Determinants of Zika virus host tropism uncovered by deep mutational scanning


Arboviruses cycle between, and replicate in, both invertebrate and vertebrate hosts, which for Zika virus (ZIKV) involves Aedes mosquitoes and primates1. The viral determinants required for replication in such obligate hosts are under strong purifying selection during natural virus evolution, making it challenging to resolve which determinants are optimal for viral fitness in each host. Herein we describe a deep mutational scanning (DMS) strategy2,3,4,5 whereby a viral cDNA library was constructed containing all codon substitutions in the C-terminal 204 amino acids of ZIKV envelope protein (E). The cDNA library was transfected into C6/36 (Aedes) and Vero (primate) cells, with subsequent deep sequencing and computational analyses of recovered viruses showing that substitutions K316Q and S461G, or Q350L and T397S, conferred substantial replicative advantages in mosquito and primate cells, respectively. A 316Q/461G virus was constructed and shown to be replication-defective in mammalian cells due to severely compromised virus particle formation and secretion. The 316Q/461G virus was also highly attenuated in human brain organoids, and illustrated utility as a vaccine in mice. This approach can thus imitate evolutionary selection in a matter of days and identify amino acids key to the regulation of virus replication in specific host environments.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.


All prices are NET prices.

Fig. 1: DMS of C-terminal region of ZIKV E protein.
Fig. 2: In vitro characterization of ZIKV mutants.
Fig. 3: Immunofluorescence and TEM of infected C6/36 or Vero cells.
Fig. 4: Molecular analysis and modelling of the S461G and K316 mutations.
Fig. 5: Infection of iPSC-derived human brain organoids.
Fig. 6: 316G/461Q mutant as an attenuated ZIKV vaccine candidate.

Data availability

Deep sequencing data are deposited on the Sequence Read Archive under PRJNA449413. Data files are also provided as Supplementary Data 1 and 2. The software suite for DMS data analysis can be found on The actual code used to run the suite in this study is at


  1. 1.

    Musso, D. & Gubler, D. J. Zika virus. Clin. Microbiol. Rev. 29, 487–524 (2016).

    CAS  Article  Google Scholar 

  2. 2.

    Ashenberg, O., Padmakumar, J., Doud, M. B. & Bloom, J. D. Deep mutational scanning identifies sites in influenza nucleoprotein that affect viral inhibition by MxA. PLoS Pathog. 13, e1006288 (2017).

    Article  Google Scholar 

  3. 3.

    Dingens, A. S., Haddox, H. K., Overbaugh, J. & Bloom, J. D. Comprehensive mapping of HIV-1 escape from a broadly neutralizing antibody. Cell Host Microbe 21, 777–787 (2017).

    CAS  Article  Google Scholar 

  4. 4.

    Doud, M. B. & Bloom, J. D. Accurate measurement of the effects of all amino-acid mutations on influenza hemagglutinin. Viruses 8, 155 (2016).

    Article  Google Scholar 

  5. 5.

    Doud, M. B., Hensley, S. E. & Bloom, J. D. Complete mapping of viral escape from neutralizing antibodies. PLoS Pathog. 13, e1006271 (2017).

    Article  Google Scholar 

  6. 6.

    Pierson, T. C. & Diamond, M. S. The emergence of Zika virus and its new clinical syndromes. Nature 560, 573–581 (2018).

    CAS  Article  Google Scholar 

  7. 7.

    Mittal, R. et al. Zika virus: an emerging global health threat. Front. Cell Infect. Microbiol. 7, 486 (2017).

    Article  Google Scholar 

  8. 8.

    Carod-Artal, F. J. Neurological complications of Zika virus infection. Expert Rev. Anti Infect. Ther. 16, 399–410 (2018).

    CAS  Article  Google Scholar 

  9. 9.

    Qi, H. et al. A quantitative high-resolution genetic profile rapidly identifies sequence determinants of hepatitis C viral fitness and drug sensitivity. PLoS Pathog. 10, e1004064 (2014).

    Article  Google Scholar 

  10. 10.

    Wu, N. C. et al. High-throughput profiling of influenza A virus hemagglutinin gene at single-nucleotide resolution. Sci. Rep. 4, 4942 (2014).

    Article  Google Scholar 

  11. 11.

    Mlakar, J. et al. Zika virus associated with microcephaly. N. Engl. J. Med. 374, 951–958 (2016).

    CAS  Article  Google Scholar 

  12. 12.

    Setoh, Y. X. et al. De novo generation and characterization of new zika virus isolate using sequence data from a microcephaly case. mSphere 2, e00190-17 (2017).

