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  1. Our take

    In this preprint that has not yet been peer reviewed, researchers surveyed coronavirus diversity in 46 species of bats in northern Laos sampled between July 2020 and January 2021. Among the diverse coronaviruses found, three viruses closely related to SARS-CoV-2 were identified in Rhinolophus species, with BANAL-52 from R. malayanus representing the closest relative of SARS-CoV-2 identified in bats to date, with 96.8% sequence identity at the genome level. Additionally, researchers were able to isolate a coronavirus related to SARS-CoV-2 from a bat in cell culture, representing a first for this lineage and only the fifth time a bat-borne coronaviruses has been isolated. The study indicates that Laos and other countries in Southeast Asia may harbor additional related viruses in Rhinolophus spp. bats that shed light on the evolutionary origin of SARS-CoV-2.

    Study design


    Study population and setting

    To investigate the presence of coronaviruses in bats in Laos, investigators captured and took samples (blood, saliva, feces, anal swabs, and urine swabs) from 645 bats of 46 species sharing caves at four sites in Vientiane and Oudomxay Provinces in northern Laos between July 2020 and January 2021. The authors used polymerase chain reaction (PCR) to identify presence of coronavirus RNA in 539 fecal samples from a subset of bats (methods did not specify number of species). Samples that were positive for betacoronavirus RNA were subjected to full genome sequencing. Betacoronavirus genomes were assessed for evidence of genetic recombination. To test the ability of viruses to enter human cells, the researchers performed molecular simulations of binding between virus spike and the human ACE2 receptor. Potential infectivity of viruses was further analyzed by measuring the entry of pseudotyped lentivirus particles expressing bat coronavirus spike into human kidney cells expressing ACE2. Finally, neutralizing sera from confirmed COVID-19 patients and non-neutralizing control sera from before the pandemic were used to evaluate restriction of pseudotyped lentivirus entry.

    Summary of main findings

    Coronavirus RNA was amplified from 24 bats of 10 species, representing multiple coronavirus subgenera. Sequences of the Sarbecovirus subgenus were only found in Rhinolophus species ­­– R. malayanus, R. marshalli, and R. pusillus – sampled from Feung District in Vientiane Province; alphacoronavirus RNA was found in a single R. affinis and eight other Rhinolophus species were all negative. Five full genomes of sarbecoviruses were sequenced from bats and all five clustered in the novel clade of bat sarbecoviruses related to SARS-CoV-2; three viruses were very closely related to SARS-CoV-2. The virus BANAL-52 from Rhinolophus malayanus is 96.8% similar to SARS-CoV-2 across its genome; two other viruses, BANAL-103 and BANAL-236 were more distant from SARS-CoV-2 but still within the same cluster with BANAL-52. BANAL-236 was successfully isolated in cell culture. Within the spike protein, 16/17 of the amino acid residues that interact with human ACE2 are the same in BANAL-52 or -103 as in SARS-CoV-2 (compared to 11/17 for RaTG13, previously the closest relative of SARS-CoV-2 sequenced from R. affinis collected in Yunnan Province in 2013). Phylogenetic analysis found extensive evidence of recombination in the bat coronaviruses and within SARS-CoV-2, suggesting that transmission of sarbecoviruses among different Rhinolophus species living in the same caves contributes to a complex evolutionary history. The spike of BANAL-52/103/236 binds to human ACE2 with high affinity similar to SARS-CoV-2 and lentivirus particles pseudotyped with BANAL-236 spike readily entered human cells expressing ACE2; entry of particles was successfully blocked by human by neutralizing sera from COVID-19 patients.

    Study strengths

    The study used a moderately large sample of bats from a diverse assemblage of species in a hotspot of bat diversity, especially of Rhinolophus spp. Historically, few surveys of coronaviruses in bats have been performed in Southeast Asia relative to China, so this study is an important contribution to the understanding of the biogeography of coronaviruses. The detailed characterization of the detected viruses, including full genome sequencing, spike protein modeling, evaluation of human cell entry, and virus isolation in cell culture, are highly valuable.


    As with other studies describing relatives of SARS-CoV-2 in bats, the viruses described in this study are still too genetically divergent to be the direct progenitor of SARS-CoV-2, representing decades of evolutionary time.

    Value added

    The successful isolation of BANAL-236 in cell culture is important because in over 15 years of research on coronaviruses in bats, only five coronaviruses have successfully been isolated in culture; most detections of coronaviruses in bats are only genetic sequences and do not represent cultured viruses, including RaTG13. Thus, this study is the first to obtain a physical specimen of a bat coronavirus related to SARS-CoV-2. The similarity of BANAL-52 to SARS-CoV-2 exceeds the previous record of 96.1% similarity between SARS-CoV-2 and virus RaTG13 from Rhinolophus affinis, making BANAL-52 the closest known ancestor to SARS-CoV-2 in an animal reservoir. The analysis of recombination indicates that the origin of SARS-CoV-2 may not point to a single progenitor virus existing in bats, but rather a complex of viruses that circulate and recombine in multiple species.

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