1. Our take

    In this preprint that has not yet been peer-reviewed, the authors report new evidence of alphacoronaviruses and betacoronaviruses in bats sampled in Yunnan, China, in 2019 and 2020. The results contribute to evidence that Rhinolophus species bats are a rich source of viruses related to SARS-CoV and SARS-CoV-2. However, a virus with sufficient similarity to SARS-CoV-2 to represent a direct ancestor in bats was not found in the study.

    Study design

    other

    Study population and setting

    To examine the diversity of coronaviruses circulating in bats in southern China, authors collected samples from 23 bat species in the Xishuangbanna Tropical Botanical Garden and nearby areas in Mengla County, Yunnan province, China between May 2019 and November 2020. In total, 411 samples from 342 bats were collected, including 283 fecal samples, 109 oral swabs, and 19 urine samples. Samples were merged into 100 pooled libraries based on sample type, species, and collection date; 18 pools were discussed in a previous study (https://doi.org/10.1016/j.cub.2020.05.023). Metatranscriptomic sequencing was performed on pools to detect viral RNA and pools that were positive for coronaviruses related to SARS-CoV-2 were further tested using PCR and sequencing to identify individual samples that were positive prior to pooling. In addition to viral detection, the authors used occurrence data and numerous environmental variables to predict the geographic distributions of 49 Rhinolophus bat species in Southeast Asia and China.

    Summary of main findings

    Coronavirus sequences were detected in 40/100 libraries, producing 26 long sequences from 20 libraries. Of the 26 sequences, 9 were identified as the genus Betacoronavirus and 17 were genus Alphacoronavirus. All 9 betacoronaviruses were related to SARS-CoV or SARS-CoV-2. PCR tests confirmed that 9 individual samples from Rhinolophus malayanus, R. pusillus, and R. stheno sampled between May and July 2020 were positive for betacoronaviruses. Two viruses (RmYN01 and RmYN02) closely related to SARS-CoV-2 from one pool of fecal samples from R. malayanus were described previously (https://doi.org/10.1016/j.cub.2020.05.023). Three viruses shared between 91.6 and 93.3% sequence identity across the genome with SARS-CoV and other related viruses from bats sampled previously in Yunnan. Four viruses (RpYN06, RsYN04, RmYN05, and RmYN08) were more similar to SARS-CoV-2. RpYN06 from R. pusillus shared 94.5% sequence identity with SARS-CoV-2 across the genome while the latter three were more distantly related to SARS-CoV-2 and were more similar to pangolin coronaviruses. Models using environmental variables showed high accuracy in their prediction of Rhinolophus bat species distributions, showing that up to 23 Rhinolophus species can coexist in Southeast Asia, particularly in Thailand, Laos, Vietnam, and southern China.

    Study strengths

    This is the first study reporting results of new sampling and detection of coronaviruses in bats in China after the beginning of the COVID-19 pandemic. Specific methodological strengths include correcting for the possibility of false positives due to sequencing errors (e.g., index hopping) by removing short sequences with high similarity to others within the same chip and same lane in the sequencer. They also confirmed via RT-PCR which bat samples were positive within the pools with amplified coronavirus sequences. Finally, the identities of bat species that were positive for coronaviruses were confirmed by sequencing mitochondrial DNA.

    Limitations

    The supplementary material is not currently available with the preprint, so it is unclear how many pools or individual samples from each bat species were positive for coronaviruses. Additionally, the authors only validated the presence of coronaviruses in individual bat samples with PCR for pools positive for betacoronaviruses. Thus, it is not clear which individual bat samples were positive for the alphacoronaviruses described.

    Value added

    The paper provides valuable new information on the circulation of coronaviruses in bat species in Yunnan sampled in 2019 and throughout 2020. The results indicate multiple betacoronaviruses related to SARS-CoV and SARS-CoV-2 can be found in Rhinolophus species bats in this region. Paired with the ecological modeling of Rhinolophus species ranges, this manuscript contributed to existing evidence that Yunnan and other parts of Southeast Asia are regions with high diversity of Rhinolophus species and are likely important hotspots for betacoronavirus diversity. Nevertheless, the viruses amplified do not provide evidence that bats harbor the direct ancestor of SARS-CoV-2. Even though the described RpYN06 virus was 94.5% similar to SARS-CoV-2 across the whole genome, this virus is still too genetically divergent to be the progenitor of SARS-CoV-2.

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