Location: Zoonotic and Emerging Disease Research
Project Number: 3022-32000-027-004-S
Project Type: Non-Assistance Cooperative Agreement
Start Date: Aug 15, 2023
End Date: Aug 14, 2026
Objective:
Viruses are our planet's most abundant biological entities; our understanding of their diversity and distribution is extremely poor. Only a fraction of the viruses circulating in wildlife have been described, despite the availability of sophisticated tools to find and characterize viral genomes. Bats (in particular) have been identified as important hosts of zoonotic viruses, motivating efforts to find and characterize the viruses they carry. These studies have yielded many important discoveries, but significant gaps remain. There are over 1400 species of bats, and very few have been comprehensively surveyed, meaning most viruses circulating in bats have not been described. As it is not feasible to comprehensively survey every bat species around the world (to do so would require a herculean effort and extraordinary financial resources), there is a need for targeted virus discovery to fill strategic host and geographic gaps and to generate predictive insights into the ecological and evolutionary forces that shape the zoonotic potential of wildlife viruses.
One notable gap is our understanding of the diversity of paramyxoviruses, and other emerging high-consequence viruses (ie coronaviruses, and filoviruses) that exist in the New World. Significant effort has gone into describing the diversity of the high-risk viral families in Africa and Asia, much less is known about viral diversity in the Americas or the Caribbean islands. Serologic evidence suggests that filoviruses circulate in bats in the Caribbean; however, no specific filovirus has yet been identified. There is the potential for viruses from families of zoonotic concern to exist in New World bats, but more focused surveillance efforts are now needed to identify these viruses and assess their potential for human infection and infection of intermediate livestock species.
The goal is of this study is to comprehensively survey the diversity of paramyxoviruses, and other emerging zoonotic threats in cave-dwelling bats in Puerto Rico. There are 13 bat species on the island, many of which form very large aggregations of individuals in the extensive cave systems found on the island. For example, the Culebrones Cave is a structurally complex cave with an estimated 300,000 individual bats from 6 species. This large number of bats from multiple co-roosting species provides a unique opportunity to exhaust the discovery of novel viruses in several bat species, but also to investigate the factors that drive or limit cross-species transmission events.
Objective 1: Describe the viral diversity in cave-dwelling bats in Puerto Rico.
Objective 2: Describe the frequency of cross-species transmission events between co-occurring bat species and evaluate the host and environmental factors that promote cross-species transmission(spatial overlap versus genetic distance between species)
Objective 3: Investigate the utility of environmental samples for describing viral diversity in bats.
Approach:
Most investments in pandemic preparedness have focused on post-emergence pandemic response, building critical tools and interventions such as vaccines and therapeutics to reduce the scale of disease outbreaks. However, there is also a need for research and investments at the pre-emergence phase; investments that will generate critical intelligence to improve pandemic forecasting and reduce the frequency of future spillover events. This study will describe the pre-emergent viral diversity circulating in bats in Puerto Rico, evaluate the genetic and ecological barriers to spillover and spread, and implement novel technologies (e.g., air sampling) to refine future surveillance efforts.
Field work: We plan to sample cave-dwelling bats in the Mata de Platano forest reserve in Puerto Rico. We will initially focus our efforts on a single cave, the Culebrones cave, to comprehensive survey the diversity of paramyxoviruses, and other viruses circulating in these bats. Based on our prior work, we have shown that approx. 1000 individuals per species are required to saturate the discovery of novel viruses. We will aim to sample 1000 bats for the two most abundant species, and approx. 500 individuals for two additional species. This will generate a comprehensive viral diversity survey for 4 bat species in this cave. While 2 additional species also roost in the Culebrones cave, they are much less abundant, making reaching the sample numbers required to saturate discovery difficult. Bats will be captured using harp nets and held in cloth bags until sampling. Oral and rectal swabs (feces) will be collected from each bat and stored in DNA/RNA shield to inactivate infectious materials and preserve nucleic acids. (In future studies as resources allow, additional samples may be collected in VTM to support virus isolation attempts). Where possible, blood will also be collected. In addition to sampling individual bats, we will use commercial air sampling devices to collect filtered air samples from inside the cave, as well as from outside the cave at the time of bat emergence. To find the optimal environmental (air) sampling conditions, we will vary the time and volume of air being filtered and the location of the filtering device. Filtered air samples will also be placed in DNA/RNA shield for downstream molecular analysis.
Laboratory work: Samples from individual bats and air samples will be transported to the Anthony Lab at UC Davis. Total nucleic acids will be extracted from the oral and rectal swabs, and from the environmental air samples. Blood samples will be stored for future analyses. Next-generation sequencing libraries will be generated, and the samples run on the Element Bio AVITI sequencer to maximize the number of reads per sample. (Alternative sequencing platforms may be used depending on the best cost per sample obtained at the sequencing time). Existing bioinformatics pipelines will be used to identify viral sequences from fastq files and, where possible, full viral genomes will be assembled and annotated. Sequences will be shared on public databases and used to support further experimental and computational work and risk assessments.
