The detection and isolation of bat sarbecoviruses from several locations in Japan

By Dr. Liji Thomas, MDReviewed by Danielle Ellis, B.Sc.May 19 2022

Betacoronaviruses have caused several major pandemics of zoonotic disease over the last few decades, the latest being the coronavirus disease 2019 (COVID-19) pandemic. Many of these viruses are endemic among bats, making the surveillance of these mammalian viruses a must to understand and prevent the spillover of these pathogens into humans. A new preprint discusses the findings of a Japanese study aimed at isolating and characterizing bat Sarbecoviruses in that country, which could contribute to the sum of knowledge about them.

This news article was a review of a preliminary scientific report that had not undergone peer-review at the time of publication. Since its initial publication, the scientific report has now been peer reviewed and accepted for publication in a Scientific Journal. Links to the preliminary and peer-reviewed reports are available in the Sources section at the bottom of this article. View Sources

Introduction

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) caused millions of deaths and hundreds of millions of cases, shutting down entire national economies, schools, businesses, and travel destinations. Yet it was just one of the many betacoronaviruses that pose a continual threat to human health. The bats that carry these viruses are therefore of great interest to scientists who are trying to understand and predict outbreaks of these viruses as they cross the species barrier into humans.

These bats mostly belong to the genus Rhinolophus, a natural reservoir for Sarbecoviruses, to which betacoronaviruses belong. They have been detected in Asian countries but also in some regions of Europe and Africa. Other Sarbecoviruses closely related to SARS-CoV-2 have been found in Asia, some of which have the receptor-binding domain for the angiotensin-converting enzyme 2 (ACE2) host receptor found in humans. These viruses may therefore infect humans as well under the right conditions.

The authors of this preprint, appearing on the bioRxiv* server, had earlier identified a Japanese bat sarbecovirus termed Rc-o319 from a bat species named Rhinolophus cornutus. This virus belonged to the same lineage as SARS-CoV-2, and pseudoviruses expressing its spike successfully infected cells expressing the R. cornutus bat but not the human ACE2, indicating that the former is the viral entry receptor.

The current paper reports the detection of bat Sarbecoviruses from multiple places in Japan with their characteristics.

What did the study show?

The investigators collected fecal samples from bats of both R. cornutus and R. ferrumequinum in three prefectures of Japan. By running reverse transcriptase polymerase chain reactions (RT-PCR) on them, they detected the sarbecovirus E gene in some samples from each site from the former but not the latter species, indicating that only the former is the host for these viruses in Japan.

They obtained four isolates from these samples using Rc-binding ACE2 (RcACE2)-expressing human cells. They found that the genetic composition of the four isolates they obtained was very similar, at 95% to 97% homology, except for the open reading frame 8 (ORF8), missing from two isolates. Again, the N-terminal domain (NTD) and the receptor binding domain (RBD) of the spike gene showed differences in the coding segments between the isolates.

The receptor-binding motif of all these isolates showed a deletion of 9 residues, making them unable to bind to human ACE2. Their replication is dependent on the presence of RcACE2. This contrasts with SARS-CoV-2, which showed replication in three types of cells used: those expressing RcACE2, human ACE2, and the serine protease TMPRSS2. It could not infect the ACE2-knockout cells.

Thus, SARS-CoV-2 can infect cells bearing multiple types of ACE2 receptors, including that of R. cornutus. The Japanese viruses thus formed a single cluster within the SARS-CoV-2 clade, dubbed the Japanese clade of Sarbecoviruses.

What are the implications?

The findings of this study show the presence of four types of Sarbecoviruses from the R. cornutus bats found in several parts of Japan, which infected cells bearing the RcACE2 receptor. Another species of Rhinopholus bat in the south of China or the Indochina peninsula is thought to have given rise to SARS-CoV-2 since the Sarbecoviruses found there show extreme homology with the latter and bind with high affinity to human ACE2.

The virus isolates found in this study were more distantly related to SARS-CoV-2, in contrast, though of the same clade. They could not bind human ACE2 and are thus not in the ancestral line directly but could belong somewhere upstream of the point where SARS-CoV branched off from SARS-CoV-2.

R. cornutus is found extensively in Japan and was the only species to yield Sarbecoviruses. In China, however, R. ferrumequinum also hosts these viruses, and larger sample collections may help clarify if this is the case in Japan as well. Two other species, R. pumilus and R. perditus, remain to be studied for the presence of Sarbecoviruses, of this kind or other sorts.

No bat sarbecovirus has yet been found to use any but the ACE2 molecule as an entry receptor. The isolates identified here had highly variable NTD and RBD regions, both with viral neutralizing epitopes. The variability could thus result from selection pressure due to neutralizing antibodies.

The high degree of conservation between the strains in most other genomic regions indicates that these are recent additions from the common ancestor. The mutations at the NTD and RBD thus accumulated quickly, supporting the effect of selection pressure. In addition to betacoronaviruses, alphacoronaviruses have also been found in Rhinopholus bats. Their recombination at the RBD or NTD regions could give rise to many novel pathogens with the ability to spread to humans.

Though these isolates did not infect and replicate in cells expressing human ACE2, other similar viruses could gain this ability via mutations, often via another intermediate host such as wild animals or farmed animals. “Epidemiological studies of sarbecoviruses in wildlife, including bats, need to be conducted on a long-term basis for risk assessment of their zoonotic potential.”

This news article was a review of a preliminary scientific report that had not undergone peer-review at the time of publication. Since its initial publication, the scientific report has now been peer reviewed and accepted for publication in a Scientific Journal. Links to the preliminary and peer-reviewed reports are available in the Sources section at the bottom of this article. View Sources