Bat influenza viruses transmit among bats but are poorly adapted to non-bat species

Article metrics

Abstract

Major histocompatibility complex class II (MHC-II) molecules of multiple species function as cell-entry receptors for the haemagglutinin-like H18 protein of the bat H18N11 influenza A virus, enabling tropism of the viruses in a potentially broad range of vertebrates. However, the function of the neuraminidase-like N11 protein is unknown because it is dispensable for viral infection or the release of H18-pseudotyped viruses. Here, we show that infection of mammalian cells with wild-type H18N11 leads to the emergence of mutant viruses that lack the N11 ectodomain and acquired mutations in H18. An infectious clone of one such mutant virus, designated rP11, appeared to be genetically stable in mice and replicated to higher titres in mice and cell culture compared with wild-type H18N11. In ferrets, rP11 antigen and RNA were detected at low levels in various tissues, including the tonsils, whereas the wild-type virus was not. In Neotropical Jamaican fruit bats, wild-type H18N11 was found in intestinal Peyer’s patches and was shed to high concentrations in rectal samples, resulting in viral transmission to naive contact bats. Notably, rP11 also replicated efficiently in bats; however, only restored full-length N11 viruses were transmissible. Our findings suggest that wild-type H18N11 replicates poorly in mice and ferrets and that N11 is a determinant for viral transmission in bats.

Access optionsAccess options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Fig. 1: Mutations in H18 and N11 enhance replication of H18N11 in vitro.
Fig. 2: H18N11 replicates in the absence of the N11 head domain in vitro.
Fig. 3: H18K170R,N250S, rather than N11 truncation, enhances infectivity in vitro.
Fig. 4: Viral replication of WT H18N11 and rP11 is constrained to the URT of infected mice.
Fig. 5: rP11 exhibits only limited replication ability in ferrets.
Fig. 6: Bat IAVs that encode full-length N11 are spread among bats.

Data availability

The data supporting the findings of this study are available within the paper and its Supplementary Information. Any further relevant data are available from the corresponding authors on reasonable request.

References

  1. 1.

    Webster, R. G., Bean, W. J., Gorman, O. T., Chambers, T. M. & Kawaoka, Y. Evolution and ecology of influenza A viruses. Microbiol. Rev. 56, 152–179 (1992).

  2. 2.

    Desselberger, U. et al. Biochemical evidence that “new” influenza virus strains in nature may arise by recombination (reassortment). Proc. Natl Acad. Sci. USA 75, 3341–3345 (1978).

  3. 3.

    Garten, R. J. et al. Antigenic and genetic characteristics of swine-origin 2009 A(H1N1) influenza viruses circulating in humans. Science 325, 197–201 (2009).

  4. 4.

    Herfst, S. et al. Airborne transmission of influenza A/H5N1 virus between ferrets. Science 336, 1534–1541 (2012).

  5. 5.

    Imai, M. et al. Experimental adaptation of an influenza H5 HA confers respiratory droplet transmission to a reassortant H5 HA/H1N1 virus in ferrets. Nature 486, 420–428 (2012).

  6. 6.

    Tong, S. et al. A distinct lineage of influenza A virus from bats. Proc. Natl Acad. Sci. USA 109, 4269–4274 (2012).

  7. 7.

    Tong, S. et al. New world bats harbor diverse influenza A viruses. PLoS Pathog. 9, e1003657 (2013).

  8. 8.

    Campos, A. C. A. et al. Bat influenza A(HL18NL11) virus in fruit bats, Brazil. Emerg. Infect. Dis. 25, 333–337 (2019).

  9. 9.

    Sun, X. et al. Bat-derived influenza hemagglutinin H17 does not bind canonical avian or human receptors and most likely uses a unique entry mechanism. Cell Rep. 3, 769–778 (2013).

  10. 10.

    Zhu, X. et al. Crystal structures of two subtype N10 neuraminidase-like proteins from bat influenza A viruses reveal a diverged putative active site. Proc. Natl Acad. Sci. USA 109, 18903–18908 (2012).

  11. 11.

    Zhu, X. et al. Hemagglutinin homologue from H17N10 bat influenza virus exhibits divergent receptor-binding and pH-dependent fusion activities. Proc. Natl Acad. Sci. USA 110, 1458–1463 (2013).

  12. 12.

    Li, Q. et al. Structural and functional characterization of neuraminidase-like molecule N10 derived from bat influenza A virus. Proc. Natl Acad. Sci. USA 109, 18897–18902 (2012).

  13. 13.

    Moreira, E. A. et al. Synthetically derived bat influenza A-like viruses reveal a cell type- but not species-specific tropism. Proc. Natl Acad. Sci. USA 113, 12797–12802 (2016).

  14. 14.

    Maruyama, J. et al. Characterization of the glycoproteins of bat-derived influenza viruses. Virology 488, 43–50 (2016).

  15. 15.

    Karakus, U. et al. MHC class II proteins mediate cross-species entry of bat influenza viruses. Nature 567, 109–112 (2019).

  16. 16.

    Maher, J. A. & DeStefano, J. The ferret: an animal model to study influenza virus. Lab Anim. 33, 50–53 (2004).

  17. 17.

