{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,8,14]],"date-time":"2026-08-14T09:39:48Z","timestamp":1786700388528,"version":"build-2736575974"},"reference-count":44,"publisher":"MDPI AG","issue":"8","license":[{"start":{"date-parts":[[2026,8,14]],"date-time":"2026-08-14T00:00:00Z","timestamp":1786665600000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100001809","name":"National Natural Science Foundation of China","doi-asserted-by":"publisher","award":["32573340"],"award-info":[{"award-number":["32573340"]}],"id":[{"id":"10.13039\/501100001809","id-type":"DOI","asserted-by":"publisher"}]},{"award":["32573340"],"award-info":[{"award-number":["32573340"]}],"id":[{"id":"https:\/\/ror.org\/01h0zpd94","id-type":"ROR","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Microorganisms"],"abstract":"<jats:p>Airborne transmission represents a primary route of influenza virus dissemination. In animal infection models, conventional intranasal instillation fails to recapitulate the physiological features of natural respiratory infection. Existing aerosol-based challenge methods\u2014including whole-body inhalation and oronasal exposure\u2014face limitations in precise quantification of the delivered infectious dose. Moreover, oronasal exposure systems require specialized, high-cost instrumentation. This study established an economical and dosing-controlled aerosol challenge method utilizing a lung fluid quantitative nebulizer, which can deliver aerosolized virus directly to the lower respiratory tract via endotracheal intubation. The count median aerodynamic diameter (CMAD) of aerosols generated by the nebulizer was 2.28 \u03bcm. We compared pulmonary deposition efficiency and pathogenicity between lung-delivery aerosol inhalation and conventional intranasal instillation of PR8 influenza virus in mice. The pulmonary deposition rate for the aerosol inhalation was 63.1%, with an LD50 of 31.1 EID50; in contrast, intranasal instillation achieved a pulmonary deposition rate of 20.0% and an LD50 of 115.0 EID50. Histopathological analysis revealed significantly more severe lesions in lungs of mice challenged via lung-delivery aerosol inhalation relative to those receiving intranasal instillation. Furthermore, using a guinea pig model and a recombinant PR8 virus expressing Gaussia luciferase (PR8-Gluc), we demonstrated that pulmonary aerosol inhalation resulted in both more homogeneous spatial distribution and higher viral loads across lung tissues. Our results demonstrate that animals are more sensitive to lung-delivery inhaled virus than virus delivered by intranasal instillation. This study provides an optimized, economical, and technically straightforward aerosol challenge method with controlled administered dosing for viral infection models.<\/jats:p>","DOI":"10.3390\/microorganisms14081793","type":"journal-article","created":{"date-parts":[[2026,8,14]],"date-time":"2026-08-14T09:04:01Z","timestamp":1786698241000},"page":"1793","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Development of a Controlled-Dose Aerosol Challenge Method Using a Lung Fluid Quantitative Nebulizer"],"prefix":"10.3390","volume":"14","author":[{"given":"Mingjie","family":"Shao","sequence":"first","affiliation":[{"name":"College of Life Sciences, Jilin Agricultural University, 2888 Xincheng Street, Changchun 130118, China"},{"name":"Key Laboratory of Jilin Province for Zoonoses Prevention and Control, State Key Laboratory of Pathogen and Biosecurity, Academy of Military Medical Sciences, 573 Tulip Street, Changchun 130122, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Huan","family":"Cui","sequence":"additional","affiliation":[{"name":"College of Veterinary Medicine, Hebei Agricultural University, 2596 Lucky South Street, Baoding 071000, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Zhaoliang","family":"Chen","sequence":"additional","affiliation":[{"name":"Key Laboratory of Jilin Province for Zoonoses Prevention and Control, State Key Laboratory of Pathogen and Biosecurity, Academy of Military Medical Sciences, 573 Tulip Street, Changchun 130122, China"},{"name":"College of Veterinary Medicine, Hebei Agricultural University, 2596 Lucky South Street, Baoding 071000, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Lei","family":"Zhang","sequence":"additional","affiliation":[{"name":"College of Veterinary Medicine, Hebei Agricultural University, 2596 Lucky South Street, Baoding 071000, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Cheng","family":"Zhang","sequence":"additional","affiliation":[{"name":"College of Veterinary Medicine, Hebei Agricultural University, 2596 Lucky