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Research Project: Detection and Characterization of Potential Emerging and Emergent Arboviruses in Mosquitoes in the Caribbean

Location: Foreign Arthropod Borne Animal Disease Research

Project Number: 3022-32000-024-011-S
Project Type: Non-Assistance Cooperative Agreement

Start Date: Sep 1, 2022
End Date: Sep 30, 2025

Over the past few decades there has been a global resurgence of mosquito-borne viral diseases. In many cases, the geographic distribution of the viruses and their mosquito vectors have expanded globally, accompanied by more frequent and larger epidemics, while other viruses have been introduced into new regions. Globalization, increased migration of people and animals and Climate Change are thought to be driving this. In the 1950s and 60s, the Collaborator demonstrated the existence of several mosquito-borne viruses in human and animal populations in the Caribbean region, including in the twin island republic of Trinidad and Tobago. The advent of new molecular sequencing technologies makes it important to follow up these earlier studies in order to understand which viruses are currently present in mosquito populations and whether these viruses pose a risk to animal and public health, following a One Health approach. With the rapid rate of urbanization and encroachment of the human population into once-forested areas, the likelihood of other mosquito-borne pathogens emerging/re-emerging in Trinidad is high. Additionally, the risk of foreign pathogens entering Trinidad and Tobago is also significant given the high levels of tourism and legal/illegal migration of animals and humans into the island from neighboring Venezuela on the South American mainland. Our main goal of this cooperative agreement is therefore to determine the identity, distribution, and biodiversity of mosquito-borne viruses in Trinidad and Tobago, and to infer their demographic and migration histories with a view to identifying whether they are emerging or potentially emergent. We plan to develop and apply new metagenomic sequencing technology to detect and characterize arboviruses present in different mosquito populations in Trinidad and Tobago that could pose emerging animal and human disease threats, and to develop a baseline of virus population variability. The areas that will be targeted for investigation will include forested, forest fringe (bordering forests), urban and farmed (livestock) areas that will inform predictive models of the effects of human and livestock encroachment on the emergence of arboviruses. The two main objectives of this collaborative study are: Objective 1: To identify human and animal mosquito-borne viruses in Trinidad and Tobago and to determine their distribution relative to human and animal habitations. Objective 2: To characterize mosquito-borne viruses currently in circulation in Trinidad and Tobago and to infer their evolutionary and migration histories, as well as the threats they pose to human and animal health nationally, regionally, and internationally, including to the USA.

Trapped mosquitoes will be used as the main source of viruses since viremia in vertebrate hosts is often short-lived. To this end, mosquito traps will be set in four (4) different ecologically distinct (forested, forest fringe, urban and farmed (livestock)) ecozones in Trinidad and Tobago throughout the rainy season from (May – November). Appropriate traps will be selected for use to maximize mosquito species richness. Traps will be set at least once every two weeks at each site and mosquitoes collected the next day. Global Positioning System (GPS) coordinates of the sample sites and information on the environmental characteristics of trap locations will be documented and analyzed through a Geographic Information System (GIS). This GIS approach will be used to determine the temporal and spatial distribution of the viruses and their vectors. Logistic regression analyses will then be used to create a model for predicting the locations of foci of infection. Mosquitoes will be removed from the traps and immediately placed on ice for transporting to the laboratory. Upon arrival, they will be sorted on a chill table according to trap, location, sex (including noting engorged status for females) and species, using standard taxonomic keys. The mosquito heads will be separated from the bodies and the latter placed in microtiter plates before storage at -80’C. Mosquito heads will be divided into pools of fifty and homogenized. The supernatant will be collected, and nucleic acids will be extracted and used for confirmation of taxonomic identification using molecular bar-coding methods and for screening for the presence of arboviruses. Unbiased metagenomic sequencing will be used for detection and characterization of viral genome sequences in mosquito pools. If the unbiased approach does not produce sufficient viral sequence reads to recover whole genomes, then targeted sequencing approaches will be developed. Phylogenetic relatedness of viruses from other regions and those previously isolated in Trinidad and Tobago will be done using IQ-TREE, maximum likelihood analysis. The Bayesian Markov Chain Monte Carlo method implemented in the BEAST software package will be used to simultaneously estimate evolutionary relationships, rates of evolution, underlying driving forces for evolution, dates of origin for lineages, past population dynamics and spatial diffusion patterns from date- and location-stamped sequences. BEAST also incorporates spatial diffusion models that quantify the rate at which viral lineages move among sampled locations or across continuous space and time. Thus, it explicitly integrates the spatial, temporal, and demographic dynamics of viral epidemics, and offers a powerful, rigorous, and flexible statistical framework for testing hypotheses about the mechanisms underlying viral emergence, epidemic expansion, and maintenance. The data generated in this collaborative project will provide important baseline information on which viruses are present in different mosquito populations from different ecozones in Trinidad and Tobago and whether the identified viruses could emerge and threaten animal and/or human health.