    Article  Google Scholar 

  13. 13.

    Fontes-Garfias, C. R. et al. Functional analysis of glycosylation of zika virus envelope protein. Cell Rep. 21, 1180–1190 (2017).

    CAS  Article  Google Scholar 

  14. 14.

    Gong, D. et al. High-throughput fitness profiling of Zika virus E protein reveals different roles for glycosylation during infection of mammalian and mosquito cells. iScience 1, 97–111 (2018).

    CAS  Article  Google Scholar 

  15. 15.

    Bloom, J. D. Software for the analysis and visualization of deep mutational scanning data. BMC Bioinformatics 16, 168 (2015).

    Article  Google Scholar 

  16. 16.

    Edmonds, J. et al. A novel bacterium-free method for generation of flavivirus infectious DNA by circular polymerase extension reaction allows accurate recapitulation of viral heterogeneity. J. Virol. 87, 2367–2372 (2013).

    CAS  Article  Google Scholar 

  17. 17.

    Amarilla, A. A. et al. Chimeric viruses between Rocio and West Nile: the role for Rocio prM-E proteins in virulence and inhibition of interferon-alpha/beta signaling. Sci. Rep. 7, 44642 (2017).

    CAS  Article  Google Scholar 

  18. 18.

    Piyasena, T. B. H. et al. Infectious DNAs derived from insect-specific flavivirus genomes enable identification of pre- and post-entry host restrictions in vertebrate cells. Sci. Rep. 7, 2940 (2017).

    Article  Google Scholar 

  19. 19.

    Setoh, Y. X. et al. Helicase domain of west nile virus NS3 protein plays a role in inhibition of type I interferon signalling. Viruses 9, 326 (2017).

    Article  Google Scholar 

  20. 20.

    Setoh, Y. X. et al. Systematic analysis of viral genes responsible for differential virulence between american and australian west nile virus strains. J. Gen. Virol. 96, 1297–1308 (2015).

    CAS  Article  Google Scholar 

  21. 21.

    Kostyuchenko, V. A. et al. Structure of the thermally stable Zika virus. Nature 533, 425–428 (2016).

    CAS  Article  Google Scholar 

  22. 22.

    Sevvana, M. et al. Refinement and analysis of the mature zika virus cryo-EM structure at 3.1 A resolution. Structure 26, 1169–1177, e1163 (2018).

    CAS  Article  Google Scholar 

  23. 23.

    Watanabe, M. et al. Self-organized cerebral organoids with human-specific features predict effective drugs to combat zika virus infection. Cell Rep. 21, 517–532 (2017).

    CAS  Article  Google Scholar 

  24. 24.

    Qian, X. et al. Brain-region-specific organoids using mini-bioreactors for modeling ZIKV exposure. Cell 165, 1238–1254 (2016).

    CAS  Article  Google Scholar 

  25. 25.

    Sutarjono, B. Can we better understand how Zika leads to microcephaly? A systematic review of the effects of the Zika virus on human brain organoids. J. Infect. Dis. 219, 734–745 (2018).

    Article  Google Scholar 

  26. 26.

    Gabriel, E. et al. Recent Zika virus isolates induce premature differentiation of neural progenitors in human brain organoids. Cell Stem Cell 20, 397–406, e395 (2017).

    CAS  Article  Google Scholar 

  27. 27.

    Prow, N. A. et al. A vaccinia-based single vector construct multi-pathogen vaccine protects against both Zika and chikungunya viruses. Nat. Commun. 9, 1230 (2018).

    Article  Google Scholar 

  28. 28.

    Rey, F. A., Stiasny, K., Vaney, M. C., Dellarole, M. & Heinz, F. X. The bright and the dark side of human antibody responses to flaviviruses: lessons for vaccine design. EMBO Rep. 19, 206–224 (2018).

    CAS  Article  Google Scholar 

  29. 29.

    Mukhopadhyay, S., Kuhn, R. J. & Rossmann, M. G. A structural perspective of the flavivirus life cycle. Nat. Rev. Microbiol. 3, 13–22 (2005).

    CAS  Article  Google Scholar 

  30. 30.

    Sirohi, D. & Kuhn, R. J. Zika virus structure, maturation, and receptors. J. Infect. Dis. 216, S935–S944 (2017).

    CAS  Article  Google Scholar 

  31. 31.

    Zhang, X. et al. Cryo-EM structure of the mature dengue virus at 3.5-A resolution. Nat. Struct. Mol. Biol. 20, 105–110 (2013).

    Article  Google Scholar 

  32. 32.

