Publications by authors named "Gerard Terradas"

9 Publications

  • Page 1 of 1

Inherently confinable split-drive systems in Drosophila.

Nat Commun 2021 03 5;12(1):1480. Epub 2021 Mar 5.

Section of Cell and Developmental Biology, University of California, San Diego, La Jolla, CA, USA.

CRISPR-based gene-drive systems, which copy themselves via gene conversion mediated by the homology-directed repair (HDR) pathway, have the potential to revolutionize vector control. However, mutant alleles generated by the competing non-homologous end-joining (NHEJ) pathway, resistant to Cas9 cleavage, can interrupt the spread of gene-drive elements. We hypothesized that drives targeting genes essential for viability or reproduction also carrying recoded sequences that restore endogenous gene functionality should benefit from dominantly-acting maternal clearance of NHEJ alleles combined with recessive Mendelian culling processes. Here, we test split gene-drive (sGD) systems in Drosophila melanogaster that are inserted into essential genes required for viability (rab5, rab11, prosalpha2) or fertility (spo11). In single generation crosses, sGDs copy with variable efficiencies and display sex-biased transmission. In multigenerational cage trials, sGDs follow distinct drive trajectories reflecting their differential tendencies to induce target chromosome damage and/or lethal/sterile mosaic Cas9-dependent phenotypes, leading to inherently confinable drive outcomes.
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http://dx.doi.org/10.1038/s41467-021-21771-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7935863PMC
March 2021

Efficient population modification gene-drive rescue system in the malaria mosquito Anopheles stephensi.

Nat Commun 2020 11 3;11(1):5553. Epub 2020 Nov 3.

Department of Microbiology & Molecular Genetics, University of California, Irvine, CA, 92697-3900, USA.

Cas9/gRNA-mediated gene-drive systems have advanced development of genetic technologies for controlling vector-borne pathogen transmission. These technologies include population suppression approaches, genetic analogs of insecticidal techniques that reduce the number of insect vectors, and population modification (replacement/alteration) approaches, which interfere with competence to transmit pathogens. Here, we develop a recoded gene-drive rescue system for population modification of the malaria vector, Anopheles stephensi, that relieves the load in females caused by integration of the drive into the kynurenine hydroxylase gene by rescuing its function. Non-functional resistant alleles are eliminated via a dominantly-acting maternal effect combined with slower-acting standard negative selection, and rare functional resistant alleles do not prevent drive invasion. Small cage trials show that single releases of gene-drive males robustly result in efficient population modification with ≥95% of mosquitoes carrying the drive within 5-11 generations over a range of initial release ratios.
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http://dx.doi.org/10.1038/s41467-020-19426-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7609566PMC
November 2020

Translating gene drive science to promote linguistic diversity in community and stakeholder engagement.

Glob Public Health 2020 10 26;15(10):1551-1565. Epub 2020 Jun 26.

The Qualcomm Institute, Calit2, UC San Diego, La Jolla, CA, USA.

Information about genetic engineering (GE) for vector control in the United States is disseminated primarily in English, though non-English speakers are equally, and in some geographic regions even more affected by such technologies. Non-English-speaking publics should have equal access to such information, which is especially critical when the technology in question may impact whole communities. We convened an interdisciplinary workgroup to translate previously developed narrated slideshows on gene drive mosquitoes from English into Spanish, reviewing each iteration for scientific accuracy and accessibility to laypeople. Using the finalised stimuli, we conducted five online, chat-based focus groups with Spanish-speaking adults from California. Overall, participants expressed interest in the topic and were able to summarise the information presented in their own words. Importantly, participants asked for clarification and expressed scepticism about the information presented, indicating critical engagement with the material. Through collaboration with Spanish-speaking scientists engaged in the development of GE methods of vector control, we translated highly technical scientific information into Spanish that successfully engaged Spanish-speaking participants in conversations about this topic. In this manuscript, we document the feasibility of consulting Spanish-speaking publics about a complex emerging technology by drawing on the linguistic diversity of the scientific teams developing the technology.
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http://dx.doi.org/10.1080/17441692.2020.1779328DOI Listing
October 2020

Using genetic variation in Aedes aegypti to identify candidate anti-dengue virus genes.

BMC Infect Dis 2019 Jul 4;19(1):580. Epub 2019 Jul 4.

School of Biological Sciences, Monash University, Melbourne, Victoria, 3800, Australia.

Background: Transcriptomic profiling has generated extensive lists of genes that respond to viral infection in mosquitoes. These gene lists contain two types of genes; (1) those that are responsible for the insect's natural antiviral defense mechanisms, including some known innate immunity genes, and (2) genes whose change in expression may occur simply as a result of infection. As genetic modification tools for mosquitoes continue to improve, the opportunities to make refractory insects via allelic replacement or delivery of small RNAs that alter gene expression are expanding. Therefore, the ability to identify which genes in transcriptional profiles may have immune function has increasing value. Arboviruses encounter a range of mosquito tissues and physiologies as they traverse from the midgut to the salivary glands. While the midgut is well-studied as the primary tissue barrier, antiviral genes expressed in the subsequent tissues of the carcass offer additional candidates for second stage intervention in the mosquito body.

Methods: Mosquito lines collected recently from field populations exhibit natural genetic variation for dengue virus susceptibility. We sought to use a modified full-sib breeding design to identify mosquito families that varied in their dengue viral load in their bodies post infection.

Results: By delivering virus intrathoracically, we bypassed the midgut and focused on whole body responses in order to evaluate carcass-associated refractoriness. We tested 25 candidate genes selected for their appearance in multiple published transcriptional profiles and were able to identify 12 whose expression varied with susceptibility in the genetic families.

Conclusions: This method, using natural genetic variation, offers a simple means to screen and reduce candidate gene lists prior to carrying out more labor-intensive functional studies. The extracted RNA from the females across the families represents a storable resource that can be used to screen subsequent candidate genes in the future. The aspect of vector competence being assessed could be varied by focusing on different tissues or time points post infection.
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http://dx.doi.org/10.1186/s12879-019-4212-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6611004PMC
July 2019

A Role for the Insulin Receptor in the Suppression of Dengue Virus and Zika Virus in Wolbachia-Infected Mosquito Cells.

Cell Rep 2019 01;26(3):529-535.e3

Infection and Immunity Program, Monash Biomedicine Discovery Institute and Department of Microbiology, Monash University, Clayton, VIC 3800, Australia. Electronic address:

Wolbachia-infected mosquitoes are refractory to super-infection with arthropod-borne pathogens, but the role of host cell signaling proteins in pathogen-blocking mechanisms remains to be elucidated. Here, we use an antibody microarray approach to provide a comprehensive picture of the signaling response of Aedes aegypti-derived cells to Wolbachia. This approach identifies the host cell insulin receptor as being downregulated by the bacterium. Furthermore, siRNA-mediated knockdown and treatment with a small-molecule inhibitor of the insulin receptor kinase concur to assign a crucial role for this enzyme in the replication of dengue and Zika viruses in cultured mosquito cells. Finally, we show that the production of Zika virus in Wolbachia-free live mosquitoes is impaired by treatment with the selective inhibitor mimicking Wolbachia infection. This study identifies Wolbachia-mediated downregulation of insulin receptor kinase activity as a mechanism contributing to the blocking of super-infection by arboviruses.
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http://dx.doi.org/10.1016/j.celrep.2018.12.068DOI Listing
January 2019

Family level variation in Wolbachia-mediated dengue virus blocking in Aedes aegypti.

Parasit Vectors 2017 12 28;10(1):622. Epub 2017 Dec 28.

School of Biological Sciences, Monash University, Clayton, Melbourne, VIC, Australia.

Background: The mosquito vector Aedes aegypti is responsible for transmitting a range of arboviruses including dengue (DENV) and Zika (ZIKV). The global reach of these viruses is increasing due to an expansion of the mosquito's geographic range and increasing urbanization and human travel. Vector control remains the primary means for limiting these diseases. Wolbachia pipientis is an endosymbiotic bacterium of insects that has the ability to block the replication of pathogens, including flaviviruses such as DENV or ZIKV, inside the body of the vector. A strain of Wolbachia called wMel is currently being released into wild mosquito populations to test its potential to limit virus transmission to humans. The mechanism that underpins the virus blocking effect, however, remains elusive.

Methods: We used a modified full-sib breeding design in conjunction with vector competence assays in wildtype and wMel-infected Aedes aegypti collected from the field. All individuals were injected with DENV-2 intrathoracically at 5-6 days of age. Tissues were dissected 7 days post-infection to allow quantification of DENV and Wolbachia loads.

Results: We show the first evidence of family level variation in Wolbachia-mediated blocking in mosquitoes. This variation may stem from either genetic contributions from the mosquito and Wolbachia genomes or environmental influences on Wolbachia. In these families, we also tested for correlations between strength of blocking and expression level for several insect immunity genes with possible roles in blocking, identifying two genes of interest (AGO2 and SCP-2).

Conclusions: In this study we show variation in Wolbachia-mediated DENV blocking in Aedes aegypti that may arise from genetic contributions and environmental influences on the mosquito-Wolbachia association. This suggests that Wolbachia-mediated blocking may have the ability to evolve through time or be expressed differentially across environments. The long-term efficacy of Wolbachia in the field will be dependent on the stability of blocking. Understanding the mechanism of blocking will be necessary for successful development of strategies that counter the emergence of evolved resistance or variation in its expression under diverse field conditions.
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http://dx.doi.org/10.1186/s13071-017-2589-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5746003PMC
December 2017

Wolbachia-mediated virus blocking in the mosquito vector Aedes aegypti.

