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      Evidence of vertical transmission of Zika virus in field-collected eggs of Aedes aegypti in the Brazilian Amazon

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          Abstract

          Background

          Arboviruses are viruses transmitted to humans and other animals by the bite of hematophagous arthropods. Infections caused by chikungunya virus (CHIKV), dengue virus (DENV), Zika virus (ZIKV), and the deadlier yellow fever virus (YFV) are current public health problems in several countries, mainly those located in tropical and subtropical regions. One of the main prevention strategies continues to be vector control, with the elimination of breeding sites and surveillance of infested areas. The use of ovitraps for Aedes mosquitos monitoring has already demonstrated promising results, and maybe be also useful for arboviral surveillance.

          Methods

          This work aimed to detect natural vertical transmission of arboviruses in Aedes aegypti and Aedes albopictus. Mosquito egg collection was carried out using ovitraps in Itacoatiara, a mid-size city in Amazonas state, Brazil. Collected eggs were allowed to hatch and larvae were tested for CHIKV, DENV, and ZIKV RNA by RT-qPCR.

          Results

          A total of 2,057 specimens (1,793 Ae. aegypti and 264 Ae. albopictus), in 154 larvae pools were processed. Results showed one positive pool for CHIKV and one positive pool for ZIKV. The active ZIKV infection was further confirmed by the detection of the negative-strand viral RNA and nucleotide sequencing which confirmed the Asian genotype. The Infection Rate per 1,000 mosquitoes tested was assessed by Maximum Likelihood Estimation (MLE) with 0.45 and 0.44 for CHIKV and ZIKV, respectively, and by Minimum Infection Rate (MIR) with 0.45 for both viruses.

          Conclusion

          To our knowledge, this is the first detection of ZIKV in natural vertical transmission in the Ae. aegypti, a fact that may contribute to ZIKV maintenance in nature during epidemics periods. Furthermore, our results highlight that the use of ovitraps and the molecular detection of arbovirus may contribute to health surveillance, directing the efforts to more efficient transmission blockade.

          Author summary

          The control of the vast majority of arbovirus infections relies on entomological measures to reduce mosquito infestation. Therefore, this study analyzed the use of ovitraps for arboviral surveillance in a mid-size city of the Amazonas state, Brazil. We found one larva pool infected with chikungunya virus, before the first human case confirmed in this municipality. Another pool was infected with Zika virus, demonstrating the first evidence that vertical transmission occurs in naturally infected Aedes aegypti mosquito populations.

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          Most cited references32

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          Purification of RNA using TRIzol (TRI reagent).

          TRIzol solubilization and extraction is a relatively recently developed general method for deproteinizing RNA. This method is particularly advantageous in situations where cells or tissues are enriched for endogenous RNases or when separation of cytoplasmic RNA from nuclear RNA is impractical. TRIzol (or TRI Reagent) is a monophasic solution of phenol and guanidinium isothiocyanate that simultaneously solubilizes biological material and denatures protein. After solubilization, the addition of chloroform causes phase separation (much like extraction with phenol:chloroform:isoamyl alcohol), where protein is extracted to the organic phase, DNA resolves at the interface, and RNA remains in the aqueous phase. Therefore, RNA, DNA, and protein can be purified from a single sample (hence, the name TRIzol). TRIzol extraction is also an effective method for isolating small RNAs, such as microRNAs, piwi-associated RNAs, or endogeneous, small interfering RNAs. However, TRIzol is expensive and RNA pellets can be difficult to resuspend. Thus, the use of TRIzol is not recommend when regular phenol extraction is practical.
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            First detection of natural infection of Aedes aegypti with Zika virus in Brazil and throughout South America

            Zika virus (ZIKV) has caused a major epidemic in Brazil and several other American countries. ZIKV is an arbovirus whose natural vectors during epidemics have been poorly determined. In this study, 1,683 mosquitoes collected in the vicinity of ZIKV suspected cases in Rio de Janeiro, Brazil, from June 2015 to May 2016 were screened for natural infection by using molecular methods. Three pools of Aedes aegypti were found with the ZIKV genome, one of which had only one male. This finding supports the occurrence of vertical and/or venereal transmission of ZIKV in Ae. aegypti in nature. None of the examined Ae. albopictus and Culex quinquefasciatus was positive. This is the first report of natural infection by ZIKV in mosquitoes in Brazil and other South American countries. So far, Ae. aegypti is the only confirmed vector of ZIKV during the ongoing Pan-American epidemics.
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              Vertical Transmission of Zika Virus in Aedes aegypti Mosquitoes

              Previous experimental studies have demonstrated that a number of mosquito-borne flavivirus pathogens are vertically transmitted in their insect vectors, providing a mechanism for these arboviruses to persist during adverse climatic conditions or in the absence of a susceptible vertebrate host. In this study, designed to test whether Zika virus (ZIKV) could be vertically transmitted, female Aedes aegypti and Aedes albopictus were injected with ZIKV, and their F1 adult progeny were tested for ZIKV infection. Six of 69 Ae. aegypti pools, comprised of a total of 1,738 F1 adults, yielded ZIKV upon culture, giving a minimum filial infection rate of 1:290. In contrast, none of 803 F1 Ae. albopictus adults (32 pools) yielded ZIKV. The MFIR for Ae. aegypti was comparable to MFIRs reported for other flaviviruses in mosquitoes, including dengue, Japanese encephalitis, yellow fever, West Nile, and St. Louis encephalitis viruses. The results suggest that vertical transmission may provide a potential mechanism for the virus to survive during adverse conditions.
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                Author and article information

