{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,29]],"date-time":"2026-09-29T23:48:56Z","timestamp":1790725736591,"version":"4.1.0"},"reference-count":44,"publisher":"MDPI AG","issue":"2","license":[{"start":{"date-parts":[[2021,2,22]],"date-time":"2021-02-22T00:00:00Z","timestamp":1613952000000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Viruses"],"abstract":"<jats:p>Infection with highly pathogenic avian H5N1 influenza virus in humans often leads to severe respiratory disease with high mortality. Experimental infection in non-human primates can provide additional insight into disease pathogenesis. However, such a model should recapitulate the disease symptoms observed in humans, such as pneumonia and inflammatory cytokine response. While previous studies in macaques have demonstrated the occurrence of typical lesions in the lungs early after infection and a high level of immune activation, progression to severe disease and lethality were rarely observed. Here, we evaluated a routinely used combined route of infection via intra-bronchial, oral, and intra-nasal virus inoculation with aerosolized H5N1 exposure, with or without the regular collection of bronchoalveolar lavages early after infection. Both combined route and aerosol exposure resulted in similar levels of virus replication in nose and throat and similar levels of immune activation, cytokine, and chemokine release in the blood. However, while animals exposed to H5N1 by combined-route inoculation developed severe disease with high lethality, aerosolized exposure resulted in less lesions, as measured by consecutive computed tomography and less fever and lethal disease. In conclusion, not virus levels or immune activation, but route of infection determines fatal outcome for highly pathogenic avian H5N1 influenza infection.<\/jats:p>","DOI":"10.3390\/v13020345","type":"journal-article","created":{"date-parts":[[2021,2,22]],"date-time":"2021-02-22T20:42:51Z","timestamp":1614026571000},"page":"345","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":14,"title":["Aerosolized Exposure to H5N1 Influenza Virus Causes Less Severe Disease Than Infection via Combined Intrabronchial, Oral, and Nasal Inoculation in Cynomolgus Macaques"],"prefix":"10.3390","volume":"13","author":[{"ORCID":"https:\/\/orcid.org\/0000-0001-8608-8629","authenticated-orcid":false,"given":"Petra","family":"Mooij","sequence":"first","affiliation":[{"name":"Department of Virology, Biomedical Primate Research Centre, Lange Kleiweg 161, 2288 GJ Rijswijk, The Netherlands"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-4261-6535","authenticated-orcid":false,"given":"Marieke A.","family":"Stammes","sequence":"additional","affiliation":[{"name":"Department of Parasitology, Biomedical Primate Research Centre, Lange Kleiweg 161, 2288 GJ Rijswijk, The Netherlands"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Daniella","family":"Mortier","sequence":"additional","affiliation":[{"name":"Department of Virology, Biomedical Primate Research Centre, Lange Kleiweg 161, 2288 GJ Rijswijk, The Netherlands"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Zahra","family":"Fagrouch","sequence":"additional","affiliation":[{"name":"Department of Virology, Biomedical Primate Research Centre, Lange Kleiweg 161, 2288 GJ Rijswijk, The Netherlands"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-3300-8204","authenticated-orcid":false,"given":"Nikki","family":"van Driel","sequence":"additional","affiliation":[{"name":"Animal Science Department, Biomedical Primate Research Centre, Lange Kleiweg 161, 2288 GJ Rijswijk, The Netherlands"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-0912-2074","authenticated-orcid":false,"given":"Ernst J.","family":"Verschoor","sequence":"additional","affiliation":[{"name":"Department of Virology, Biomedical Primate Research Centre, Lange