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    In this preprint that has not yet been peer reviewed, researchers surveyed coronavirus diversity in 46 species of bats in northern Laos sampled between July 2020 and January 2021.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

    other

    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.

    Limitations

    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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  2. Our take

    In this study, available as a preprint and thus not yet peer reviewed, the authors reported a massive effort to describe the diversity of coronaviruses in bats across 14 provinces of China sampled between 2016 and 2021. In 13,064 samples from bats, 146 sarbecoviruses were detected predominantly in Rhinolophus species. While no relatives of SARS-CoV-2 were detected, the closest relative of SARS-CoV in bats was discovered in R. sinicus sampled in 2020, with 95.8% similarity to SARS-CoV at the genome level. Swab samples taken from the Huanan Seafood Market in Wuhan in February 2020 detected animal coronaviruses reflective of animals previously reported being sold in the market, but none of the viruses were related to SARS-CoV-2. This study highlights that China possesses a diverse assemblage of SARS-related coronaviruses in bats, with southern China being a notable hotspot. Additional sampling in this region and in neighboring areas of Southeast Asia could shed more light on the evolutionary origin of SARS coronaviruses.

    Study design

    other

    Study population and setting

    In an effort to describe the diversity of coronaviruses in bats across China, the authors collected 13,064 samples from 56 bat species in 703 locations across 14 Chinese provinces between 2016 and January 2021. Pharyngeal and anal swabs were collected from live bats and then pooled by collection date, species, and site. The field team also had brief access to the Huanan Seafood Market in Wuhan in February 2020, a location where some of the earliest COVID-19 cases visited, and may have been a site where spillover of SARS-CoV-2 from animals occurred. Environmental swab samples (n = 22) were collected from cold storage areas that contained animal products, and 80 swab samples were taken from the environment around stalls selling animal products (ground, walls, sewers, door handles, chopping blocks, knives, and scissors). All samples from bats and the market were tested for the presence of coronavirus RNA using PCR targeting the RdRp gene and with next-generation sequencing. Phylogenetic analysis was then performed to identify different clades of sarbecoviruses in the samples, identity evidence of recombination, and infer whether identified viruses could use human ACE2 based on similarity of their spike protein with other viruses with known ability to enter human cells.

    Summary of main findings

    In the samples collected from bats, 199 of 372 pools were positive for coronavirus RNA: 113 with alphacoronaviruses, 64 with betacoronaviruses, and 22 with both genera. Samples within 44 pools containing sarbecovirus RNA (n = 1,068) were rescreened individually, yielding 146 positive samples mainly from seven Rhinolophus species; 69 of the positive samples produced full genomes from next-generation sequencing. Phylogenetic analysis showed that none of the sarbecoviruses from bats were related to SARS-CoV-2, and instead fell into multiple clades more closely related to SARS-CoV. Six identical genomes (YN2020B-G) from R. sinicus collected in Yunnan Province in 2020 had the highest sequence identity shared with SARS-CoV detected in a bat to date (95.8% across genome, 93.3% within spike); these viruses and another, YN2020H from the same species and year, were predicted to be capable of using human ACE2 based on the phylogenetic clustering with SARS-CoV. In the samples collected from the Huanan Market, three of 11 pools were positive for coronavirus RNA and four coronaviruses were detected, but none were related to SARS-CoV-2 or other sarbecoviruses. Viruses included hedgehog HKU31-related coronavirus in the subgenus Merbecovirus, rabbit HKU14-related coronavirus in the subgenus Embecovirus, canine coronavirus in the subgenus Tegacovirus, and rat coronavirus in the subgenus Embecovirus; these findings were consistent with animal species reported being sold (https://doi.org/10.1038/s41598-021-91470-2 [https://doi.org/10.1038/s41598-021-91470-2]) in the market up to 2019 (e.g., hedgehog, rabbit, bamboo rat).

    Study strengths

    This study reports an enormous sampling effort to describe coronaviruses circulating in bats in China, including during 2020 and 2021, and the team had very privileged access to the Huanan Market shortly after it was closed to the public.

    Limitations

    Due to the pooling strategy, the authors only know the individual-level prevalence of samples containing sarbecovirus RNA that were screened individually; for most other bat species that were not screened individually, these data are unavailable. Such information would have been useful to know if prevalence changed over time, especially in commonly sampled species over the long time period of the study. The inference by the authors about whether viruses could enter human cells was based solely on phylogenetic clustering with other spike sequences that were evaluated in previous studies. In vitro experiments would be needed to evaluate whether other factors beyond ACE2 binding, such as the presence of key proteases, influence infectivity of human cells. Finally, a very small area of the Huanan Market was sampled in the study, so it is unclear what proportion of the animal-selling stalls were sampled in February 2020, and whether or not they were representative of the animal species sold prior to the start of the pandemic.

    Value added

    This study provides additional data that highlights southern China as a hotspot for sarbecovirus diversity. The absence of relatives of SARS-CoV-2 in the sampled bats, despite sampling in the same areas where previous studies have detected such relatives, suggests that this lineage may not be common in most parts of China and may be more restricted to the southern provinces of China and in neighboring countries in Southeast Asia.

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