RaTG13

Various SARS-CoV-2 like viruses have been isolated from bats in China, Thailand, and Japan, but none have a spike protein that can bind ACE2 and allow entry into human cells. Sampling of bats in Laos has now revealed the presence of such viruses.

The genome of a virus called RaTG13, from Rhinolophus affinis bats in China, is the closest to SARS-CoV-2 (although infectious RaTG13 has never been isolated). However the spike receptor binding domain (RBD) encoded in this genome has low sequence similarity with that of SARS-CoV-2 and its affinity for ACE2 is very limited. RaTG13 is clearly not the proximal ancestor of SARS-CoV-2.

Sampling of 645 bats from limestone caves in Northern Laos (see map) yielded three Sarbecovirus genomes, called BANAL-52, -103, and -236, with high sequence similarity to SARS-CoV-2 and RaTG13. These three viral genomes were obtained from three different Rhinolophus species. Of the 17 amino acids that interact with the receptor binding domain of ACE2, 16 are conserved between SARS-CoV-2 and BANAL-52 or -103, and 15/17 conserved with BANAL-236. In contrast, only 11/17 RBD amino acids are conserved among SARS-CoV-2 and RaTG13. In other words, the RBD encoded in these BANAL genomes are closer to SARS-CoV-2 than that of any other known bat virus.

Binding affinity assays done with purified proteins revealed that the BANAL-52/103 and -236 spikes bind ACE2 with affinities in the low nanomolar range, comparable to reported values for the SARS-CoV-2 spike.

Lentiviral particles with the spike protein from BANAL-236 were able to bind and enter human cells producing ACE2. Entry of this virus was blocked by human sera containing antibodies to SARS-CoV-2 but not by control sera.

Infectious BANAL-236 virus was recovered from a bat fecal swab after inoculation of Vero cells, providing a rare virus isolate for a bat SARS-CoV-2 like virus. The virus will be useful for studying the biology of infection.

Recombination analysis demonstrated that the SARS-CoV-2 genome is a mosaic of at least five different genomes, including BANAL-52, -103, and -236, and the previously published RmYN02, RpYN06, and RaTG13, the latter all discovered in China. The implication of these observations is clear: SARS-CoV-2 likely arose from recombination of viruses circulating in different species of Rhinolophus bats in the limestone caves of South China and Southeast Asia. Because the RBD of the BANAL isolates can mediate binding to and entry into cells that produce ACE2, no host to host passage need be hypothesized to explain increased RBD affinity in an intermediate host before spillover into humans.

The spike proteins encoded in the BANAL isolates do not have the furin cleavage site found in the protein from SARS-CoV-2. Such sites might be present in viruses within these bat communities, but were missed due to insufficient sampling. Alternatively, it is possible that selection for the furin cleavage site occurred after spillover into humans or another intermediate host.

These findings further emphasize and demonstrate that SARS-CoV-2 came from Nature, not from a lab.