    Belser, J. A., Katz, J. M. & Tumpey, T. M. The ferret as a model organism to study influenza A virus infection. Dis. Model Mech. 4, 575–579 (2011).

  18. 18.

    Moore, I. N. et al. Severity of clinical disease and pathology in ferrets experimentally infected with influenza viruses is influenced by inoculum volume. J. Virol. 88, 13879–13891 (2014).

  19. 19.

    Brandtzaeg, P. Food allergy: separating the science from the mythology. Nat. Rev. Gastroenterol. Hepatol. 7, 380–400 (2010).

  20. 20.

    Webster, R. G., Yakhno, M., Hinshaw, V. S., Bean, W. J. & Murti, K. G. Intestinal influenza: replication and characterization of influenza viruses in ducks. Virology 84, 268–278 (1978).

  21. 21.

    Barclay, W. S. Receptor for bat influenza virus uncovers potential risk to humans. Nature 567, 35–36 (2019).

  22. 22.

    Hughes, M. T., Matrosovich, M., Rodgers, M. E., McGregor, M. & Kawaoka, Y. Influenza A viruses lacking sialidase activity can undergo multiple cycles of replication in cell culture, eggs, or mice. J. Virol. 74, 5206–5212 (2000).

  23. 23.

    Kalthoff, D. et al. Truncation and sequence shuffling of segment 6 generate replication-competent neuraminidase-negative influenza H5N1 viruses. J. Virol. 87, 13556–13568 (2013).

  24. 24.

    Samson, M. et al. Characterization of drug-resistant influenza virus A(H1N1) and A(H3N2) variants selected in vitro with laninamivir. Antimicrob. Agents Chemother. 58, 5220–5228 (2014).

  25. 25.

    Ann, J. et al. Impact of a large deletion in the neuraminidase protein identified in a laninamivir-selected influenza A/Brisbane/10/2007 (H3N2) variant on viral fitness in vitro and in ferrets. Influenza Other Respir. Virus. 10, 122–126 (2016).

  26. 26.

    Juozapaitis, M. et al. An infectious bat-derived chimeric influenza virus harbouring the entry machinery of an influenza A virus. Nat. Commun. 5, 4448 (2014).

  27. 27.

    Martin, M. Cutadapt removes adapter sequences from high-throughput sequencing reads. EMBnet J. 17, 10 (2011).

  28. 28.

    Langmead, B. & Salzberg, S. L. Fast gapped-read alignment with Bowtie 2. Nat. Methods 9, 357–359 (2012).

  29. 29.

    Li, H. et al. The Sequence Alignment/Map format and SAMtools. Bioinformatics 25, 2078–2079 (2009).

  30. 30.

    Wilm, A. et al. LoFreq: a sequence-quality aware, ultra-sensitive variant caller for uncovering cell-population heterogeneity from high-throughput sequencing datasets. Nucleic Acids Res. 40, 11189–11201 (2012).

  31. 31.

    Klupp, B. G., Granzow, H. & Mettenleiter, T. C. Primary envelopment of pseudorabies virus at the nuclear membrane requires the UL34 gene product. J. Virol. 74, 10063–10073 (2000).

  32. 32.

    Grund, C. et al. A novel European H5N8 influenza a virus has increased virulence in ducks but low zoonotic potential. Emerg. Microbes Infect. 7, 132 (2018).

  33. 33.

    Yewdell, J., Frank, E. & Gerhard, W. Expression of influenza A virus internal antigens on the surface of infected P815 cells. J. Immunol. 126, 1814–1819 (1981).

Download references

Acknowledgements

We thank S. Schuparis and G. Czerwinski for histotechnological support and S. Giese and H. Bolte for reading the manuscript. This work was supported by grants from the DFG to M.S. (SCHW 632/17-2) and M.B. (BE 5187/4-2); the CRIP and the Saint Jude CEIRS, two NIAID-funded CEIRS to A.G.-S. (HHSN272201400008C) and W.M. (HHSN272201400006C), respectively; NIH NIAID grants to G.D.E. (AI067380), R.A.M. (AI134108), W.M. and T.S. (1R01AI134768); the CSU Vice President for Research, College of Veterinary Medicine and Biomedical Sciences, and Department of Microbiology, Immunology and Pathology to T.S.; and the China Scholarships Council (201506170046) to W.R. The funders had no role in study design, data collection and analysis, decision to publish or preparation of the manuscript.

Author information

K.C., M.S., M.B., W.M. and T.S. conceived and designed the experiments. K.C. and W.R. performed in vitro and mouse experiments. M.G. and D.H. performed ferret experiments. T.S., A.M., M.E., C.L.C., W.M., J.L. and J.M. performed bat experiments. J.S. and R.U. performed pathology, immunohistochemistry and in situ hybridization. A.P. and R.A.M. performed NGS. K.F. performed electron microscopy. K.C., M.B., T.S., A.G.-S., J.S., R.U., T.A.A., G.D.E., W.M. and M.S. analysed the data. K.C. and M.S. wrote the paper with input from all of the other authors.

Correspondence to Wenjun Ma or Tony Schountz or Martin Beer or Martin Schwemmle.

Ethics declarations

Competing interests

The authors declare no competing interests.

Additional information

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary information

Supplementary Information

Supplementary Tables 1–6, Supplementary Figs. 1–6.

Reporting Summary

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark