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      Wolbachia-Based Dengue Virus Inhibition Is Not Tissue-Specific in Aedes aegypti

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      PLoS Neglected Tropical Diseases
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          Abstract

          Background

          Dengue fever, caused by the dengue virus (DENV), is now the most common arbovirus transmitted disease globally. One novel approach to control DENV is to use the endosymbiotic bacterium, Wolbachia pipientis, to limit DENV replication inside the primary mosquito vector, Aedes aegypti. Wolbachia that is naturally present in a range of insects reduces the capacity for viruses, bacteria, parasites and fungi to replicate inside insects. Wolbachia’s mode of action is not well understood but may involve components of immune activation or competition with pathogens for limited host resources. The strength of Wolbachia-based anti DENV effects appear to correlate with bacterial density in the whole insect and in cell culture. Here we aimed to determine whether particular tissues, especially those with high Wolbachia densities or immune activity, play a greater role in mediating the anti DENV effect.

          Methodology/findings

          Ae. aegypti mosquito lines with and without Wolbachia (Wildtype) were orally fed DENV 3 and their viral loads subsequently measured over two time points post infection in the midgut, head, salivary glands, Malpighian tubules, fat body and carcass. We did not find correlations between Wolbachia densities and DENV loads in any tissue, nor with DENV loads in salivary glands, the endpoint of infection. This is in contrast with strong positive correlations between DENV loads in a range of tissues and salivary gland loads for Wildtype mosquitoes. Lastly, there was no evidence of a heightened role for tissues with known immune function including the fat body and the Malpighian tubules in Wolbachia’s limitation of DENV.

          Conclusion/significance

          We conclude that the efficacy of DENV blocking in Wolbachia infected mosquitoes is not reliant on any particular tissue. This work therefore suggests that the mechanism of Wolbachia-based antiviral effects is either systemic or acts locally via processes that are fundamental to diverse cell types. We further conclude that the relationship between DENV blocking and Wolbachia density is not linear in mosquito tissues

          Author Summary

          Dengue fever caused by the dengue virus (DENV) is transmitted by the mosquito, Aedes aegypti. To control the disease, an intracellular bacterium called Wolbachia has been introduced into Ae. aegypti where it blocks/limits success of infection of DENV. The mechanistic basis of blocking is not well understood but may involve Wolbachia activating the host immune system or competing with DENV for host resources. The strength of blocking appears to correlate with Wolbachia density. Here, we aimed to determine if any particular tissues inside the mosquito play a greater role in blocking. Tissues were chosen based on their Wolbachia density and their roles in infection and immunity. Wolbachia infected and uninfected mosquitoes were orally infected with DENV and Wolbachia density and DENV load were assessed in midgut, salivary gland, head, Malpighian tubules, fat body and carcass. Wolbachia density did not correlate with DENV loads in the same tissues nor with DENV loads in the salivary glands. We also showed that no one tissue appeared to play a greater role in blocking. In summary, these finding suggest that in the mosquito a threshold Wolbachia density may be required for DENV blocking. Our findings also suggest that blocking may involve mechanisms that are fundamental to all cells.

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          Most cited references33

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          Housekeeping genes as internal standards: use and limits.

          Quantitative studies are commonly realised in the biomedical research to compare RNA expression in different experimental or clinical conditions. These quantifications are performed through their comparison to the expression of the housekeeping gene transcripts like glyceraldehyde-3-phosphate dehydrogenase (G3PDH), albumin, actins, tubulins, cyclophilin, hypoxantine phsophoribosyltransferase (HRPT), L32. 28S, and 18S rRNAs are also used as internal standards. In this paper, it is recalled that the commonly used internal standards can quantitatively vary in response to various factors. Possible variations are illustrated using three experimental examples. Preferred types of internal standards are then proposed for each of these samples and thereafter the general procedure concerning the choice of an internal standard and the way to manage its uses are discussed.
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            Dengue virus type 2: replication and tropisms in orally infected Aedes aegypti mosquitoes

            Background To be transmitted by its mosquito vector, dengue virus (DENV) must infect midgut epithelial cells, replicate and disseminate into the hemocoel, and finally infect the salivary glands, which is essential for transmission. The extrinsic incubation period (EIP) is very relevant epidemiologically and is the time required from the ingestion of virus until it can be transmitted to the next vertebrate host. The EIP is conditioned by the kinetics and tropisms of virus replication in its vector. Here we document the virogenesis of DENV-2 in newly-colonized Aedes aegypti mosquitoes from Chetumal, Mexico in order to understand better the effect of vector-virus interactions on dengue transmission. Results After ingestion of DENV-2, midgut infections in Chetumal mosquitoes were characterized by a peak in virus titers between 7 and 10 days post-infection (dpi). The amount of viral antigen and viral titers in the midgut then declined, but viral RNA levels remained stable. The presence of DENV-2 antigen in the trachea was positively correlated with virus dissemination from the midgut. DENV-2 antigen was found in salivary gland tissue in more than a third of mosquitoes at 4 dpi. Unlike in the midgut, the amount of viral antigen (as well as the percent of infected salivary glands) increased with time. DENV-2 antigen also accumulated and increased in neural tissue throughout the EIP. DENV-2 antigen was detected in multiple tissues of the vector, but unlike some other arboviruses, was not detected in muscle. Conclusion Our results suggest that the EIP of DENV-2 in its vector may be shorter that the previously reported and that the tracheal system may facilitate DENV-2 dissemination from the midgut. Mosquito organs (e.g. midgut, neural tissue, and salivary glands) differed in their response to DENV-2 infection.
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              Wolbachia invades Anopheles stephensi populations and induces refractoriness to Plasmodium infection.