South Street, Baoding 071000, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-8705-7744","authenticated-orcid":false,"given":"Zhendong","family":"Guo","sequence":"additional","affiliation":[{"name":"Key Laboratory of Jilin Province for Zoonoses Prevention and Control, State Key Laboratory of Pathogen and Biosecurity, Academy of Military Medical Sciences, 573 Tulip Street, Changchun 130122, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Peng","family":"Han","sequence":"additional","affiliation":[{"name":"College of Life Sciences, Jilin Agricultural University, 2888 Xincheng Street, Changchun 130118, China"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2026,8,14]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"196","DOI":"10.1038\/nrg2053","article-title":"The evolution of epidemic influenza","volume":"8","author":"Nelson","year":"2007","journal-title":"Nat. Rev. Genet."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"422","DOI":"10.1016\/j.jinf.2017.01.003","article-title":"Human infection with an avian influenza A\/H9N2 virus in Guangdong in 2016","volume":"74","author":"Yuan","year":"2017","journal-title":"J. Infect."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"51","DOI":"10.4103\/1735-1995.187253","article-title":"The avian influenza H9N2 at avian-human interface: A possible risk for the future pandemics","volume":"21","author":"RahimiRad","year":"2016","journal-title":"J. Res. Med. Sci."},{"key":"ref_4","first-page":"291","article-title":"Increased risk of A(H1N1)pdm09 influenza infection in UK pig industry workers compared to a general population cohort. Influenza Other Respir","volume":"10","author":"Fragaszy","year":"2016","journal-title":"Viruses"},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"311","DOI":"10.4110\/in.2016.16.5.311","article-title":"Genetic Characteristics and Immunogenicity of Pandemic H1N1 Influenza Virus Isolate from Pig in Korea","volume":"16","author":"Moon","year":"2016","journal-title":"Immune. Netw."},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"133","DOI":"10.1007\/s00430-002-0132-3","article-title":"Antigenic drift and variability of influenza viruses","volume":"191","author":"Schweiger","year":"2002","journal-title":"Med. Microbiol. Immunol."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"3","DOI":"10.1038\/s41572-018-0002-y","article-title":"Influenza","volume":"4","author":"Krammer","year":"2018","journal-title":"Nat. Rev. Dis. Primers"},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"19780","DOI":"10.2807\/ese.16.05.19780-en","article-title":"Group A streptococcal infections during the seasonal influenza outbreak 2010\/11 in South East England","volume":"16","author":"Scaber","year":"2011","journal-title":"Eur. Surveill."},{"key":"ref_9","doi-asserted-by":"crossref","unstructured":"Bowen, L.E., Rivers, K., Trombley, J.E., Bohannon, J.K., Li, S.X., Boydston, J.A., and Eichelberger, M.C. (2012). Development of a murine nose-only inhalation model of influenza: Comparison of disease caused by instilled and inhaled A\/PR\/8\/34. Front. Cell Infect. Microbiol., 2.","DOI":"10.3389\/fcimb.2012.00074"},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"1657","DOI":"10.3201\/eid1211.060426","article-title":"Review of aerosol transmission of influenza A virus","volume":"12","author":"Tellier","year":"2006","journal-title":"Emerg. Infect. Dis."},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"131","DOI":"10.1089\/vim.2010.0085","article-title":"Aerosol inoculation with a sub-lethal influenza virus leads to exacerbated morbidity and pulmonary disease pathogenesis","volume":"24","author":"Smith","year":"2011","journal-title":"Viral Immunol."},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"S783","DOI":"10.1098\/rsif.2009.0302.focus","article-title":"Aerosol transmission of influenza A virus: A review of new studies","volume":"6","author":"Tellier","year":"2009","journal-title":"J. R. Soc. Interface"},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"215","DOI":"10.1016\/j.epidem.2010.10.001","article-title":"High infectivity and pathogenicity of influenza A virus via aerosol and droplet transmission","volume":"2","author":"Teunis","year":"2010","journal-title":"Epidemics"},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"171","DOI":"10.1586\/ers.10.15","article-title":"Aerosol and nasal delivery of vaccines and antiviral drugs against seasonal and pandemic influenza","volume":"4","author":"Wong","year":"2010","journal-title":"Expert Rev. Respir. Med."