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

Abstract

D uring the past 20 years, coronaviruses belonging to the genus Betacoronavirus have caused multiple human epidemic or pandemic diseases, including severe acute respiratory syndrome (SARS), Middle East respiratory syndrome (MERS), and coronavirus disease (COVID-19). Two viruses of the subgenus Sarbecovirus are severe acute respiratory syndrome coronavirus (SARS-CoV), which causes SARS, and SARS-CoV-2, which causes COVID-19. Although Rhinolophus spp. bats in Asia, Europe, and Africa are considered natural reservoirs of sarbecoviruses (1-3), the epidemiology and distribution of these viruses remain largely unknown, especially outside China. Previously, partial RNA-dependent RNA polymerase (RdRp) genes of betacoronaviruses were detected in little Japanese horseshoe bats (Rhinolophus cornutus) (4). However, limited sequence information left the genetic and virological properties unclear. We detected and determined the entire genome sequence of a bat sarbecovirus belonging to a phylogenetic clade that includes SARS-CoV-2 from R. cornutus bats in Japan. Further, we used a pseudotyped virus system to characterize an entry step of this virus into cells. The Study R. cornutus is an endemic bat species in Japan and is found nationwide. These bats primarily inhabit caves and abandoned tunnels in the countryside during daytime and capture insects at night outside their roosts. R. cornutus bats often cohabit with other insectivorous bats, such as R. ferrumequinum or Myotis macrodactylus, and occasionally with wild animals, such as the masked palm civet (Paguma larvata), in their daytime roosts. In 2013, we captured 4 R. cornutus bats in a cave in Iwate prefecture, Japan, and extracted RNA from fresh feces. Then, we used real-time reverse transcription PCR (rRT-PCR) to detect the partial RdRp gene of sarbecovirus from 2 samples by using a pair of primers designed to detect betacoronavirus. In 2020, we performed RNA sequencing and determined the full genome sequence of 1 sample, Rc-o319, which exhibited lower cycle threshold value by rRT-PCR. We performed a BLAST (https://blast.ncbi.nlm. nih.gov/Blast.cgi) analysis of the full genome of Rc-o319, which showed Rc-o319 had the highest nucleotide homology to SARS-CoV-2 HKG/HKU-904a/2020 strain (GenBank accession no. MT365032) with a query cover of 96% and sequence identity of 81.47%. The maximum-likelihood analysis with sarbecoviruses demonstrated that the full genome and spike protein (S) gene of Rc-o319 were positioned within a specific clade that included SARS-CoV-2 (Figure 1, panels A, B). Amino acid sequences of open reading frame 1ab (ORF1ab) and S of Rc-o319 also were positioned within the SARS-CoV-2 clade (Figure 1 panels C and D). The phylogenetic trees maintained the same topology between ORF1ab and S, indicating that no recombination event occurred in Rc-0319, which was supported by similarity plot analysis (Appendix Fig

Evidence for SARS-CoV-2 related coronaviruses circulating in bats and pangolins in Southeast Asia

“…An early report by Zhou et al identified a closely related SARSr-CoV genome sequence, RaTG13, which shared a 96% whole-genome sequence identity with SARS-CoV-2, indicating a probable bat origin of SARS-CoV-2 2 . Since then, more SARS-CoV-2-related viral genome sequences from bats have been reported from Eastern China 7 and Japan 8 , and from pangolins in China 9 , 10 . However, the immediate animal ancestor or progenitor virus, the equivalent of the >99% identical SARS-CoV sequences identified in civets during the SARS outbreak in 2003 11 , remains elusive for SARS-CoV-2.…”

Progressing adaptation of SARS-CoV-2 to humans

“…The evolutionary trajectory of an evolved virus is difficult to analyse from nucleotide sequences because the data have complex multivariate structures with numerous dimensions 2 . Phylogenetic trees have long been used to present relationships in among sequences 3 and many studies on severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) 4 have employed them 5–8 , but they have two drawbacks. One is the decisive lack of falsifiability that ruins objectivity 9 , and the other is a lack of generality that makes it difficult to integrate with other data sources.…”

Characterization and structural prediction of the putative ORF10 protein in SARS-CoV-2

“…9 Although there exists sequences homologous to the ORF10 protein in other closely related bat and pangolin CoVs, there are no experimentally derived crystallographic structures for the ORF10 protein. 10,11 Studies attempting to predict the secondary structural elements of this protein indicate one α-helix and, depending on the study, two β-strands. 12,13 The ORF10 protein also lacks significant levels of disorder; however, a short molecular recognition feature (MoRF) likely spans residues 3-7.…”