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SARS-CoV-2-related bat viruses evade human intrinsic immunity but lack efficient transmission capacity

Nature Microbiology volume 9, pages 2038–2050 (2024)Cite this article

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Abstract

Circulating bat coronaviruses represent a pandemic threat. However, our understanding of bat coronavirus pathogenesis and transmission potential is limited by the lack of phenotypically characterized strains. We created molecular clones for the two closest known relatives of SARS-CoV-2, BANAL-52 and BANAL-236. We demonstrated that BANAL-CoVs and SARS-CoV-2 have similar replication kinetics in human bronchial epithelial cells. However, BANAL-CoVs have impaired replication in human nasal epithelial cells and in the upper airway of mice. We also observed reduced pathogenesis in mice and diminished transmission in hamsters. Further, we observed that diverse bat coronaviruses evade interferon and downregulate major histocompatibility complex class I. Collectively, our study demonstrates that despite high genetic similarity across bat coronaviruses, prediction of pandemic potential of a virus necessitates functional characterization. Finally, the restriction of bat coronavirus replication in the upper airway highlights that transmission potential and innate immune restriction can be uncoupled in this high-risk family of emerging viruses.

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Fig. 1: Molecular clones of BANAL-CoVs exhibit similar kinetics and drug susceptibility to SARS-CoV-2.
Fig. 2: Bat SARS-related viruses evade human innate immunity and downregulate MHC-I.
Fig. 3: BANAL-52, BANAL-236 and SARS-CoV-2 display a gradient of pathogenesis in K18-hACE2 mice.
Fig. 4: BANAL-CoVs have impaired transmission capacity in a hamster model.
Fig. 5: The S1/S2 FCS promotes replication in HNECs and hamsters.

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Data availability

All data related to this manuscript were included in the source data files. GenBank accession codes for the molecular clones of BANAL-52 and BANAL-236 are PP856573 and PP856574, respectively. Viral stocks and plasmids are available with a material transfer agreement (MTA) and confirmation of appropriate biosafety facilities and procedures. Since potential experiments of concern could be performed with these viral strains, we will require a list of planned experimental procedures, to be reviewed before the MTA is executed. Yale University will review and determine the MTA timeline. Correspondence should be sent to A.I. (akiko.iwasaki@yale.edu) and C.B.W. (craig.wilen@yale.edu).

Code availability

The NGS sequencing raw data and the code to reproduce the analysis of the SARS-CoV-2 FCS from the Extended Data can be found on Zenodo at https://zenodo.org/records/11519958 (ref. 56).

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Acknowledgements

We thank M. Linehan, J. Klein, J. Frank, I. Ott and J. Wei (Yale) for technical assistance and helpful discussions; R. Baric from UNC-Chapel Hill for kindly providing WIV1 and SHC014 stocks; B. Graham from VRC-NIAID for providing Vero-ACE2/TMPRSS2 cells; D. Brackney from Connecticut Agricultural Experiment Section for providing BHK-21 clone 15 cells; B. Fontes and Yale EH&S for assistance in performing enhanced biosafety level 3 research. D.R.M. was funded by a Hanna H. Gray Fellowship from the Howard Hughes Medical Institute. B.L.M. was supported by NIH T32 HL007974. This study was partly funded by Howard Hughes Medical Institute (to A.I.). C.B.W. was supported by Burroughs Wellcome Fund, NIH R21 AI173821 and the Smith Family Foundation. This study was partially funded by ASAP and MAVDA Development Research programme grants (B.D.L, C.B.W.). The funders had no role in study design, data collection and analysis, the decision to publish or the preparation of the manuscript. This manuscript is dedicated in loving memory of Brett D. Lindenbach, who will be deeply missed as a mentor, friend and colleague in the scientific and virology community.

Author information

Authors and Affiliations

  1. Department of Immunobiology, Yale University School of Medicine, New Haven, CT, USA

    Mario A. Peña-Hernández, Miyu Moriyama, Yong Kong, Tianyang Mao, Madeleine C. Mankowski, David R. Martinez, Benjamin Israelow, Akiko Iwasaki & Craig B. Wilen

  2. Department of Microbial Pathogenesis, Yale University School of Medicine, New Haven, CT, USA

    Mario A. Peña-Hernández, Emma L. Keeler, Shuo Zhang & Brett D. Lindenbach

  3. Department of Laboratory Medicine, Yale School of Medicine, New Haven, CT, USA

    Mario A. Peña-Hernández, Mia Madel Alfajaro, Renata B. Filler, Emma L. Keeler, Zara E. Ranglin, Bridget L. Menasche, Madeleine C. Mankowski, Zhe Zhao, Shuo Zhang & Craig B. Wilen

