The Keystone Symposia conference on positive-strand RNA viruses provides an international forum for research on human, animal, insect, plant and bacterial viruses with positive-strand RNA genomes. This group of viruses contains many clinically relevant and well-known pathogens (e.g., poliovirus, hepatitis C virus, Dengue and West Nile virus). Furthermore, the enormous diversity of positive-strand RNA viruses in animals and insects, combined with their evolutionary and adaptive potential following a species-jump, poses a threat to the human population as demonstrated by the growing list of emerging viruses, including zoonotic as well as arbovirus-transmitted pathogens such as MERS-CoV, Chikungunya and Zika virus. Detailed insight into the “virosphere”, the “virome”, viral evolution, as well as in the molecular details of viral replication and spreading, tissue tropism, and viral recognition by the host immune system is critical to understand virus transmission, viral pathogenesis as well as to develop novel therapeutic and preventive measures. This conference brings together experienced and junior experts to discuss the latest developments in molecular biology, cell biology, vector biology, immunology, vaccinology, and antiviral drug development applied to the field of positive-strand RNA viruses.