Location: Zoonotic and Emerging Disease Research
Project Number: 3022-32000-027-041-S
Project Type: Non-Assistance Cooperative Agreement
Start Date: Jul 1, 2026
End Date: Jun 30, 2028
Objective:
Objective 1: Characterize viral diversity in cave-dwelling bats in Puerto Rico. Conduct comprehensive metagenomic surveillance of viruses circulating among the six co-roosting bat species in Culebrones Cave, with emphasis on paramyxoviruses, filoviruses, and other viral families of zoonotic concern — including those with potential to threaten U.S. livestock systems and agricultural biosecurity. Generate sequence data and metadata to assess pathogen diversity, evolutionary relationships, and the geographic distribution of viruses with zoonotic potential in the New World, a region historically underrepresented in wildlife virus discovery efforts.
Objective 2: Investigate drivers of cross-species transmission within the cave system using the closed, multi-species roosting system as a natural laboratory, evaluate the host and environmental factors that promote or limit viral sharing between co-occurring bat species. Assess the relative contributions of spatial overlap, roost microstructure, interspecies contact rates, and phylogenetic distance between host species to cross-species transmission frequency. This objective will generate predictive insights into the ecological and evolutionary forces that shape spillover potential.
Objective 3: Assess the role of feral pigs as a bridge host at the cave interface. Characterize the nature and frequency of interactions between feral pigs and the Culebrones Cave system, including roost entrances and surrounding habitat. Evaluate serological and molecular evidence of viral exposure in feral pigs consistent with bat-origin viruses detected under Objective 1 and assess whether feral pigs may serve as bridge hosts capable of amplifying or transmitting bat-origin viruses to livestock or human populations.
Objective 4: Evaluate environmental sampling as a scalable surveillance tool. Assess the utility of environmental samples — including cave substrate, water, and air — for detecting and characterizing bat viruses without direct animal capture. Compare environmental detection sensitivity and viral community composition against paired individual animal samples to determine whether environmental surveillance can serve as a practical, lower-disturbance approach for ongoing monitoring of high-consequence viral families in cave ecosystems.
Approach:
Most investments in pandemic preparedness have focused on post-emergence response, building vaccines and therapeutics to reduce outbreak scale. However, there is also a critical need for pre-emergence research that generates intelligence to improve pandemic forecasting and reduce the frequency of future spillover events. This study will describe pre-emergent viral diversity circulating in cave-dwelling bats in Puerto Rico, evaluate genetic and ecological barriers to spillover, assess the role of feral pigs as potential bridge hosts, and implement novel environmental sampling technologies to refine future surveillance efforts.
Field Work Bat surveillance will be conducted at the Culebrones Cave in the Mata de Plátano forest reserve. Based on prior work demonstrating that approximately 1,000 individuals per species are required to saturate novel virus discovery, we will target 1,000 individuals from the two most abundant species and approximately 500 from two additional species, generating a comprehensive viral diversity survey across four species. Two additional low-abundance species roosting in the cave will be sampled opportunistically. Bats will be captured using harp nets and held in cloth bags until sampling. Oral and rectal swabs will be collected and stored in DNA/RNA shield to inactivate infectious material and preserve nucleic acids. Blood will be collected where feasible for serological analyses. Feral pig activity around cave entrances and surrounding habitat will be documented, and specimens collected to evaluate serological and molecular evidence of exposure to bat-origin viruses. Air sampling devices will collect filtered samples from inside the cave and at the cave entrance during bat emergence. Sampling conditions — including time, volume, and device placement — will be varied to optimize environmental detection protocols. Air samples will be stored in DNA/RNA shield for downstream molecular analysis.
Laboratory Work - Total nucleic acids will be extracted from oral and rectal swabs, air samples, and feral pig specimens. Blood samples will be stored for future analyses. Next-generation sequencing libraries will be prepared and run on the Element Bio AVITI sequencer to maximize reads per sample, with alternative platforms considered based on cost. Existing bioinformatics pipelines will identify viral sequences from fastq files, and full viral genomes will be assembled and annotated where possible. Sequences will be deposited in public databases to support further experimental, computational, and risk assessment work.