  4. Department of Epidemiology of Microbial Diseases, Yale School of Public Health, New Haven, CT, USA

    Chantal B. F. Vogels, Anne M. Hahn, Chaney C. Kalinich, Nathan D. Grubaugh & Akiko Iwasaki

  5. Department of Molecular, Cellular and Developmental Biology, Yale University, New Haven, CT, USA

    Nicholas Huston, Han Wan, Rafael Araujo-Tavares, Anna Marie Pyle & Akiko Iwasaki

  6. Department of Pathology, Yale School of Medicine, New Haven, CT, USA

    Robert Homer

  7. Department of Chemistry, Yale University, New Haven, CT, USA

    Anna Marie Pyle

  8. Howard Hughes Medical Institute, Chevy Chase, MD, USA

    Anna Marie Pyle & Akiko Iwasaki

  9. Department of Internal Medicine, Section of Infectious Diseases, Yale School of Medicine, New Haven, CT, USA

    Benjamin Israelow

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  1. Mario A. Peña-Hernández
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Contributions

M.A.P-H., A.I. and C.B.W. conceptualized the project. M.A.P.-H., M.M.A., R.B.F., B.L.M., M.M., E.L.K., Z.E.R., Y.K., C.B.F.V., A.M.H., C.C.K., B.D.L, R.H., A.M.P., D.R.M., N.D.G., B.I., A.I. and C.B.W. developed the methodology. M.A.P.-H., M.M.A., R.B.F., B.L.M., M.M., Z.E.R., Y.K., T.M., M.C.M., Z.Z., C.B.F.V., S.Z., N.H., H.W., R.A.-T., R.H., N.D.G. and B.I. conducted investigations. M.A.P.-H., M.M., Z.E.R., Y.K., C.B.F.V., R.H., A.I. and C.B.W. performed visualization. A.I. and C.B.W. acquired funding. M.A.P.-H., A.I. and C.B.W. wrote the original draft. All authors reviewed and edited the manuscript.

Corresponding authors

Correspondence to Akiko Iwasaki or Craig B. Wilen.

Ethics declarations

Competing interests

A.I. serves as a consultant for RIGImmune, Xanadu Bio, Paratus Bio and Invisishield, and serves on the Board of Directors for Roche Holdings Ltd. A.I. is an investigator of the Howard Hughes Medical Institute. C.B.W. is on the Scientific Advisory Board for ExcepGen. The remaining authors declare no competing interests.

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Nature Microbiology thanks Zheng-Li Shi, Youchun Wang and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 BANAL-236 spike NTD is divergent from SARS-2 and BANAL-52.

(A) Protein alignment of BANAL-CoV and SARS-2 spikes. The S1 NTD and S1/S2 FCS are highlighted with red boxes.

Extended Data Fig. 2 BANAL-CoVs mNGs are susceptible to small molecule inhibitors and anti-SARS-2 sera.

(A) Representative image of an agarose gel showing all the BANAL-CoVs fragments synthesized and amplified before cloning them to their respective vectors. (B) Representative image of an agarose gel showing full-length genome ligation of BANAL-52 and BANAL-236 cDNA used for in vitro transcription and to produce infectious viruses. (C) Bat CoV titres in Vero-ACE2/TMPRSS2 cells. (D) BANAL-CoV and BANAL-CoV mNG titres in Vero-ACE2/TMPRSS2 cells. (E) Representative images of Vero-ACE2/TMPRSS2 cells infected with mNeonGreen viruses. (F-H) Inhibition of the three viruses by two doses of Pfizer (F) of Moderna (G) vaccinated pooled sera assessed at 48 hpi (MOI 0.1) as determined by cell viability measured by Cell-Titer Glo. Inhibition of the three viruses by convalescent pooled sera (H) assessed at 48 hpi (MOI 0.1) as determined by cell viability measured by Cell-Titer Glo. Color-coded cures represent a non-linear regression least square fit of biological replicates (n = 3) and shading represents 95% confidence intervals of the fit. For panels F-H, data are represented by mean ± SD. All figure panels are representative of two independent experiments.

Source data

Extended Data Fig. 3 BANAL-CoVs utilize similar entry factors than SARS-2.