Kleiweg 161, 2288 GJ Rijswijk, The Netherlands"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ivanela","family":"Kondova","sequence":"additional","affiliation":[{"name":"Animal Science Department, Biomedical Primate Research Centre, Lange Kleiweg 161, 2288 GJ Rijswijk, The Netherlands"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-4563-8513","authenticated-orcid":false,"given":"Willy M. J. M.","family":"Bogers","sequence":"additional","affiliation":[{"name":"Department of Virology, Biomedical Primate Research Centre, Lange Kleiweg 161, 2288 GJ Rijswijk, The Netherlands"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-3428-5978","authenticated-orcid":false,"given":"Gerrit","family":"Koopman","sequence":"additional","affiliation":[{"name":"Department of Virology, Biomedical Primate Research Centre, Lange Kleiweg 161, 2288 GJ Rijswijk, The Netherlands"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2021,2,22]]},"reference":[{"key":"ref_1","first-page":"1","article-title":"A review of H5Nx avian influenza viruses","volume":"7","author":"Nunez","year":"2019","journal-title":"Ther. Adv. Vaccines Immunother"},{"key":"ref_2","unstructured":"(2020, December 02). Cumulative Number of Confirmed Human Cases for Avian Influenza A (H5N1) Reported to WHO, 2003\u20132020. Available online: https:\/\/www.who.int\/influenza\/human_animal_interface\/H5N1_cumulative_table_archives\/en\/."},{"key":"ref_3","unstructured":"Avian Influenza A Virus Infection in Humans (2020, December 02). Atlanta, GA: Centers for Disease Control and Prevention, Available online: https:\/\/www.cdc.gov\/flu\/avianflu\/avian-in-humans.htm."},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"11294","DOI":"10.1128\/JVI.01192-08","article-title":"H5N1 avian influenza virus induces apoptotic cell death in mammalian airway epithelial cells","volume":"82","author":"Daidoji","year":"2008","journal-title":"J. Virol."},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"1203","DOI":"10.1038\/nm1477","article-title":"Fatal outcome of human influenza A (H5N1) is associated with high viral load and hypercytokinemia","volume":"12","author":"Simmons","year":"2006","journal-title":"Nat. Med."},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"242","DOI":"10.1002\/1096-9071(200103)63:3<242::AID-JMV1007>3.0.CO;2-N","article-title":"Pathology of fatal human infection associated with avian influenza A H5N1 virus","volume":"63","author":"To","year":"2001","journal-title":"J. Med. Virol."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"708","DOI":"10.3201\/eid1305.060572","article-title":"Apoptosis and pathogenesis of avian influenza A (H5N1) virus in humans","volume":"13","author":"Uiprasertkul","year":"2007","journal-title":"Emerg. Infect. Dis."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"1530","DOI":"10.3390\/v20801530","article-title":"Animal Models for Influenza Virus Pathogenesis and Transmission","volume":"2","author":"Bouvier","year":"2010","journal-title":"Viruses"},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"6895","DOI":"10.1016\/j.vaccine.2018.10.024","article-title":"Workshop report: Experimental animal models for universal influenza vaccines","volume":"36","author":"Koopman","year":"2018","journal-title":"Vaccine"},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"75","DOI":"10.1016\/j.antiviral.2015.02.011","article-title":"The use of nonhuman primates in research on seasonal, pandemic and avian influenza, 1893\u20132014","volume":"117","author":"Davis","year":"2015","journal-title":"Antivir. Res."