Barcoded SARS-CoV- 2 viruses define the impact of duration and route of exposure on the transmission bottleneck in a hamster model

The transmission bottleneck, defined as the number of viruses shed from one host to infect another, is an important determinant of the rate of virus evolution and the level of immunity required to protect against virus transmission. Despite its importance, the severe acute respiratory syndrome coronavirus 2 (SARS-CoV- 2) transmission bottleneck remains poorly characterized. We adapted a SARS-CoV- 2 reverse genetics system to generate a pool of >200 isogenic SARS-CoV- 2 viruses harboring specific 6-nucleotide barcodes, infected donor hamsters with this pool, and exposed contact hamsters to paired infected donors, varying the duration and route of exposure. Following exposure, the nasal turbinates, trachea, and lungs were collected and the number of barcodes in each tissue was enumerated. We found that longer and more direct exposures increased the transmission bottleneck and that the upper airway is the primary source of transmitted virus in this model. Together, these findings highlight the utility of barcoded viruses as tools to rigorously study virus transmission.