},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"106606","DOI":"10.1016\/j.ejps.2023.106606","article-title":"Influence of mesh nebulizer characteristics on aerosol delivery in non-human primates","volume":"191","author":"Cabrera","year":"2023","journal-title":"Eur. J. Pharm. Sci."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"e250","DOI":"10.1097\/PCC.0b013e31828a7f79","article-title":"Influences of cannula size and flow rate on aerosol drug delivery through the Vapotherm humidified high-flow nasal cannula system","volume":"14","author":"Perry","year":"2013","journal-title":"Pediatr. Crit. Care Med."},{"key":"ref_17","doi-asserted-by":"crossref","unstructured":"Chang, K.H., Moon, S.H., Yoo, S.K., Park, B.J., and Nam, K.C. (2020). Aerosol Delivery of Dornase Alfa Generated by Jet and Mesh Nebulizers. Pharmaceutics, 12.","DOI":"10.3390\/pharmaceutics12080721"},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"91","DOI":"10.1164\/ajrccm.161.1.9812094","article-title":"Lung delivery of aerosolized dextran","volume":"161","author":"Finlay","year":"2000","journal-title":"Am. J. Respir. Crit. Care Med."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1016\/j.ijpharm.2010.12.040","article-title":"Influence of particle size on regional lung deposition\u2014What evidence is there?","volume":"406","author":"Carvalho","year":"2011","journal-title":"Int. J. Pharm."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"314","DOI":"10.1016\/j.ijpharm.2007.08.013","article-title":"Effect of particle size of dry powder mannitol on the lung deposition in healthy volunteers","volume":"349","author":"Glover","year":"2008","journal-title":"Int. J. Pharm."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"317","DOI":"10.1089\/jamp.2018.1508","article-title":"Devices for Improved Delivery of Nebulized Pharmaceutical Aerosols to the Lungs","volume":"32","author":"Longest","year":"2019","journal-title":"J. Aerosol Med. Pulm. Drug Deliv."},{"key":"ref_22","doi-asserted-by":"crossref","unstructured":"Kotta, S., Aldawsari, H.M., Badr-Eldin, S.M., Binmahfouz, L.S., Bakhaidar, R.B., Sreeharsha, N., Nair, A.B., and Ramnarayanan, C. (2021). Aerosol Delivery of Surfactant Liposomes for Management of Pulmonary Fibrosis: An Approach Supporting Pulmonary Mechanics. Pharmaceutics, 13.","DOI":"10.3390\/pharmaceutics13111851"},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"10","DOI":"10.1208\/s12249-022-02460-0","article-title":"In Vitro Evaluation of Nebulized Pharmaceutical Aerosol Delivery to the Lungs Using a New Heated Dryer System (HDS)","volume":"24","author":"Spence","year":"2022","journal-title":"AAPS PharmSciTech"},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"227","DOI":"10.1016\/S0378-5173(99)00341-5","article-title":"Variability in lung deposition of inhaled drug, within and between asthmatic patients, with a pMDI and a dry powder inhaler, Turbuhaler","volume":"193","author":"Bengtsson","year":"2000","journal-title":"Int. J. Pharm."},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"e12910","DOI":"10.1002\/adma.202512910","article-title":"Cytosol-Targeting Delivery of Non-Nucleotide STING Agonist Achieves Inhalable Nanoparticle-Based Anthrax Vaccine","volume":"38","author":"Zhang","year":"2026","journal-title":"Adv. Mater."},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"1703","DOI":"10.1007\/s11095-024-03740-z","article-title":"Development of a New Dry Powder Aerosol Synthetic Lung Surfactant Product for Neonatal Respiratory Distress Syndrome (RDS)\u2014Part I: In Vitro Testing and Characterization","volume":"41","author":"Momin","year":"2024","journal-title":"Pharm. Res."},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"457","DOI":"10.1016\/j.ijpharm.2003.09.041","article-title":"Formulation and characterization of spray-dried powders containing nanoparticles for aerosol delivery to the lung","volume":"269","author":"Sham","year":"2004","journal-title":"Int. J. Pharm."},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"630","DOI":"10.1038\/s41586-023-06809-8","article-title":"Inhaled SARS-CoV-2 vaccine for single-dose dry powder aerosol immunization","volume":"624","author":"Ye","year":"2023","journal-title":"Nature"},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"213","DOI":"10.1586\/14760584.6.2.213","article-title":"Pulmonary vaccine delivery","volume":"6","author":"Lu","year":"2007","journal-title":"Expert Rev. Vaccines"},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"102370","DOI":"10.1016\/j.coi.2023.102370","article-title":"Viral-vectored respiratory mucosal vaccine strategies","volume":"84","author":"Jeyanathan","year":"2023","journal-title":"Curr. Opin. Immunol."