},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"3455","DOI":"10.1073\/pnas.0813234106","article-title":"Early and sustained innate immune response defines pathology and death in nonhuman primates infected by highly pathogenic influenza virus","volume":"106","author":"Baskin","year":"2009","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"227","DOI":"10.1007\/s00705-008-0277-5","article-title":"Pathological lesions and viral localization of influenza A (H5N1) virus in experimentally infected Chinese rhesus macaques: Implications for pathogenesis and viral transmission","volume":"154","author":"Chen","year":"2009","journal-title":"Arch. Virol."},{"key":"ref_13","doi-asserted-by":"crossref","unstructured":"Cilloniz, C., Shinya, K., Peng, X., Korth, M.J., Proll, S.C., Aicher, L.D., Carter, V.S., Chang, J.H., Kobasa, D., and Feldmann, F. (2009). Lethal influenza virus infection in macaques is associated with early dysregulation of inflammatory related genes. PLoS Pathog., 5.","DOI":"10.1371\/journal.ppat.1000604"},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"6687","DOI":"10.1128\/JVI.75.14.6687-6691.2001","article-title":"Pathogenesis of influenza A (H5N1) virus infection in a primate model","volume":"75","author":"Rimmelzwaan","year":"2001","journal-title":"J. Virol."},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"6055","DOI":"10.1128\/JVI.00365-12","article-title":"Integrated clinical, pathologic, virologic, and transcriptomic analysis of H5N1 influenza virus-induced viral pneumonia in the rhesus macaque","volume":"86","author":"Shinya","year":"2012","journal-title":"J. Virol."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"844","DOI":"10.3389\/fmicb.2018.00844","article-title":"The Marmoset as an Animal Model of Influenza: Infection With A (H1N1)pdm09 and Highly Pathogenic A(H5N1) Viruses via the Conventional or Tracheal Spray Route","volume":"9","author":"Nakajima","year":"2018","journal-title":"Front Microbiol."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"2012","DOI":"10.1093\/infdis\/jiu009","article-title":"Massive mobilization of dendritic cells during influenza A virus subtype H5N1 infection of nonhuman primates","volume":"209","author":"Soloff","year":"2014","journal-title":"J. Infect. Dis."},{"key":"ref_18","doi-asserted-by":"crossref","unstructured":"Fan, S., Gao, Y., Shinya, K., Li, C.K., Li, Y., Shi, J., Jiang, Y., Suo, Y., Tong, T., and Zhong, G. (2009). Immunogenicity and protective efficacy of a live attenuated H5N1 vaccine in nonhuman primates. PLoS Pathog., 5.","DOI":"10.1371\/journal.ppat.1000409"},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"562","DOI":"10.1016\/j.vaccine.2007.11.031","article-title":"A vaccine prepared from a non-pathogenic H5N1 avian influenza virus strain confers protective immunity against highly pathogenic avian influenza virus infection in cynomolgus macaques","volume":"26","author":"Itoh","year":"2008","journal-title":"Vaccine"},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"8981","DOI":"10.1128\/JVI.00907-14","article-title":"Disease severity is associated with differential gene expression at the early and late phases of infection in nonhuman primates infected with different H5N1 highly pathogenic avian influenza viruses","volume":"88","author":"Muramoto","year":"2014","journal-title":"J. Virol."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"1562","DOI":"10.1093\/infdis\/jis232","article-title":"A computationally optimized hemagglutinin virus-like particle vaccine elicits broadly reactive antibodies that protect nonhuman primates from H5N1 infection","volume":"205","author":"Giles","year":"2012","journal-title":"J. Infect. Dis."},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"405","DOI":"10.1086\/595984","article-title":"Recombinant modified vaccinia virus Ankara expressing the hemagglutinin gene confers protection against homologous and heterologous H5N1 influenza virus infections in macaques","volume":"199","author":"Kreijtz","year":"2009","journal-title":"J. Infect. Dis."