Detection and Characterization of Bat Sarbecovirus Phylogenetically Related to SARS-CoV-2, Japan

Abstract

Epidemiology of bat Betacoronavirus, subgenus Sarbecovirus is largely unknown, especially outside China. We detected a sarbecovirus phylogenetically related to severe acute respiratory syndrome coronavirus 2 from Rhinolophus cornutus bats in Japan. The sarbecovirus' spike protein specifically recognizes angiotensin-converting enzyme 2 of R. cornutus, but not humans, as an entry receptor.

Keywords: COVID-19; Japan; Rhinolophus cornutus; SARS; SARS-CoV-2; bat sarbecovirus; betacoronavirus; coronavirus; coronavirus disease; respiratory infections; severe acute respiratory syndrome coronavirus 2; viruses; zoonoses.

Figure 1

Phylogenetic analysis of sarbecovirus sequenced from little Japanese horseshoe bats (Rhinolophus cornutus) and genetically related to human SARS-CoV-2, Japan. A–D) Phylogenetic trees were generated by using maximum-likelihood analysis combined with 500 bootstrap replicates and show relationships between bat-, human-, and pangolin-derived sarbecoviruses. Phylogenetic trees are shown for nucleotide sequences of the full genome (A), the S protein gene and amino acid sequences (B), the ORF1ab (C), and the S protein (D). Red text indicates positions of Rc-o319, the sarbecovirus sequenced in this study. For panels B and D, magenta bands indicate viruses with S proteins that bind to human ACE2; blue bands indicate viruses with S proteins that do not bind to human ACE2. Bootstrap values are shown above and to the left of the major nodes. Scale bars indicate nucleotide or amino acid substitutions per site. E) Amino acid sequence alignment of the RBM of S proteins that are able or unable to bind to human ACE2. Amino acid residues of the RBM that contact human ACE2 of SARS-CoV-2 and SARS-CoV are indicated in the upper side by red circles. The 2 regions of S protein RBM known to interact with human ACE2 are indicated by boxes labeled region 1 and region 2. ACE2, angiotensin-converting enzyme 2; hACE2, human angiotensin-converting enzyme 2; ORF1ab, open reading frame 1ab; RBM, receptor-binding motif; S, spike protein; SARS-CoV, severe acute respiratory syndrome coronavirus; SARS-CoV-2, severe acute respiratory syndrome coronavirus 2.

Figure 2

Infectivity titers of sarbecoviruses from bats and humans used to investigate bat sarbecovirus Rc-o319, which is genetically related to human SARS-CoV-2, Japan. Cells expressing each host-origin angiotensin-converting enzyme 2 were inoculated with VSV pseudotyped with spike proteins of Rc-o319 (A), SARS-CoV (B), SARS-CoV-2 (C), or glycoprotein of VSV (D). At 20 hour postinfection, GFP-positive cells were counted and the infectivity titers were calculated. Error bars indicate SDs from 3 independent experiments. CoV, coronavirus; GFP, green fluorescent protein; Rc/Rf, chimera of Rhinolophus cornutus and R. ferrumequinum; SARS, severe acute respiratory syndrome; VSV, vesicular stomatitis virus.