Yuemei Dong, Shengzhang Dong, Nahid BorhaniDizaji, Natalie Rutkowski, Tyler Pohlenz, Kevin Myles, George Dimopoulos
Abstract
The mosquito’s innate immune system defends against a variety of pathogens, and the conserved siRNA pathway plays a central role in the control of viral infections. Here, we show that transgenic overexpression of Dicer2 (Dcr2) or R2d2 resulted in an accumulation of 21-nucleotide viral sequences that was accompanied by a significant suppression of dengue virus (DENV), Zika virus (ZIKV), and chikungunya virus (CHIKV) replication, thus indicating the broad-spectrum antiviral response mediated by the siRNA pathway that can be applied for the development of novel arbovirus control strategies. Interestingly, overexpression of Dcr2 or R2d2 regulated the mRNA abundance of a variety of antimicrobial immune genes, pointing to additional functions of DCR2 and R2D2 as well as cross-talk between the siRNA pathway and other immune pathways. Accordingly, transgenic overexpression of Dcr2 or R2d2 resulted in a lesser proliferation of the midgutmicrobiota and increased resistance to bacterial and fungal infections.
Introduction
Mosquito-transmitted arboviruses are increasingly causing reemergent epidemic disease worldwide, despite significant efforts to control their vectors over the past few decades. Dengue remains the major vector-borne viral disease and is responsible for over 390 million infections per year [1,2], and the Zika epidemics in 2015 and 2016 resulted in a tremendous public health burden. The lack of specific drugs and limited availability of a partially effective vaccine has accentuated the need for the development of novel control strategies for arbovirus transmission.
Materials and methods
Ethics statement
This study has been carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. Mice were used according to the approved animal protocol for rearing mosquitoes as a blood source for the maintenance of the mosquito colonies. The protocol (permit # MO15H144) was approved by the Animal Care and Use Committee of the Johns Hopkins University. Commercially obtained anonymous human blood type O+ and untyped human serum (Interstate Blood Bank, Inc.) were used for DENV and ZIKV infection assays in mosquitoes, and informed consent was therefore not required. Defibrinated sheep’s blood (Colorado Serum) was used for mosquito oral infection with CHIKV.
Results
Generation of blood meal–inducible Dcr2- and R2d2-expressing transgenic Ae. aegypti
Studies have demonstrated the production of DENV-derived siRNAs and piwi-interacting RNAs in virally infected mosquitoes, suggesting that the RNA interference (RNAi) machinery is potent in controlling viral infection and replication (Fig 1A) [12,14]. In the siRNA immune pathway, both DCR2 and R2D2 are key players in assuring proper RNA recognition and activation of this pathway.
Discussion
Although the function of the siRNA pathway has been studied in cultured cells, several published studies have linked the siRNA pathway to the restriction of DENV in Ae. aegypti mosquitoes, but the mechanism of siRNA-mediated viral restriction, and the translational potential of the control remain poorly understood. In the present study, overexpression of Dcr2 and R2d2 in the midgut through a blood meal–inducible promoter resulted in significant suppression of 3 human viral pathogens, DENV2, ZIKV, and CHIKV, both in the midgut tissue and systemically, with significant reduction of virus dissemination to the salivary glands. These results suggest that both DCR2 and R2D2 are restriction factors of the siRNA pathway, mediating an antiviral response that is important in controlling these viral infections. Furthermore, the broad-spectrum activity of this pathway against multiple viruses underscores its translational utility for disease control.
Citation: Dong Y, Dong S, Dizaji NB, Rutkowski N, Pohlenz T, Myles K, et al. (2022) The Aedesaegypti siRNA pathway mediates broad-spectrum defense against human pathogenic viruses and modulates antibacterial and antifungal defenses. PLoSBiol 20(6): e3001668. https://doi.org/10.1371/journal.pbio.3001668
Academic Editor: Ronald P. Van Rij, Radboud University Medical Center, NETHERLANDS
Received: July 19, 2021; Accepted: May 11, 2022; Published: June 9, 2022
Copyright: © 2022 Dong et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: All data are available in the main text or the supplementary materials. All of the RNA-seq data sets that were used in this study are available in the supplementary data or the National Center for Biotechnology Information (NCBI) Sequence Read Archive (PRJNA838236).
Funding: This work was supported by the National Institutes of Health / National Institute of Allergy and Infectious Disease R01AI141532 (to GD and KM) and the Bloomberg Philanthropies (to GD). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Abbreviations: AMP, antimicrobial peptide; CHIKV, chikungunya virus; CPE, cytopathic effect; CFU, colony-forming unit; DEG, differentially expressed gene; DENV, dengue virus; DENV2, DENV serotype 2; dpi, days post-infection; dsRBP, double-stranded RNA binding protein; dsRNA, double-stranded RNA; FBS, fetal bovine serum; gDNA, genomic DNA; GO, gene ontology; IFA, immunofluorescence microscopy assay; miRNA, microRNA; MOI, multiplicity of infection; PAMP, pathogen-associated molecular pattern; PBM, post-blood meal; PFU, plaque-forming unit; PIBM, post-infectious blood meal; PRR, pattern recognition receptor; qRT-PCR, quantitative real-time PCR; RISC, RNA-induced-silencing-complex; siRNA, small interfering RNA; WT, wild type; ZIKV, Zika virus
https://journals.plos.org/plosbiology/article?id=10.1371/journal.pbio.3001668#abstract0