(A-C) Inhibition of the three sarbecoviruses by (A) E64d assessed at 24 hpi in Vero-E6 as determined by % mNG expressing cells, (B) Camostat mesylate and (C) E64d assessed at 48 hpi in Vero-ACE2/TMPRSS2 cells as determined by cell viability measured by Cell-Titer Glo. Color-coded curves represent a non-linear regression least square fit of 4 biological replicates and shading represents 95% confidence intervals of the fit. Panels A-C are representative of two independent experiments. (D-E). Inhibition of the three sarbecoviruses by (D) camostat mesylate and (E) E64d in HBECs culture at 48 hpi. Error bars illustrate standard deviation. Significance between groups was assessed by two-way ANOVA corrected to multiple comparisons. For panel D, p values of mock and camostat treated cells were all p < 0.0001 for SARS-2, BANAL-52 and BANAL-236. Panels D and E are pooled from two independent experiments with n = 3 biological replicates each (n = 6 total). Mock treated wells are same for panels D and E. For all panels, data are represented by mean ± SD.

Source data

Extended Data Fig. 4 ISG induction in primary human airway cells after BANAL-CoV infection.

(A-B) mRNA fold change of ISG induction relative to uninfected cells measured by RT-qPCR from infected HBECs (A) or HNECs (B). Fold change was assessed by 2–ΔΔCt method using gapdh as a control. Significance of expression between viruses was assessed by one-way ANOVA. Panels A-B are pooled from two-independent experiments with a total of n = 6 biological replicates. For panel A, exact p values for ISG15 SARS-2 vs BANAL-52 p = .0105 and SARS-2 vs BANAL.236 p = .0162. For IFIT3 SARS-2 vs BANAL-52 p = .0129 and SARS-2 vs BANAL.236 p = .0207. For panel B, exact p values for IFIT3 SARS-2 vs BANAL-52 p = .0125 and SARS-2 vs BANAL-236 p = .0386. For RSAD2 SARS-2 vs BANAL-52 p = .0449. (C-E) Reduction of infectious viral titres of IFN-pretreated HBECs normalized to the median of the mock group of SARS-CoV-2, BANAL-52 and BANAL-236 infection using (C) IFN-α, (D) IFN-γ, and (E) IFN-λ. Graphs represent pooled data from two independent experiments (n = 5 biological replicates for IFN pre-treated cells, n = 6 biological replicates for IFN untreated infected cells). Significance between viral titres was assessed by one-way ANOVA corrected for multiple comparisons. For panel C SARS-2 vs BANAL-52 and SARS-2 vs BANAL-236 p < 0.0001. For panel D, SARS-2 vs BANAL-52 p = .0001 and SARS-2 vs BANAL-236 p < 0.0001. For panel E, SARS-2 vs BANAL-52 p = .003 and SARS-2 vs BANAL-236 p = 0.007. (F) Infectious viral titres and (G) normalization values of SARS-CoV-2 and BANAL-CoVs in HNECs pre-treated with IFN-α. Graphs F-G represent pooled data from two independent experiments (n = 6). Significance between viruses in IFN normalized to mock values (second graph) was assessed by one-way ANOVA corrected for multiple comparisons. For panel F, Mock treated cells SARS-2 vs BANAL-52 p < 0.0001 and SARS-2 vs BANAL-236 p = .0006. For IFN treated cells, p = .0350. For panel G, SARS-2 vs BANAL-52 p = .0006 and SARS-2 vs BANAL-236 p = .0045. (H) Normalization of pseudovirus infectivity following IFN-α pre-treatment in Vero-ACE2/TMPRSS2 cells for SARS-CoV-2 and BANAL-CoVs. One out of three independent experiments are shown. N = 6 biological replicates for SARS-2 and BANAL-52, and n = 5 for BANAL-236. Data from panels C-H are represented by mean ± SD. (I) Gating strategy for flow cytometry analysis. (J) Representative graphs of detection efficiency of A549-ACE2/TMPRSS2 SARS-2- spike positive cells by flow cytometry. N = 4 biological replicates out of 2 independent experiments. Significance between viruses was assessed by one-way ANOVA corrected for multiple comparisons. P values for panel J of SARS-2 vs mock infected, SARS-2 vs BANAL-52, and SARS-2 vs BANAL-236 were all p < 0.0001. For all panels, error bars illustrate standard deviation, and ** indicates p < 0.01, *** indicates p < 0.001, **** indicates p < 0.0001.

