Journal: Scientific Reports
Article Title: Transgenic refractory Aedes aegypti lines are resistant to multiple serotypes of dengue virus
doi: 10.1038/s41598-021-03229-4
Figure Lengend Snippet: Sequence of the designed antiviral miRNAs was based on DENV consensus genomic sequence. ( a ) DENV target genes. Through the mariner ( Mos1 ) transposase, ubiquitous and midgut-specific induction promoters drive the expression of a polycistronic cluster of eight synthetic small RNAs that were engineered to target conserved genes in the DENV genome. A genomic integration system was used to produce a series of transgenic mosquitoes that can express antiviral miRNAs. The DENV-8miR plasmid contains eight miRNAs: miR_8-1 targeting the 5′UTR, miR_8-7 targeting the 3′UTR, and miR_8-2 to miR_8-6 and miR_8-8 targeting the capsid, as indicated with colored circles. ( b ) The binding site coverage and similarity are presented in the table. Five types of miRNAs for DENV-1 (Myanmar strain), two types of miRNAs for DENV-2 (NGC strain), five types of miRNAs for DENV-3 (98TW503 strain), and two types of miRNAs for DENV-4 (H241 strain) were complementary targets to DENV. The viral genome sequences of each serotype were downloaded from the Viral Bioinformatic Resources Center ( http://athena.bioc.uvic.ca/ ) and then aligned using CLC bio-Genomics software version 9.5.3 (CLC Bio, https://www.qiagenbioinformatics.com/ ). ( c ) Schematic of anti-DENV miRNA production. Schematic of the anti-DENV transgene, consisting of an AeCPA / AePUB promoter that drives expression of the polycistronic cluster of eight synthetic small RNAs that were engineered to target conserved genes in the DENV genome. ( d ) Screening of transgenic mosquitoes under fluorescence microscopy. The EGFP reporter driven by the 3 × P3 promoter was expressed in the eyes or anal papillae of transgenic larvae lines. Examples of EGFP reporter expression in larval and adult stages under natural (left) and blue light (right) conditions. Arrows indicate eyes and asterisks indicate anal papillae regions with EGFP expression.
Article Snippet: In order to design artificial miRNAs targeting the viral genomic RNA of the four serotypes (1, 2, 3, and 4) of DENV, the viral genome sequences of each serotype were downloaded from the Viral Bioinformatic Resources Center ( http://athena.bioc.uvic.ca/ ) and then aligned using CLC bio-Genomics software version 9.5.3 (CLC Bio, QIAGEN, https://www.qiagenbioinformatics.com/ ).
Techniques: Sequencing, Expressing, Transgenic Assay, Plasmid Preparation, Binding Assay, Software, Fluorescence, Microscopy
