The appearance and spread of diseases, such as Ebola, have had profound global health impacts and have heightened awareness regarding the interconnectedness of wildlife, domestic animal, and human health. The impacts have illustrated our vulnerability to infectious diseases affecting all species and the disappearing boundaries between the less developed and more developed parts of the world. Further, population growth and environmental change are facilitating contact with wildlife in unprecedented ways and increasing frequency, yet many nations lack the resources and infrastructure necessary to detect and respond. Management of zoonotic diseases therefore warrants vigilant attention but also provides a challenge for wildlife managers to protect species. USAID initiated the Emerging Pandemic Threats program in 2009 with the goal of strengthening capacities in developing countries to prevent, detect, and control infectious diseases. PREDICT, a wildlife surveillance and virus discovery component of the program, focused on building capacity and applying a One Health approach to this challenge. Through a consortium of partners, PREDICT’s efforts focused on early detection and response to potentially high-consequence viruses in regional “hotspots” for infectious pathogen spillover. Implemented in over 20 countries, we improved early detection and response to disease threats by: 1) strengthening viral surveillance in wildlife; 2) improving virus detection and discovery capacities; 3) characterizing high-risk wildlife-human interfaces, behaviors, and drivers of pathogen spillover; 4) optimizing predictive models for disease emergence and spread; and 5) deploying cutting-edge information management and communication. In just five years, the consortium humanely sampled more than 56,000 wild animals, while training 2,500 wildlife professional in optimal field and laboratory practices, and detected more than 815 novel viruses in addition to 169 known ones. PREDICT has played a key role in investigating the cause of human and wildlife diseases and is identifying high-risk human behaviors for the protection of wildlife and global health.