},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"2565","DOI":"10.1128\/JVI.01928-07","article-title":"Vaccination of macaques with adjuvanted formalin-inactivated influenza A virus (H5N1) vaccines: Protection against H5N1 challenge without disease enhancement","volume":"82","author":"Ruat","year":"2008","journal-title":"J. Virol."},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"879","DOI":"10.1016\/S1473-3099(11)70142-6","article-title":"Potential role of human challenge studies for investigation of influenza transmission","volume":"11","author":"Killingley","year":"2011","journal-title":"Lancet. Infect. Dis."},{"key":"ref_25","doi-asserted-by":"crossref","unstructured":"Mooij, P., Mortier, D., Stammes, M., Fagrouch, Z., Verschoor, E.J., Bogers, W., and Koopman, G. (2020). Aerosolized pH1N1 influenza infection induces less systemic and local immune activation in the lung than combined intrabronchial, nasal and oral exposure in cynomolgus macaques. J. Gen. Virol.","DOI":"10.1099\/jgv.0.001489"},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"4801","DOI":"10.1038\/s41598-018-23022-0","article-title":"Experimental infection of Cynomolgus Macaques with highly pathogenic H5N1 influenza virus through the aerosol route","volume":"8","author":"Watanabe","year":"2018","journal-title":"Sci. Rep."},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"1616","DOI":"10.4049\/jimmunol.1601770","article-title":"Widespread Virus Replication in Alveoli Drives Acute Respiratory Distress Syndrome in Aerosolized H5N1 Influenza Infection of Macaques","volume":"198","author":"Wonderlich","year":"2017","journal-title":"J. Immunol."},{"key":"ref_28","doi-asserted-by":"crossref","unstructured":"Mooij, P., Koopman, G., Mortier, D., van Heteren, M., Oostermeijer, H., Fagrouch, Z., de Laat, R., Kobinger, G., Li, Y., and Remarque, E.J. (2015). Pandemic Swine-Origin H1N1 Influenza Virus Replicates to Higher Levels and Induces More Fever and Acute Inflammatory Cytokines in Cynomolgus versus Rhesus Monkeys and Can Replicate in Common Marmosets. PLoS ONE, 10.","DOI":"10.1371\/journal.pone.0126132"},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"7391","DOI":"10.1016\/j.vaccine.2012.10.008","article-title":"Consecutive CT in vivo lung imaging as quantitative parameter of influenza vaccine efficacy in the ferret model","volume":"30","author":"Stittelaar","year":"2012","journal-title":"Vaccine"},{"key":"ref_30","first-page":"389","article-title":"Thoracic radiography as a refinement methodology for the study of H1N1 influenza in cynomologus macaques (Macaca fascicularis)","volume":"60","author":"Brining","year":"2010","journal-title":"Comp. Med."},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"1023","DOI":"10.1128\/JVI.02757-15","article-title":"Correlation between Virus Replication and Antibody Responses in Macaques following Infection with Pandemic Influenza A Virus","volume":"90","author":"Koopman","year":"2016","journal-title":"J. Virol."},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"275","DOI":"10.1089\/jam.1999.12.275","article-title":"Intrapulmonary distribution of deposited particles","volume":"12","author":"Brand","year":"1999","journal-title":"J. Aerosol. Med."},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"153","DOI":"10.1186\/1465-9921-12-153","article-title":"Aerosolized adenovirus-vectored vaccine as an alternative vaccine delivery method","volume":"12","author":"Roy","year":"2011","journal-title":"Respir. Res."},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"1555","DOI":"10.1373\/clinchem.2009.130229","article-title":"Molecular detection of a novel human influenza (H1N1) of pandemic potential by conventional and real-time quantitative RT-PCR assays","volume":"55","author":"Poon","year":"2009","journal-title":"Clin. Chem."