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Ancestral sequence reconstruction pinpoints adaptations that enable avian influenza virus transmission in pigs

Abstract

Understanding the evolutionary adaptations that enable avian influenza viruses to transmit in mammalian hosts could allow better detection of zoonotic viruses with pandemic potential. We applied ancestral sequence reconstruction to gain viruses representing different adaptive stages of the European avian-like (EA) H1N1 swine influenza virus as it transitioned from avian to swine hosts since 1979. Ancestral viruses representing the avian-like precursor virus and EA swine influenza viruses from 1979–1983, 1984–1987 and 1988–1992 were reconstructed and characterized. Glycan-binding analyses showed stepwise changes in the haemagglutinin receptor–binding specificity of the EA swine influenza viruses—that is, from recognition of both α2,3- and α2,6-linked sialosides to recognition of α2,6-linked sialosides only; however, efficient transmission in piglets was enabled by adaptive changes in the viral polymerase protein and nucleoprotein, which have been fixed since 1983. PB1-Q621R and NP-R351K increased viral replication and transmission in piglets when introduced into the 1979–1983 ancestral virus that lacked efficient transmissibility. The stepwise adaptation of an avian influenza virus to a mammalian host suggests that there may be opportunities to intervene and prevent interspecies jumps through strategic coordination of surveillance and risk assessment activities.

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Fig. 1: Avian and EA swine influenza viruses differed in receptor binding profiles, in vitro and ex vivo replication efficiencies and contact transmissibility in pigs.
Fig. 2: Characterization of reconstructed EA influenza viruses representing different evolutionary stages of EA swine influenza viruses.
Fig. 3: EA swine H1N1 viruses acquired efficient pig-to-pig transmissibility after 1983.
Fig. 4: Introduction of HA and NA genes derived from the RG-EA3 virus did not enhance the efficient contact transmissibility of the RG-EA2 virus in pigs.
Fig. 5: The NP-R351K mutation in EA swine influenza viruses was the minimal molecular change required to facilitate the transmission of the non-transmissible RG-EA2 virus.

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Data availability

Sequences of Sanger sequencing validated in the study were deposited to the public database GISAID under the following accession numbers: DK/77, EPI_ISL_539860; A/swine/Arnsberg/6554/1979, EPI_ISL_539869; Be01-lung, EPI_ISL_1055769; Be02-lung, EPI_ISL_1055773; A/swine/Netherlands/3/1980, EPI_ISL_539861; SW/81, EPI_ISL_539862; A/swine/Netherlands/12/1985, EPI_ISL_539863; SW/92, EPI_ISL_539864; A/swine/Hong Kong/8512/2001, EPI_ISL_539865; A/swine/Hong Kong/72/2007, EPI_ISL_539866; A/swine/Hong Kong/1559/2008, EPI_ISL_539867; A/swine/Hong Kong/NS4848/2011, EPI_ISL_539868; RG-EA1, EPI_ISL_539852; RG-EA2, EPI_ISL_539853; RG-EA3, EPI_ISL_539854; and RG-EA4, EPI_ISL_539855. Samples of next-generation sequencing have been deposited in the Sequence Read Archive of the NCBI under project accession number PRJNA725802. Source data are provided with this paper.

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Acknowledgements

We thank the St. Jude core facilities, including the Animal Resources Centre and the Veterinary Pathology Core for technical support. This study was supported by contract HHSN272201400006C from NIAID, NIH, USA, and the Theme-Based Research Scheme (grant nos T11-705/14N and T11-712/19-N) from the Research Grants Council, Hong Kong SAR, China. Y.C.F.S., U.J., J.J. and G.J.D.S. are supported by the Duke–NUS Signature Research Programme funded by the Ministry of Health, Singapore.

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Contributions

H.-L.Y., G.J.D.S. and R.J.W. conceived and designed the experiments. W.S., Y.C.F.S., U.J., J.J. and G.J.D.S. performed the sequence analyses. W.S., Y.Z., M.Z. and Y.J. performed in vitro experiments. W.S. and H.T.B. performed ex vivo experiments. W.S., R.H., J.D., J.-C.C., T.J., A.R., J.F., P.N.Q.P., C.K., J.C.J. and L.K. performed in vivo animal experiments. S.K., S.P., M.C.W.C., R.G.W., C.-Y.W., K.V.R. and M.P. provided novel reagents to support the study. W.S., Y.C.F.S., G.J.D.S., R.J.W., M.P. and H.-L.Y. wrote the draft manuscript, and all authors reviewed the paper.

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Correspondence to Richard J. Webby, Gavin J. D. Smith or Hui-Ling Yen.

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The authors declare no competing interests.

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Peer review information Nature Microbiology thanks Eric Gaucher, Larisa Rudenko and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Peer reviewer reports are available.

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Extended data

Extended Data Fig. 1 Receptor binding profile of avian and EA swine influenza A viruses.

An ELISA-based assay was employed to investigate receptor binding preference of avian and EA swine H1N1 influenza viruses. 3'SLN (shown in cyan circles) and 6'SLN (shown in pink squares) were coated in 96-well plates at concentrations of 5 to 0.1 µg/ml. Infectious viruses (diluted to 64 HAU/50µL) were added to each well and co-incubated with 3'SLN and 6'SLN. The plates were washed with PBS and the detection of bound virus was done using a rabbit polyclonal antibody against HA from A/Duck/NZL/160/1976(H1N3) (Sino Biological Inc., Beijing, China) followed by polyclonal Goat Anti-Rabbit Immunoglobulins/HRP (Dako, Denmark). The absorbance at 450 nm was plotted against glycan concentration. Results are shown with the mean and s.d. absorbance from 3 replicates in one of two independently performed experiment.

Source data

Extended Data Fig. 2 EA swine virus sequence from archived pig-lung homogenates in 1979 shared high homology with the ancestral sequence of RG-EA2 that genetically resembles 1979–1983 era EA swine viruses.

Full genome of A/swine/Belgium/2/1979 (Be02-lung) and a partial genome of A/swine/Belgium/1/1979 (Be01-lung) EA swine influenza viruses were sequenced from two archived pig-lung homogenates from 1979. Four recombinant viruses were resurrected using ancestral sequence reconstruction, with RG-EA1 representing the precursor virus of EA swine viruses, RG-EA2 representing the early evolutionary stage of EA swine viruses in 1979–1983, and RG-EA3 and RG-EA4 representing EA swine viruses in 1984–1987 and 1988–1992, respectively. Among the 33 amino acids that differentiate the non-transmissible RG-EA2 virus and transmissible RG-EA3 virus, the sequences of Be01 and Be02 showed high homology to RG-EA2. /, unsuccessfully determined.

Extended Data Fig. 3 Structure of glycans used for glycan array.

A synthesised glycan microarray comprised of 38 glycans of 𝛼2,3-linked sialosides (glycan 1–38), 3 glycans containing both 𝛼2,3 & 𝛼2,6-linked sialosides (glycan 39–41) and 32 glycans of 𝛼2,6-linked sialosides (glycan 42–73) was utilized for glycan array assay.

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Su, W., Harfoot, R., Su, Y.C.F. et al. Ancestral sequence reconstruction pinpoints adaptations that enable avian influenza virus transmission in pigs. Nat Microbiol 6, 1455–1465 (2021). https://doi.org/10.1038/s41564-021-00976-y

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