                Contributors
                Role: ConceptualizationRole: Data curationRole: Formal analysisRole: InvestigationRole: MethodologyRole: Project administrationRole: ResourcesRole: SupervisionRole: ValidationRole: VisualizationRole: Writing – original draft
                Role: InvestigationRole: Methodology
                Role: InvestigationRole: Methodology
                Role: InvestigationRole: Methodology
                Role: InvestigationRole: Methodology
                Role: Formal analysis
                Role: ConceptualizationRole: Data curationRole: Formal analysisRole: InvestigationRole: MethodologyRole: Project administrationRole: ValidationRole: VisualizationRole: Writing – original draft
                Role: InvestigationRole: Methodology
                Role: ConceptualizationRole: Data curationRole: Formal analysisRole: Funding acquisitionRole: InvestigationRole: MethodologyRole: Project administrationRole: ResourcesRole: SupervisionRole: ValidationRole: VisualizationRole: Writing – original draft
                Role: ConceptualizationRole: Data curationRole: Formal analysisRole: Funding acquisitionRole: InvestigationRole: MethodologyRole: Project administrationRole: ResourcesRole: SupervisionRole: ValidationRole: VisualizationRole: Writing – original draft
                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
                16 July 2018
                July 2018
                : 12
                : 7
                : e0006594
                Affiliations
                [1 ] Health Surveillance Foundation of Amazonas State FVS, Department of Environmental Surveillance, Manaus, Amazonas, Brazil
                [2 ] Laboratório de Ecologia de Doenças Transmissíveis na Amazônia, Instituto Leônidas e Maria Deane – Fiocruz Amazônia, Manaus, Amazonas, Brazil
                [3 ] Programa de Iniciação Científica, Instituto Leônidas e Maria Deane – Fiocruz Amazônia, Manaus, Amazonas, Brazil
                [4 ] Programa de Pós-Graduação em Biologia Celular e Molecular, Instituto Oswaldo Cruz, Fiocruz, Rio de Janeiro, Brazil
                [5 ] Programa de Pós-Graduação em Imunologia Básica e Aplicada, Universidade Federal do Amazonas, Manaus, Amazonas, Brazil
                [6 ] Laboratory of Physiology and Control of Arthropod Vectors - Oswaldo Cruz Institute - FIOCRUZ, Rio de Janeiro, Brazil
                [7 ] Programa de Pós-Graduação em Biologia da Interação Patógeno-Hospedeiro, Instituto Leônidas e Maria Deane – Fiocruz Amazônia, Manaus, Amazonas, Brazil
                Independent Researcher, UNITED STATES
                Author notes

                The authors have declared that no competing interests exist.

                ‡ JBPL and FGN also contributed equally to this work.

                Author information
                http://orcid.org/0000-0002-5015-3019
                Article
                PNTD-D-17-01951
                10.1371/journal.pntd.0006594
                6075777
                30011278
                3fca67e8-5d5c-4143-864d-32edbb3dce18
                © 2018 Costa 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.

                History
                : 11 December 2017
                : 7 June 2018
                Page count
                Figures: 1, Tables: 1, Pages: 12
                Funding
                Funded by: funder-id http://dx.doi.org/10.13039/501100003593, Conselho Nacional de Desenvolvimento Científico e Tecnológico;
                Award ID: 440856/2016-7
                Award Recipient :
                Funded by: Fundação de Amparo à Pesquisa do Estado do Amazonas – FAPEAM
                Award ID: 001/2013 – PPSUS / 062.00656/2014
                Award Recipient :
                Funded by: Fundação de Amparo à Pesquisa do Estado do Amazonas – FAPEAM
                Award ID: 001/2014 – PROEP / 062.01939/2014
                Award Recipient :
                Funded by: Fundação de Amparo à Pesquisa do Estado do Amazonas – FAPEAM
                Award ID: visiting senior researcher fellow PVS PECTI-SAÚDE
                Award Recipient :
                Funded by: Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (BR)
                Award ID: 88881.130825/2016-01
                Award Recipient :
                Funded by: funder-id http://dx.doi.org/10.13039/501100002322, Coordenação de Aperfeiçoamento de Pessoal de Nível Superior;
                Award ID: 88887.130823/2016-00
                Award Recipient :
                Funded by: funder-id http://dx.doi.org/10.13039/501100003593, Conselho Nacional de Desenvolvimento Científico e Tecnológico;
                Award ID: MCTIC/FNDCT -CNPq / MEC-CAPES/ MS-Decit 14/2016
                Award Recipient :
                JBPL has a visiting senior researcher fellow PVS PECTI-SAÚDE (Amazonas State Research Support Foundation - FAPEAM). FGN is funded by Fundação de Amparo à Pesquisa do Estado do Amazonas – FAPEAM ( www.fapeam.am.gov.br, call 001/2013 – PPSUS / 062.00656/2014 and call 001/2014 – PROEP / 062.01939/2014); Conselho Nacional de Desenvolvimento Científico e Tecnológico ( http://www.cnpq.br, grant 440856/2016-7) and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior ( http://www.capes.gov.br, grants 88881.130825/2016-01 and 88887.130823/2016-00) call MCTIC/FNDCT -CNPq / MEC-CAPES/ MS-Decit 14/2016 – Prevenção e Combate ao vírus Zika. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
                Categories
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                Custom metadata
                vor-update-to-uncorrected-proof
                2018-08-03
                The final ZIKV sequence is available in GenBank under the accession number MG279550.

                Infectious disease & Microbiology
                Infectious disease & Microbiology

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