Curr Opin Insect Sci 2017 08 10;22:37-44. Epub 2017 May 10.

School of Biological Sciences, Monash University, Clayton VIC 3800, Melbourne, Australia. Electronic address:

Viruses transmitted by mosquitoes such as dengue, Zika and West Nile cause a threat to global health due to increased geographical range and frequency of outbreaks. The bacterium Wolbachia pipientis may be the solution reducing disease transmission. Though commonly missing in vector species, the bacterium was artificially and stably introduced into Aedes aegypti to assess its potential for biocontrol. When infected with Wolbachia, mosquitoes become refractory to infection by a range of pathogens, including the aforementioned viruses. How the bacterium is conferring this phenotype remains unknown. Here we discuss current hypotheses in the field for the mechanistic basis of pathogen blocking and evaluate the evidence from mosquitoes and related insects.
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http://dx.doi.org/10.1016/j.cois.2017.05.005DOI Listing
August 2017

The RNAi pathway plays a small part in Wolbachia-mediated blocking of dengue virus in mosquito cells.

Sci Rep 2017 03 6;7:43847. Epub 2017 Mar 6.

School of Biological Sciences, Monash University, Clayton VIC 3800, Melbourne, Australia.

Wolbachia pipientis is an insect endosymbiont known to limit the replication of viruses including dengue and Zika in their primary mosquito vector, Aedes aegypti. Wolbachia is being released into mosquito populations globally in a bid to control the diseases caused by these viruses. It is theorized that Wolbachia's priming of the insect immune system may confer protection against subsequent viral infection. Other hypotheses posit a role for competition between Wolbachia and viruses for host cellular resources. Using an A. aegypti cell line infected with Wolbachia, we tested the effects of targeting siRNAs against the major innate immune pathways on dengue virus loads. We show that while Wolbachia infection induces genes in the Toll, JAK/STAT and RNAi pathways, only reduced expression of RNAi leads to a rebound of dengue virus loads in Wolbachia-infected cells. The magnitude of the effect explained less than 10% of the total DENV load, demonstrating that blocking must be dependent on other factors in addition to the expression of RNAi. The findings bode well for the long-term stability of blocking given that immunity gene expression would likely be highly plastic and susceptible to rapid evolution.
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http://dx.doi.org/10.1038/srep43847DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5338330PMC
March 2017

SAMHD1 specifically affects the antiviral potency of thymidine analog HIV reverse transcriptase inhibitors.

Antimicrob Agents Chemother 2014 Aug 9;58(8):4804-13. Epub 2014 Jun 9.

AIDS Research Institute-IrsiCaixa, Hospital Germans Trias i Pujol, Universitat Autònoma de Barcelona, Badalona, Spain

Sterile alpha motif and histidine-aspartic domain-containing protein 1 (SAMHD1) is a deoxynucleoside triphosphate (dNTP) triphosphohydrolase recently recognized as an antiviral factor that acts by depleting dNTP availability for viral reverse transcriptase (RT). SAMHD1 restriction is counteracted by the human immunodeficiency virus type 2 (HIV-2) accessory protein Vpx, which targets SAMHD1 for proteosomal degradation, resulting in an increased availability of dNTPs and consequently enhanced viral replication. Nucleoside reverse transcriptase inhibitors (NRTI), one of the most common agents used in antiretroviral therapy, compete with intracellular dNTPs as the substrate for viral RT. Consequently, SAMHD1 activity may be influencing NRTI efficacy in inhibiting viral replication. Here, a panel of different RT inhibitors was analyzed for their different antiviral efficacy depending on SAMHD1. Antiviral potency was measured for all the inhibitors in transformed cell lines and primary monocyte-derived macrophages and CD4(+) T cells infected with HIV-1 with or without Vpx. No changes in sensitivity to non-NRTI or the integrase inhibitor raltegravir were observed, but for NRTI, sensitivity significantly changed only in the case of the thymidine analogs (AZT and d4T). The addition of exogenous thymidine mimicked the change in viral sensitivity observed after Vpx-mediated SAMHD1 degradation, pointing toward a differential effect of SAMHD1 activity on thymidine. Accordingly, sensitivity to AZT was also reduced in CD4(+) T cells infected with HIV-2 compared to infection with the HIV-2ΔVpx strain. In conclusion, reduction of SAMHD1 levels significantly decreases HIV sensitivity to thymidine but not other nucleotide RT analog inhibitors in both macrophages and lymphocytes.
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http://dx.doi.org/10.1128/AAC.03145-14DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4136047PMC
August 2014
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