ACE2-Independent Bat Sarbecovirus Entry and Replication in Human and Bat Cells

Guo, Hua; Li, Ang; Dong, Tian-Yi; Su, Jia; Yao, Yudong; Zhu, Yan; Z, Shi; Letko, Michael

doi:10.1128/mbio.02566-22

Cited by 17 publications

(73 citation statements)

References 58 publications

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“…Bat-derived cell lines RSI and RSL were stored at the Wuhan Institute of Virology according to the previously described (13,34). HEK 293T, HEK 293T/17, BHK-21, VeroE6, Huh-7, and Hela were maintained in Dulbecco's modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS).…”

Section: Cellsmentioning

confidence: 99%

“…The spike or RBD coding sequences for SARS-CoV/-2, RsWIV1, Rs4081, As6526, Rp3, 4105， 163173, B288, 141341, 151491, 190366, and 190366-S-V976L were codon optimized for human cells as previously described (13). Plasmid encoding T7-promoter driven dual reporter GFP and luciferase was generated by cloning firefly luciferase downstream of GFP in pUC19-T7-IRES-GFP.…”

Section: Plasmidsmentioning

confidence: 99%

“…The inoculated plates were centrifuged at 1200 g at 4 °C for 1 hours and continued to incubate in a 37 °C incubator for 72 h. Cell-free supernatants (50 μL each time) were collected at 0, 24, 48 and 72 hours post-infection and stored at -80 °C for future use. Viral RNA was extracted and subjected to RT-PCR as previously described (13). Viral replication was quantified by RT-PCR using primers targeting the RdRp gene, forward primer: 5'-TTGTTCTTGCTCGCAAACATA-3'; reverse primer: 5'-CACACATGACCATCTCACTTAA-3'.…”

Section: Viral Replication Detected By Real-time Rt-pcrmentioning

confidence: 99%

“…The first bat sarbecoviruses discovered were clade 2 viruses and any attempts to isolate them from field samples have failed (1,2). We recently showed that a high concentration of trypsin could facilitate in vitro entry and replication of viral pseudotyped and recombinant sarbecoviruses containing clade 2 RBD spike proteins (4,13). Many other viruses have been shown to replicate in the presence of trypsin, including several gastrointestinal coronaviruses with uncharacterized host receptors (14)(15)(16)(17).…”

Section: Introductionmentioning

confidence: 99%

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Isolation of ACE2-dependent and -independent sarbecoviruses from Chinese horseshoe bats

Guo

Dong

et al. 2023

Preprint

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While the spike proteins from SARS-CoV and SARS-CoV-2 bind to host ACE2 to infect cells, the majority of bat sarbecoviruses cannot use ACE2 from any species. Despite their discovery almost 20 years ago, ACE2-independent sarbecoviruses have never been isolated from field samples, leading to the assumption these viruses pose little risk to humans. We have previously shown how spike proteins from a small group of ACE2-independent bat sarbecoviruses may possess the ability to infect human cells in the presence of exogenous trypsin. Here, we adapted our earlier findings into a virus isolation protocol, and recovered two new ACE2-dependent viruses, RsYN2012 and RsYN2016, as well as an ACE2-independent virus, RsHuB2019. Although our stocks of RsHuB2019 rapidly acquired a tissue-culture adaption that rendered the spike protein resistant to trypsin, trypsin was still required for viral entry, suggesting limitations on the exogenous entry factors that support bat sarbecoviruses. Electron microscopy revealed ACE2-independent sarbecoviruses have a prominent spike corona and share similar morphology to other coronaviruses. Our findings demonstrate a broader zoonotic threat posed by sarbecoviruses and shed light onto the intricacies of coronavirus isolation and propagationin vitro.SIGNIFICANCESeveral coronaviruses have transmitted from animals to people and 20 years of virus discovery studies have uncovered thousands of new coronavirus sequences in nature. Most of the animal-derived sarbecoviruses have never been isolated in culture due to cell incompatibilities and a poor understanding of thein vitrorequirements for their propagation. Here, we built on our growing body of work characterizing viral entry mechanisms of bat sarbecoviruses in human cells and have developed a virus isolation protocol that allows for exploration of these understudied viruses. Our protocol is robust and practical, leading to successful isolation of more sarbecoviruses than previous approaches and from field samples that had been collected over a 10-year longitudinal study.

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Section: Cellsmentioning

confidence: 99%

Section: Plasmidsmentioning

confidence: 99%

Section: Viral Replication Detected By Real-time Rt-pcrmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Isolation of ACE2-dependent and -independent sarbecoviruses from Chinese horseshoe bats

Guo

Dong

et al. 2023

Preprint

Self Cite

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“…We assume that the risk of emerging new SARS-like outbreaks is associated with social, biological and environmental components and, because there are unobserved dynamics for emerging viruses ( 30 ), we evaluated four nested spillover pathway scenarios based on landscape change and potential hosts ( 31 ): Scenario 1 (direct - known bat hosts) represents direct transmission from bats to people, facilitated by the landscape condition, human population, and known bat hosts. Although molecular investigations suggest that direct transmission of sarbecoviruses from bats to humans may be possible ( 32 ), it has yet to be better documented ( 33 ). Rather, the involvement of an intermediary or bridging host appears more likely, perhaps because this allows for recombination and viral evolution, and/or leads to greater exposure to human populations.…”

Section: Introductionmentioning

confidence: 99%

Using drivers and transmission pathways to identify SARS-like coronavirus spillover risk hotspots

Muylaert

Wilkinson

Kingston

et al. 2022

Preprint

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The emergence of SARS-like coronaviruses is a multi-stage process from wildlife reservoirs to people. Here we characterize multivariate indicators associated with the risk of zoonotic spillover of SARS-like coronaviruses in different areas to help inform surveillance and mitigation activities. We consider direct and indirect transmission pathways by modeling four scenarios with livestock and mammalian wildlife as potential and known reservoirs, before examining how access to healthcare varies within areas. We found 19 multivariate clusters that had differing risk factor contributions. High-risk areas were mostly close (11-20%) rather than far (<1%) from healthcare. With the presented framework, areas with the highest estimated risk can be priority intervention targets in which risk management strategies can be implemented, such as land use planning and preventive measures to reduce contact between people and potential hosts.

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