},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"114831","DOI":"10.1016\/j.addr.2023.114831","article-title":"Aerosol pulmonary immune engineering","volume":"199","author":"Sudduth","year":"2023","journal-title":"Adv. Drug Deliv. Rev."},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"588","DOI":"10.1046\/j.1365-2125.2003.01892.x","article-title":"Pulmonary drug delivery. Part I: Physiological factors affecting therapeutic effectiveness of aerosolized medications","volume":"56","author":"Labiris","year":"2003","journal-title":"Br. J. Clin. Pharmacol."},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"126246","DOI":"10.1016\/j.ijpharm.2025.126246","article-title":"Spray freeze-dried doxycycline nanoparticles for macrophage-targeted lung delivery","volume":"685","author":"Magdy","year":"2025","journal-title":"Int. J. Pharm."},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"43","DOI":"10.1016\/j.jaerosci.2017.05.008","article-title":"Comparison of traditional intranasal and aerosol inhalation inoculation of guinea pigs with visualizing influenza virus","volume":"110","author":"Zhang","year":"2017","journal-title":"J. Aerosol Sci."},{"key":"ref_35","unstructured":"Mercer, T.T., American Industrial Hygiene Association, and U.S. Atomic Energy Commission (1973). Aerosol Technology in Hazard Evaluation, Academic Press."},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"829","DOI":"10.3109\/08958378.2014.935535","article-title":"Computational modeling of nanoscale and microscale particle deposition, retention and dosimetry in the mouse respiratory tract","volume":"26","author":"Asgharian","year":"2014","journal-title":"Inhal. Toxicol."},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"147","DOI":"10.1111\/j.1749-6632.1980.tb18917.x","article-title":"Viruses as agents of airborne contagion","volume":"353","author":"Knight","year":"1980","journal-title":"Ann. N. Y. Acad. Sci."},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"171","DOI":"10.1136\/thx.52.2.171","article-title":"Aerosol kinetics and bronchodilator efficacy during continuous positive airway pressure delivered by face mask","volume":"52","author":"Parkes","year":"1997","journal-title":"Thorax"},{"key":"ref_39","doi-asserted-by":"crossref","first-page":"1238","DOI":"10.4187\/respcare.03667","article-title":"Radioaerosol Pulmonary Deposition Using Mesh and Jet Nebulizers During Noninvasive Ventilation in Healthy Subjects","volume":"60","author":"Ramos","year":"2015","journal-title":"Respir. Care"},{"key":"ref_40","doi-asserted-by":"crossref","first-page":"354","DOI":"10.1089\/jam.2005.18.354","article-title":"Variation in pediatric aerosol delivery: Importance of facemask","volume":"18","author":"Smaldone","year":"2005","journal-title":"J. Aerosol Med."},{"key":"ref_41","doi-asserted-by":"crossref","first-page":"73","DOI":"10.1007\/s41030-018-0054-x","article-title":"In Vitro Determination of the Main Effects in the Design of High-Flow Nasal Therapy Systems with Respect to Aerosol Performance","volume":"4","author":"Bennett","year":"2018","journal-title":"Pulm. Ther."},{"key":"ref_42","doi-asserted-by":"crossref","first-page":"1186","DOI":"10.4187\/respcare.05127","article-title":"Aerosol Delivery Through Adult High Flow Nasal Cannula With Heliox and Oxygen","volume":"62","author":"Dailey","year":"2017","journal-title":"Respir. Care"},{"key":"ref_43","doi-asserted-by":"crossref","first-page":"657","DOI":"10.1007\/s12275-010-0120-z","article-title":"Replication and pathogenesis of the pandemic (H1N1) 2009 influenza virus in mammalian models","volume":"48","author":"Kwon","year":"2010","journal-title":"J. Microbiol."},{"key":"ref_44","doi-asserted-by":"crossref","first-page":"4995","DOI":"10.1016\/j.vaccine.2013.06.071","article-title":"Low pathogenic avian influenza A(H7N9) virus causes high mortality in ferrets upon intratracheal challenge: A model to study intervention strategies","volume":"31","author":"Kreijtz","year":"2013","journal-title":"Vaccine"}],"container-title":["Microorganisms"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/2076-2607\/14\/8\/1793\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,8,14]],"date-time":"2026-08-14T09:20:16Z","timestamp":1786699216000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/2076-2607\/14\/8\/1793"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,8,14]]},"references-count":44,"journal-issue":{"issue":"8","published-online":{"date-parts":[[2026,8]]}},"alternative-id":["microorganisms14081793"],"URL":"https:\/\/doi.org\/10.3390\/microorganisms14081793","relation":{},"ISSN":["2076-2607"],"issn-type":[{"value":"2076-2607","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,8,14]]}}}