Location: Mosquito and Fly Research
Project Number: 6036-32000-052-099-I
Project Type: Interagency Reimbursable Agreement
Start Date: Aug 1, 2022
End Date: Sep 30, 2023
Objective:
The goals of this proposal are to determine if non-sylvatic and urban MAYV transmission cycle exists via Ae. aegypti and Ae. albopictus, and the new invasive mosquito species Ae. vittatus. We will also determine the infection rates with other arbo-pathogens (e.g. DENV, Madariaga virus (MADV), and others) and insecticide resistance genotypes in select peri-urban sites in high priority Caribbean countries as identified by USSOUTHCOM. These data would provide insights for operational planning for military deployments in USSOUTHCOM and potential for the emergence of MAYV and other arbo-viruses in the U.S.
Approach:
Study site: Surveillance sites in Haiti, Dominican Republic, Trinidad & Tobago.
Study period: This study is a retrospective study using samples collected from 2017-2019 that are currently frozen.
Sample and data collection: Targeted mosquito collections: Adult mosquito trapping were conducted using BG-Sentinel-2TM traps (Biogents AG, Regensburg, Germany) with lure. Traps will be deployed to each site and set three 24 hour periods per week over a six month sampling period that will span dry and wet season. BG-Sentinel traps will be positioned peripheral to urban areas, and samples will be pool by sampling location.
Laboratory analysis: Molecular analysis of vector samples: For the molecular work, we will use a combination of traditional PCR and existing protocols as well as nanopore sequencing for more in depth analysis of detected pathogens. Nanopore sequencing allows use of a field deployable device that can allow for rapid sequencing and molecular identification of pathogens identification, vector species, bloodmeal and Insecticide resistance status.
Virus detection: The pre-identified and sorted mosquitos will be pooled by collection site and tested for MAYV, DENV and other arboviruses at the USUHS and Smithsonian Institute and WRAIR. Mosquito tissue will be pulverized in lysis buffer and total RNA extracted using the Qiagen RNA extraction kit following the protocol provided by the manufacturer. A one-step RT-PCR will be conducted using Qiagen QuantiTect Probe RT-PCR kit to detect viral RNA.
Insecticide resistance: For insecticide resistance analysis, we will use molecular genetic markers targeting single nucleotide polymorphisms (SNPs) in Ae. aegypti. We will target insecticide resistance genetic markers in the voltage gated sodium channel (VGSC) including F1534C andV1016I. We will also assess the presence of traditionally Indopacific kdr mutations (S989P and V1016G) which have recently been collected in the Caribbean and are becoming a priority target for surveillance(Murcia et al., 2019).