              Wolbachia is a maternally transmitted symbiotic bacterium of insects that has been proposed as a potential agent for the control of insect-transmitted diseases. One of the major limitations preventing the development of Wolbachia for malaria control has been the inability to establish inherited infections of Wolbachia in anopheline mosquitoes. Here, we report the establishment of a stable Wolbachia infection in an important malaria vector, Anopheles stephensi. In A. stephensi, Wolbachia strain wAlbB displays both perfect maternal transmission and the ability to induce high levels of cytoplasmic incompatibility. Seeding of naturally uninfected A. stephensi populations with infected females repeatedly resulted in Wolbachia invasion of laboratory mosquito populations. Furthermore, wAlbB conferred resistance in the mosquito to the human malaria parasite Plasmodium falciparum.
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                Author and article information

                Contributors
                Role: Editor
                Journal
                PLoS Negl Trop Dis
                PLoS Negl Trop Dis
                plos
                plosntds
                PLoS Neglected Tropical Diseases
                Public Library of Science (San Francisco, CA USA )
                1935-2727
                1935-2735
                17 November 2016
                November 2016
                : 10
                : 11
                : e0005145
                Affiliations
                [001]School of Biological Sciences, Monash University, Clayton, Victoria, Australia
                Colorado State University, UNITED STATES
                Author notes

                The authors have declared that no competing interests exist.

                • Conceptualization: EAM.

                • Formal analysis: EAM HEA.

                • Funding acquisition: EAM.

                • Investigation: HEA.

                • Methodology: EAM HEA.

                • Resources: EAM.

                • Supervision: EAM.

                • Validation: HEA.

                • Visualization: HEA.

                • Writing – original draft: HEA.

                • Writing – review & editing: EAM HEA.

                Article
                PNTD-D-16-01583
                10.1371/journal.pntd.0005145
                5113870
                27855218
                bd06010e-6a7f-4e0e-93b8-d0e691532b53
                © 2016 Amuzu, McGraw

                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.

                History
                : 25 August 2016
                : 27 October 2016
                Page count
                Figures: 7, Tables: 0, Pages: 18
                Funding
                Funded by: funder-id http://dx.doi.org/10.13039/501100000925, National Health and Medical Research Council;
                Award ID: APP1020607
                Award Recipient :
                This research was supported by the National Health and Medical Research Council of Australia through a project (APP1020607) and a program (1037003) grant to EAM. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
                Categories
                Research Article
                Biology and Life Sciences
                Organisms
                Bacteria
                Wolbachia
                Medicine and Health Sciences
                Epidemiology
                Disease Vectors
                Insect Vectors
                Mosquitoes
                Biology and Life Sciences
                Organisms
                Animals
                Invertebrates
                Arthropoda
                Insects
                Mosquitoes
                Biology and Life Sciences
                Anatomy
                Digestive System
                Salivary Glands
                Medicine and Health Sciences
                Anatomy
                Digestive System
                Salivary Glands
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                Anatomy
                Exocrine Glands
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                Medicine and Health Sciences
                Anatomy
                Exocrine Glands
                Salivary Glands
                Biology and Life Sciences
                Biochemistry
                Lipids
                Fats
                Research and analysis methods
                Extraction techniques
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                Medicine and Health Sciences
                Epidemiology
                Disease Vectors
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                Mosquitoes
                Aedes Aegypti
                Biology and Life Sciences
                Organisms
                Animals
                Invertebrates
                Arthropoda
                Insects
                Mosquitoes
                Aedes Aegypti
                Medicine and Health Sciences
                Infectious Diseases
                Infectious Disease Control
                Biology and life sciences
                Organisms
                Viruses
                RNA viruses
                Flaviviruses
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                Microbiology
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                Microbial Pathogens
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                Medicine and Health Sciences
                Pathology and Laboratory Medicine
                Pathogens
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                Biology and Life Sciences
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                Custom metadata
                All raw data files are available from the Figshare database (DOI 10.4225/03/57BFAE2B85A57).

                Infectious disease & Microbiology
                Infectious disease & Microbiology

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