Light shed on SARS-CoV-2 replication in bat cells

Bats are reservoir species for numerous viruses including coronaviruses. Given that they do not appear to be affected by diseases transmitted by these viruses, there is potential benefit in understanding how their immune system regulates infection. Scientists at the Institut Pasteur and the CNRS studied SARS-CoV-2 replication in bat cells, which involved using real-time imaging techniques for the first time. They demonstrated the existence of species-specific and cell-specific immune responses. These findings were published in The Journal of Virology on July 5, 2022.

Bats are reservoir species for numerous viruses. Unlike humans and other mammals, they are able to carry certain viruses without becoming ill following infection. Bats' immune systems therefore appear capable of controlling certain viral infections effectively. Scientists at the Institut Pasteur and the CNRS examined SARS-CoV-2 replication in bat cells to understand how these animals' cells respond to viral infection.

Their first task was to develop cellular models. This entailed gathering cell lines previously generated by other laboratories and producing new ones. Among the contributor labs were those involved in the collaborator network of the National Reference Center for Rabies at the Institut Pasteur. Bat cell cultures were derived from various bat species and various organs (brain, skin, digestive tract). "These animals are the world's second largest group of mammals with nearly 1,400 different species; it's important to have the widest possible range of bat cellular models so that we can compare and extrapolate our results. These cellular models are, moreover, essential for any research on bats," explains Laurent Dacheux, a scientist in the Lyssavirus, Epidemiology and Neuropathology Unit, Deputy Director of the National Reference Center for Rabies at the Institut Pasteur, and co-last author of the study.

The scientific teams adapted these cell cultures by modifying them to express the viral receptor, human ACE2 protein. This enabled them to develop a collection of cell lines relevant to research on SARS-CoV-2 and other related viruses.

The cell lines were then infected with SARS-CoV-2 and observed using different microscopy techniques including real-time optical microscopy. "This is the first time it has been possible to visualize SARS-CoV-2 replication in living bat cells," comments Nolwenn Jouvenet, Research Director at the CNRS, Head of the Virus Sensing and Signaling Unit at the Institut Pasteur, and co-last author of the study. Through time-lapse microscopy, scientists were able to determine the speed at which cells were infected. Cells from some cell lines died quickly, with syncytia[1] forming rapidly.

Video of cells derived from greater mouse brain (Myotis myotis) infected with SARS-CoV-2 (right) or uninfected (left). The cells are cultured in a medium containing propidium iodide as a cell death marker. Cells were photographed every 15 minutes for 48 hours. © Delphine Planas, Sophie Aicher, Nolwenn Jouvenet (Institut Pasteur)

"By installing a microscope in a BSL3 laboratory in the midst of the COVID-19 pandemic, we were able to visualize the consequences of SARS-CoV-2 infection on cell functioning in real time," adds Olivier Schwartz, Head of the Virus and Immunity Unit at the Institut Pasteur[2] and co-author of the study.

Using these ACE2-expressing bat cells and a wide range of virology, biochemistry, and optical and electron imaging techniques, the scientists demonstrated that viral replication was controlled over time in some bat cell lines, and that this control was linked to a powerful, immediate immune response that was impossible for the virus to evade.

Viral replication was blocked at different stages depending on the cell line. In some cell lines, the virus was unable to replicate even after penetrating cells. In other lines, although the virus was able to replicate, no viral particles were released into the extracellular environment. On the whole, these findings suggest the existence of species-specific molecular barriers affecting coronavirus replication in bat cells.

"Our task is now to identify and characterize proteins induced by the immune response in bats that SARS-CoV-2 is unable to block," adds Nolwenn Jouvenet. To achieve this goal, the scientists will use the large collection of cell lines they have developed and high-throughput transcriptomic techniques to identify immunity genes with antiviral properties. "We still don't know why bats' immune system is so effective at fighting off coronaviruses. The challenge lies in improving our understanding of how this animal reservoir is able to carry viruses that affect global health," she concludes.