Passaging of Wuhan-Hu-1 strain of SARS-CoV-2 Spike D614G in BHK-21 cells expressing animal ACE2

Experimental evolution of SARS-CoV-2 was performed to identify putative human- and animal-adaptive viral residues. The Wuhan-Hu-1 strain of SARS-CoV-2 bearing the Spike D614G mutation was serially passaged 10 times in BHK-21 cells expressing human, dog, cat, mink, or white-tailed deer ACE2. All passaging in dog, cat, mink, or white-tailed deer ACE2-expressing cells was performed in quadruplicate while passaging with human ACE2-expressing cells was performed in duplicate. Passage 0 virus (inoculum) was sequenced, and all passage 5 and 10 populations were sequenced. RNA was extracted from the passage 0, 5, and 10 virus populations. Library preparation was performed using an Illumina COVIDseq assay kit with ARTIC V3 primers. Paired-end 150bp Illumina sequencing was performed.