},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"715","DOI":"10.1148\/radiol.2020200370","article-title":"Time Course of Lung Changes at Chest CT during Recovery from Coronavirus Disease 2019 (COVID-19)","volume":"295","author":"Pan","year":"2020","journal-title":"Radiology"},{"key":"ref_36","doi-asserted-by":"crossref","unstructured":"Finch, C.L., Crozier, I., Lee, J.H., Byrum, R., Cooper, T.K., Liang, J., Sharer, K., Solomon, J., Sayre, P.J., and Kocher, G. (2020). Characteristic and quantifiable COVID-19-like abnormalities in CT- and PET\/CT-imaged lungs of SARS-CoV-2-infected crab-eating macaques (Macaca fascicularis). bioRxiv.","DOI":"10.1101\/2020.05.14.096727"},{"key":"ref_37","unstructured":"B\u00f6sz\u00f6rm\u00e9nyi, K.P.S.M.A., Fagrouch, Z.C., Kiemenyi-Kayere, G., Niphuis, H., Mortier, D., van Driel, N., Nieuwenhuis, I., Zuiderwijk-Sick, E., Meijer, L., and Mooij, P. (2020). Comparison of SARS-CoV-2 infection in two non-human primate species: Rhesus and cynomolgus macaques. bioRxiv."},{"key":"ref_38","doi-asserted-by":"crossref","unstructured":"Mooij, P., Grodeland, G., Koopman, G., Andersen, T.K., Mortier, D., Nieuwenhuis, I.G., Verschoor, E.J., Fagrouch, Z., Bogers, W.M., and Bogen, B. (2019). Needle-free delivery of DNA: Targeting of hemagglutinin to MHC class II molecules protects rhesus macaques against H1N1 influenza. Vaccine.","DOI":"10.1016\/j.vaccine.2018.12.049"},{"key":"ref_39","doi-asserted-by":"crossref","first-page":"399","DOI":"10.1126\/science.1125548","article-title":"H5N1 Virus Attachment to Lower Respiratory Tract","volume":"312","author":"Munster","year":"2006","journal-title":"Science"},{"key":"ref_40","doi-asserted-by":"crossref","first-page":"1215","DOI":"10.2353\/ajpath.2007.070248","article-title":"Human and avian influenza viruses target different cells in the lower respiratory tract of humans and other mammals","volume":"171","author":"Munster","year":"2007","journal-title":"Am. J. Pathol."},{"key":"ref_41","doi-asserted-by":"crossref","unstructured":"Marriott, A.C., Dennis, M., Kane, J.A., Gooch, K.E., Hatch, G., Sharpe, S., Prevosto, C., Leeming, G., Zekeng, E.G., and Staples, K.J. (2016). Influenza A Virus Challenge Models in Cynomolgus Macaques Using the Authentic Inhaled Aerosol and Intra-Nasal Routes of Infection. PLoS ONE, 11.","DOI":"10.1371\/journal.pone.0157887"},{"key":"ref_42","doi-asserted-by":"crossref","first-page":"11115","DOI":"10.1128\/JVI.01571-12","article-title":"Increased viral loads and exacerbated innate host responses in aged macaques infected with the 2009 pandemic H1N1 influenza A virus","volume":"86","author":"Josset","year":"2012","journal-title":"J. Virol."},{"key":"ref_43","doi-asserted-by":"crossref","unstructured":"Pham, V.L., Nakayama, M., Itoh, Y., Ishigaki, H., Kitano, M., Arikata, M., Ishida, H., Kitagawa, N., Shichinohe, S., and Okamatsu, M. (2013). Pathogenicity of pandemic H1N1 influenza A virus in immunocompromised cynomolgus macaques. PLoS ONE, 8.","DOI":"10.1371\/journal.pone.0075910"},{"key":"ref_44","doi-asserted-by":"crossref","unstructured":"Weinfurter, J.T., Brunner, K., Capuano, S.V., Li, C., Broman, K.W., Kawaoka, Y., and Friedrich, T.C. (2011). Cross-reactive T cells are involved in rapid clearance of 2009 pandemic H1N1 influenza virus in nonhuman primates. PLoS Pathog., 7.","DOI":"10.1371\/journal.ppat.1002381"}],"container-title":["Viruses"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1999-4915\/13\/2\/345\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,11]],"date-time":"2025-10-11T05:26:41Z","timestamp":1760160401000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1999-4915\/13\/2\/345"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2021,2,22]]},"references-count":44,"journal-issue":{"issue":"2","published-online":{"date-parts":[[2021,2]]}},"alternative-id":["v13020345"],"URL":"https:\/\/doi.org\/10.3390\/v13020345","relation":{},"ISSN":["1999-4915"],"issn-type":[{"value":"1999-4915","type":"electronic"}],"subject":[],"published":{"date-parts":[[2021,2,22]]}}}