Selection of viral variants during persistent infection of insectivorous bat cells with Middle East respiratory syndrome coronavirus

Coronaviruses that cause severe acute respiratory syndrome (SARS) and Middle East respiratory syndrome (MERS) are speculated to have originated in bats. The mechanisms by which these viruses are maintained in individuals or populations of reservoir bats remain an enigma. Mathematical models have predicted long-term persistent infection with low levels of periodic shedding as a likely route for virus maintenance and spillover from bats. In this study, we tested the hypothesis that bat cells and MERS coronavirus (CoV) can co-exist in vitro. To test our hypothesis, we established a long-term coronavirus infection model of bat cells that are persistently infected with MERS-CoV. We infected cells from Eptesicus fuscus with MERS-CoV and maintained them in culture for at least 126 days. We characterized the persistently infected cells by detecting virus particles, protein and transcripts. Basal levels of type I interferon in the long-term infected bat cells were higher, relative to uninfected cells, and disrupting the interferon response in persistently infected bat cells increased virus replication. By sequencing the whole genome of MERS-CoV from persistently infected bat cells, we identified that bat cells repeatedly selected for viral variants that contained mutations in the viral open reading frame 5 (ORF5) protein. Furthermore, bat cells that were persistently infected with ΔORF5 MERS-CoV were resistant to superinfection by wildtype virus, likely due to reduced levels of the virus receptor, dipeptidyl peptidase 4 (DPP4) and higher basal levels of interferon in these cells. In summary, our study provides evidence for a model of coronavirus persistence in bats, along with the establishment of a unique persistently infected cell culture model to study MERS-CoV-bat interactions.