint protein MAD2A (AAEL008316) and mitotic checkpoint serine/threonine protein kinases bub1 and bubr1 (AAEL011596), which are involved in cell mitosis. The genes interacting with TOLL7, including MYD, IMD, PELLE, TUBE, FADD, and TOLL6, participate in the Toll and Imd signaling pathways. According to the results, these genes and their interacting genes are mainly involved in processes such as metabolism, biosynthesis, and cell development, and this finding is consistent with the results from the above-described analyses of the DEGs. Among the DEGs, ubiq uitin-mediated proteolysis, which was the most significantly enriched pathway, plays wide range of roles in basic cellular processes, including the cell cycle, development and differentiation, the regulation of immune and inflammatory responses, and the control of signal transduction pathways . TBX6 mainly interacted with the homeobox gene, which was first discovered to be the main gene in Drosophila melanogaster that controls development and plays a key role in organogenesis and the regulation of cell proliferation and differentiation . These results further confirm that the Toll and Imd signaling pathways play an important role in the regulation of DENV-2-infected Aag2 cells and that genes and pathways involved in regulating the cell cycle, proliferation differentiation, and apoptosis also act synergistically in this process.