Abstract
Since early 2022, highly pathogenic avian influenza (HPAI) H5N1 virus infections have been reported in wild aquatic birds and poultry throughout the USA with spillover into several mammalian species1,2,3,4,5,6. In March 2024, HPAIV H5N1 clade 2.3.4.4b was first detected in dairy cows in Texas, USA, and continues to circulate on dairy farms in many states7,8. Milk production and quality are diminished in infected dairy cows, with high virus titres in milk raising concerns of exposure to mammals including humans through consumption9,10,11,12. Here we investigated routes of infection with bovine HPAIV H5N1 clade 2.3.4.4b in cynomolgus macaques, a surrogate model for human infection13. We show that intranasal or intratracheal inoculation of macaques could cause systemic infection resulting in mild and severe respiratory disease, respectively. By contrast, infection by the orogastric route resulted in limited infection and seroconversion of macaques that remained subclinical.
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Data availability
Raw data are available at Figshare (https://doi.org/10.6084/m9.figshare.27763944)37. Viral sequences were deposited into GenBank under submission number SUB14944743 (accession numbers PQ790100–PQ790155).
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Acknowledgements
The work was funded by the Intramural Research Program of NIAID, NIH. We thank R. Webby from St. Jude Children Hospital and A. Bowman from Ohio State University for providing the bovine HPAIV H5N1 clade 2.3.4.4b isolate. We thank K. O’Donnell and A. Marzi (both NIAID, NIH) for serological reagents. We thank the Visual and Medical Arts Unit, NIAID, NIH (A. Mora, R. Kissinger, A. Stewart and S. Tudor) for graphical assistance. We thank the staff of the Research Technology Branch, NIAID, NIH for assistance with sequencing and sequence analysis. We also thank the staff of the Rocky Mountain Veterinary Branch, NIAID, NIH for animal care and histopathology service. Finally, we thank the Office of the Chief, LV, NIAID, NIH for general biocontainment services.
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K.R., E.d.W., V.J.M. and H.F. designed the study. V.J.M., E.W. and K.C.Y. acquired virus isolates, grew virus stock and confirmed virus sequences. K.R., A.G., F.K., E.A., R.M., T.B., M.F., T.T., K.G., A.W., B.N.W., S.G., S.S.L., T.L., A.O., M.C.L., K.C.Y., D.R., B.J.S., K.K., C.M., C.S., G.S., P.H., N.v.D., E.d.W., V.J.M. and H.F. performed experiments and data analysis. K.R., E.d.W., V.J.M. and H.F. wrote the manuscript. E.d.W., V.J.M. and H.F. obtained funding. All authors reviewed and contributed to preparation of the final manuscript.
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Nature thanks Lineke Begeman, Malik Peiris, Douglas Reed and Guelsah Gabriel for their contribution to the peer review of this work. Peer reviewer reports are available.
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Extended data figures and tables
Extended Data Fig. 1 Weights and Temperatures in Cynomolgus macaques following inoculation with HPAIV H5N1.
Weights (A) and Temperatures (B) were collected at each clinical exam and at the time of euthanasia. Data represent 3 animals per inoculation route with the line at the mean with standard deviation displayed.
Extended Data Fig. 2 Blood Chemistries.
Serum was collected at each clinical exam and at the time of euthanasia to assess organ function over the course of the study. Data represent 3 animals per inoculation route with the line at the mean with standard deviation displayed.
Extended Data Fig. 3 Hematology.
Whole blood was collected at each clinical exam and at the time of euthanasia to assess the effects of inoculation over the course of the study. Data represent 3 animals per inoculation route with the line at the mean with standard deviation displayed.
Extended Data Fig. 4 Tissue titers.
Infectious virus was measured in tissues at D4/D5. Data points represent individual animals per inoculation route (n = 3) with the bar at the mean with standard deviation displayed.
Extended Data Fig. 5 Acute Disease.
Pulmonary lesions observed in two lobes of one orogastric (oral cavity and gavage) and one lobe in two intranasally inoculated animals. Mild signs of pneumonia was observed with limited macrophage and neutrophil infiltration. HE: Asterisk = edema, IHC: Arrows = macrophages, arrowhead = bronchiolar epithelium. 200X; bar = 50 µm.
Extended Data Fig. 6 Cytokine and Chemokine levels.
Serum was collected during the first 5 days of the study to compare cytokine and chemokine levels across the three inoculation groups. Data points represent individual animals per inoculation route (n = 6) with the bar at the mean with standard deviation displayed. P-values = * <0.05, ** <0.01, *** <0.001, **** <0.0001.
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Rosenke, K., Griffin, A., Kaiser, F. et al. Pathogenesis of bovine H5N1 clade 2.3.4.4b infection in macaques. Nature 640, 1017–1021 (2025). https://doi.org/10.1038/s41586-025-08609-8
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DOI: https://doi.org/10.1038/s41586-025-08609-8
