Abstract
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.
Highlights
Coronaviruses that cause severe acute respiratory syndrome (SARS) and Middle East respiratory syndrome (MERS) are speculated to have originated in bats
To determine if Interferon regulatory factor 3 (IRF3)-mediated innate antiviral responses play a role in the maintenance of persistent infection in bat cells, we reduced IRF3 protein levels (Fig. 6b) in these cells using small-interfering RNA. siRNA-mediated reduction of IRF3 in persistently infected bat cells increased MERS-CoV titers by ten-fold (Fig. 6a)
Successful spillover of a virus to a new species requires viruses to adapt to the use of viral receptors and circumvent innate antiviral defense mechanisms that are unique to each host species
Summary
Coronaviruses that cause severe acute respiratory syndrome (SARS) and Middle East respiratory syndrome (MERS) are speculated to have originated in bats. 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. We established a long-term coronavirus infection model of bat cells that are persistently infected with MERS-CoV. 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. Bats are speculated to be reservoirs of several emerging viruses, including coronaviruses (CoVs) that cause severe acute respiratory syndrome (SARS) and Middle East respiratory syndrome (MERS) in humans, and porcine epidemic diarrhea (PED) and swine acute diarrhoea syndrome (SADS) in pigs[3,4,5,6]. Persistence of viruses in bats and increase in virus shedding during ‘stressful’ events has been observed with Henipavirus infections[27,28]
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