    Allison, S. L., Stadler, K., Mandl, C. W., Kunz, C. & Heinz, F. X. Synthesis and secretion of recombinant tick-borne encephalitis virus protein E in soluble and particulate form. J. Virol. 69, 5816–5820 (1995).

    CAS  PubMed  PubMed Central  Google Scholar 

  33. 33.

    Allison, S. L., Stiasny, K., Stadler, K., Mandl, C. W. & Heinz, F. X. Mapping of functional elements in the stem-anchor region of tick-borne encephalitis virus envelope protein E. J. Virol. 73, 5605–5612 (1999).

    CAS  PubMed  PubMed Central  Google Scholar 

  34. 34.

    Yu, I. M. et al. Association of the pr peptides with dengue virus at acidic pH blocks membrane fusion. J. Virol. 83, 12101–12107 (2009).

    CAS  Article  Google Scholar 

  35. 35.

    Prasad, V. M. et al. Structure of the immature Zika virus at 9 A resolution. Nat. Struct. Mol. Biol. 24, 184–186 (2017).

    CAS  Article  Google Scholar 

  36. 36.

    Yu, I. M. et al. Structure of the immature dengue virus at low pH primes proteolytic maturation. Science 319, 1834–1837 (2008).

    CAS  Article  Google Scholar 

  37. 37.

    Li, L. et al. The flavivirus precursor membrane-envelope protein complex: structure and maturation. Science 319, 1830–1834 (2008).

    CAS  Article  Google Scholar 

  38. 38.

    Xie, D. Y. et al. A single residue in the alphaB helix of the E protein is critical for Zika virus thermostability. Emerg. Microbes Infect. 7, 5 (2018).

    PubMed  PubMed Central  Google Scholar 

  39. 39.

    Bloom, J. D. An experimentally determined evolutionary model dramatically improves phylogenetic fit. Mol. Biol. Evol. 31, 1956–1978 (2014).

    CAS  Article  Google Scholar 

  40. 40.

    Lancaster, M. A. et al. Cerebral organoids model human brain development and microcephaly. Nature 501, 373–379 (2013).

    CAS  Article  Google Scholar 

  41. 41.

    Dumevska, B., Bosman, A., McKernan, R., Schmidt, U. & Peura, T. Derivation of human embryonic stem cell line Genea022. Stem Cell Res. 16, 472–475 (2016).

    CAS  Article  Google Scholar 

  42. 42.

    McLean, B. J. et al. A novel insect-specific flavivirus replicates only in Aedes-derived cells and persists at high prevalence in wild Aedes vigilax populations in Sydney, Australia. Virology 486, 272–283 (2015).

    CAS  Article  Google Scholar 

Download references


This work was supported by the National Health and Medical Research Council (NHMRC) of Australia (grant No. APP1144950). A.K. and A.S. are Research Fellows with the NHMRC. E.N. was supported in part by the Daiichi Sankyo Foundation of Life Science, Japan. We thank R. Sullivan from the Queensland Brain Institute Microscopy and Histology Facility for help with preparation and imaging of the brain organoid slides. Organoid confocal microscopy work was performed in part at the Queensland node of the Australian National Fabrication Facility, a company established under the National Collaborative Research Infrastructure Strategy to provide nano- and microfabrication facilities for Australia’s researchers.

Author information




Y.S. and A.K. conceptualized the study. A.K., A.S. and Y.S. performed funding acquisition. Y.S., A.A, N.P., R.G., J.C., E.N., S.O., D.W., N.M., M.F., B.T., A.S., F.T., F.N. and P.P. performed the experiments. Y.S., A.A., R.G., J.H., E.W., S.O., J.H., A.K., J.B. and J.M. analysed the data. J.B. developed the software. T.C. established the computing resources. J.P., R.H., N.P., R.G., J.M., E.W., P.Y. and J.W. provided critical reagents and models. Y.S. and A.K. wrote the original draft. Y.S., A.K., A.S., D.W., J.B., E.W., R.H. and A.A. reviewed and edited the article.

Corresponding authors

Correspondence to Yin Xiang Setoh or Alexander A. Khromykh.

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 Notes, Supplementary Tables 1–5 and Supplementary Figures 1–9.

Reporting Summary

Supplementary Table 1

DMS mutagenesis primers.

Supplementary Data 1

Data analysis Jupyter Notebook.

Supplementary Data 2

Raw data files.

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Setoh, Y.X., Amarilla, A.A., Peng, N.Y.G. et al. Determinants of Zika virus host tropism uncovered by deep mutational scanning. Nat Microbiol 4, 876–887 (2019).

Download citation

Further reading