Source data

Extended Data Fig. 5 BANAL-CoVs do not induce robust innate immune response in mouse nasal turbinates.

(A) mRNA fold change of ISGs and inflammatory cytokines from lungs of infected mice. (B) mRNA fold change of ISGs and inflammatory cytokines from nasal turbinates of infected mice. Panels A-B were measured by RT-qPCR at 2 dpi. Fold change was assessed by 2–ΔΔCt method using b-actin as a control. Significance of expression between viruses was assessed by one-way ANOVA corrected for multiple comparisons. * indicates p < 0.05, ** indicates p < 0.01, **** indicates p < 0.0001. Exact p values for panel A are: ISG15 (BANAL-52 vs BANAL-236 p = .0374), Mx1 (SARS-2 vs BANAL-52 p = .0213, BANAL-52 vs BANAL-236 p = .0172), IFIT1 (SARS-2 vs BANAL-52 p = .0035, BANAL-52 vs BANAL-236 p = .0049). OAS1 (BANAL-52 vs BANAL-236 p = .0062), TNF (SARS-2 vs BANAL-52 p < 0.0001, BANAL-52 vs BANAL-236 p = .0423), IL-6 (SARS-2 vs BANAL-52 p = .0166, BANAL-52 vs BANAL-236 p = .0109), CXCL10 (SARS-2 vs BANAL-52 p = .0047, BANAL-52 vs BANAL-236 p = .0116). For panel B, exact p values are: IFIT1 (SARS-2 vs BANAL-52 p = .0240), CXCL10 (SARS-2 vs BANAL-52 p = .04), CXCL19 (SARS-2 vs BANAL-52 p = .0118 and SARS-2 vs BANAL-236 p = .0123). (C) Heatmap of ISGs and cytokines expression from nasal turbinates of K18-mice infected with the three sarbecoviruses measured by qPCRs. Values are from panel S5B. For panel Ain this figure the n of mice is: SARS-2 n = 11, BANAL-52 n = 12, BANAL-236 n = 13. For panels B and C the n of mice is: SARS-2 n = 8, BANAL-52 n = 8, BANAL-236 n = 8. Figure is pooled from three-independent experiments.

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Extended Data Fig. 6 BANAL-CoVs do not induce lung inflammation in hamsters.

(A) Measurement of hamster ACE2 usage by the three sarbecovirus spikes using lentiviral-based pseudoviruses (n = 4 biological replicates). Significance was assessed by two-way ANOVA. Data is represented by mean ± SD. Graphs represent one out of two independent experiments. Dashed line represents limit of detection. (B) H&E staining from infected hamster lungs with each of the three sarbecoviruses or uninfected control. Images are representative from 5 hamsters after 2 dpi.

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Extended Data Fig. 7 SARS-2 FCS is enriched upon infection in hamsters.

(A) Nucleotide and aminoacid alignment of 7 most abundant reads from SARS-CoV-2 ΔFCS stock (Reads 1-7) and reads that have mutations in the FCS region but were not between the top 20 most abundant reads (Reads 8-10). (B) Raw reads from the S1/S2 spike junction region highlighting total number reads and reads with FCS sequence. (C) Percent of reads that possess FCS from virus stock and experimental samples from hamster lungs after NGS sequencing. (D) Fold change of the number of reads containing the FCS sequence comparing SARS-2 ΔFCS stock vs hamster infected with SARS-2 ΔFCS at 2 dpi. For sequencing analyses, 4 different tubes of SARS-2 ΔFCS stock and 2 samples from hamsters infected with SARS-2 WT and SARS-2 ΔFCS were used. For panels B and C, data are represented by mean ± SD. For panel D, individual values are shown.

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Supplementary information

Supplementary Information

Uncropped gels for Extended Data Fig. 2, and raw images for Extended Data Figs. 2e and 6b.

Reporting Summary

Supplementary Tables

Supplementary Tables 1–3.

Supplementary Data 1

Molecular clone design of BANAL-CoVs.

Supplementary Data 2

Code for analysing SARS-2 ΔFCS sequences.

Supplementary Data 3

BioRender license agreement for Figs. 1b, 3a and 4a.

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Peña-Hernández, M.A., Alfajaro, M.M., Filler, R.B. et al. SARS-CoV-2-related bat viruses evade human intrinsic immunity but lack efficient transmission capacity. Nat Microbiol 9, 2038–2050 (2024). https://doi.org/10.1038/s41564-024-01765-z

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