Screening And Function Study Of Related Genes Susceptible To DENV2 In Aedes Aegypti

Mosquitoes,as an important vector of pathogens,spread a variety of vector-borne infectious diseases such as malaria,dengue fever,yellow fever,and Zika,posing a huge threat to global public health.As the main transmission vector of dengue virus,Aedes mosquito has the characteristics of wide distribution and high transmission efficiency.Dengue virus infection can cause dengue fever,and even more serious diseases such as dengue hemorrhagic fever and dengue shock syndrome.Currently,there are no effective vaccines and antiviral drugs,and the prevention and treatment of infection with this virus is more difficult than those of other viral infections because the risk of secondary infection is caused by enhanced antibody-mediated immunity.The research on the susceptibility mechanism of mosquitoes to viruses can not only clarify the molecular biological mechanism of vector effectiveness and study the immune mechanism of mosquito-borne virus infection,but also provide a theoretical basis for targeted regulation and even blocking of virus replication and transmission in mosquitoes.The research of this subject first used Ae.aegypti Aag2 cell line and Ae.albopictus C6/36 cell line as a model for studying mosquitoes with virus infection.RNA-Seq technology was used to identify the transcripts of Aag2 and C6/36 cells before and after infection with DENV2.Then through bioinformatics analysis,describe the biological function of its DEGs.After that,qRT-PCR was used to verify the sequencing data.In combination with the transcriptome results of the midgut and salivary glands of Ae.aegypti infected with DENV2 in our laboratory,some DEGs were selected to construct an overexpression model in the Aag2 cell line to study the interaction with DENV2 infection.Subsequently,the expression levels of the genes that affect DENV2 replication in the above results were re-verified in infected and uninfected Aedes aegypti individuals and tissue samples.The main results of this research include:1.Transcriptome sequencing analysis of Aedes aegypti Aag2 cells infected with DENV2 identified a total of 1199 DEGs,of which 185 were upregulated and 1014 were downregulated.Bioinformatics analysis showed that the upregulated DEGs were enriched in 39 pathways,mainly involved in DNA replication,longevity regulating pathway,FoxO signaling pathway,pyrimidine,retinol,drugs,starch and sucrose metabolism pathways.The downregulated DEGs were enriched in 41 pathways,mainly involved in longevity regulating pathway,circadian rhythm,ABC transporter,light transduction,MAPK signaling pathway,purine,tyrosine metabolism and other pathways.The qRT-PCR verification results of 12 DEGs showed that the FC range was 1.05-1.43,and 3 genes was upregulated after infection was statistically significant(P<0.05).2.Transcriptome sequencing analysis of Aedes albopictus C6/36 cells infected with DENV2 identified a total of 1239 DEGs,of which 1133 were upregulated and 106 were downregulated.Bioinformatics analysis showed that the upregulated DEGs were enriched in 82 pathways,mainly involved in a variety of signaling pathways,including MAPK,Hippo,FoxO,Wnt,mTOR and Notch signaling pathways;glycerophospholipids,Cysteine and methionine metabolic pathways;cellular physiological processes,including autophagy,endocytosis,and apoptosis;and other processes,such as circadian rhythms,mRNA surveillance pathway,mitochondria,RNA degradation,ubiquitin-mediated proteolysis,and back Abdominal axis formation and so on.The downregulated DEGs were enriched in 24 pathways,mainly involved in DNA replication,pyrimidine metabolism,base excision repair,nucleotide excision repair,mismatch repair,drug metabolism and folic acid biosynthesis.The qRT-PCR verification results of 13 DEGs showed that the FC range was 0.84-6.41,and 8 genes was upregulated after infection was statistically significant(P<0.05).3.The comparative analysis of the transcriptome data of Aag2 and C6/36 cells showed that 16 DEGs overlapped.The GO enrichment analysis of upregulated DEGs shows that there are 7 overlapping terms,mainly including the phosphate-containing compound and phosphorus metabolic processes;nucleobase containing compound,aromatic compound,heterocycle,the organic cyclic compound biosynthetic process and DNA binding.The GO enrichment results of downregulated DEGs contained 65 overlapping terms,mainly involved in aromatic compound,nucleobase-containing compound,cellular nitrogen compound,heterocycle,organic ring compound,organic substance catabolic process and so on.According to the results of KEGG enrichment,the upregulated DEGs were enriched into 23 overlapping pathways,including pyrimidine metabolism,longevity regulation pathway,drug metabolism,starch and sucrose metabolism,FoxO signaling pathway,circadian rhythm and so on.The downregulated DEGs were enriched into 8 overlapping pathways,which are involved in drug metabolism,folic acid biosynthesis,Toll and Imd signaling pathways,valine,leucine and isoleucine degradation,spliceosome,mRNA surveillance pathway,purine metabolism and RNA transport.4.According to the transcriptome and qRT-PCR verification results,24 DEGs were selected to construct an overexpression model,and Aag2 cells after gene overexpression were infected with DENV2.Among them,the expression levels of 19 genes had statistically significant differences in virus copies after gene overexpression(P<0.05),which directly indicate that the expression of these 19 genes affects the replication of DENV2 in mosquito s and changes the viral load.At 2dpi,the DENV2 RNA copies after overexpression of 9 target genes was statistically significant compared with the overexpression EGFP control group,and the FC was between 0.971-1.345.At 4dpi,the DENV2 RNA copies was statistically significant after 10 genes were overexpressed,and the FC was between 0.657 and 1.574.At 6dpi,there were also statistically significant differences in DENV2 RNA copies after 9 genes were overexpressed,and the FC was between 0.551-1.095.The expression levels of these 19 genes were verified in Aedes aegypti mosquitoes,midgut,salivary glands,and ovaries infected and uninfected with DENV2,respectively.The differences in expression levels of these 19 genes were statistically(P<0.05)distributed among different tissues,indicating that there is a certain interaction between these genes and virus replication.This article is the first to study the changes in the transcription levels of Aedes aegypti Aag2 cells and Aedes albopictus C6/36 cells infected with DENV2.It provides a faster and more effective method for mining pathogen-susceptible genes of Aedes mosquitoes.At the same time,it provides a research direction for further research on the complex mechanism of vector-pathogen interaction.Through the construction of related gene overexpression models in Aag2 cells and the detection of gene expression levels in Aedes aegypti infected with DENV2,the interaction between mosquito genes and DENV2 infection was studied,and some target genes involved in viral infection related processes were discovered.It provides a theoretical basis for the targeted regulation of mosquito-borne virus infection and transmission.