{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,5,15]],"date-time":"2026-05-15T21:17:52Z","timestamp":1778879872167,"version":"3.51.4"},"reference-count":38,"publisher":"MDPI AG","issue":"2","license":[{"start":{"date-parts":[[2024,2,9]],"date-time":"2024-02-09T00:00:00Z","timestamp":1707436800000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"Science Development Fund of the Warsaw University of Life Sciences\u2014SGGW"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Pathogens"],"abstract":"<jats:p>Human adenovirus (HAdV) is a common pathogen, which can lead to various clinical symptoms and\u2014in some cases\u2014central nervous system (CNS) dysfunctions, such as encephalitis and meningitis. Although the initial events of virus entry have already been identified in various cell types, the mechanism of neuronal uptake of adenoviruses is relatively little understood. The aim of this study was to investigate early events during adenoviral infection, in particular to determine the connection between cellular coxsackievirus and adenovirus receptor (CAR), clathrin, caveolin, and early endosomal proteins (EEA1 and Rab5) with the entry of HAdVs into primary murine neurons in vitro. An immunofluorescence assay and confocal microscopy analysis were carried out to determine HAdV4, 5, and 7 correlation with CAR, clathrin, caveolin, and early endosomal proteins in neurons. The quantification of Pearson\u2019s coefficient between CAR and HAdVs indicated that the HAdV4 and HAdV5 types correlated with CAR and that the correlation was more substantial for HAdV5. Inhibition of clathrin-mediated endocytosis using chlorpromazine limited the infection with HAdV, whereas inhibition of caveolin-mediated endocytosis did not affect virus entry. Thus, the entry of tested HAdV types into neurons was most likely associated with clathrin but not caveolin. It was also demonstrated that HAdVs correlate with the Rab proteins (EEA1, Rab5) present in early vesicles, and the observed differences in the manner of correlation depended on the serotype of the virus. With our research, we strove to expand knowledge regarding the mechanism of HAdV entry into neurons, which may be beneficial for developing potential therapeutics in the future.<\/jats:p>","DOI":"10.3390\/pathogens13020158","type":"journal-article","created":{"date-parts":[[2024,2,9]],"date-time":"2024-02-09T11:42:05Z","timestamp":1707478925000},"page":"158","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":5,"title":["Human Adenovirus Entry and Early Events during Infection of Primary Murine Neurons: Immunofluorescence Studies In Vitro"],"prefix":"10.3390","volume":"13","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-9872-3017","authenticated-orcid":false,"given":"Anna","family":"S\u0142o\u0144ska","sequence":"first","affiliation":[{"name":"Division of Microbiology, Department of Preclinical Sciences, Institute of Veterinary Medicine, Warsaw University of Life Sciences, 02-786 Warsaw, Poland"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Aleksandra","family":"Miedzi\u0144ska","sequence":"additional","affiliation":[{"name":"Division of Microbiology, Department of Preclinical Sciences, Institute of Veterinary Medicine, Warsaw University of Life Sciences, 02-786 Warsaw, Poland"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Marcin","family":"Chodkowski","sequence":"additional","affiliation":[{"name":"Military Institute of Hygiene and Epidemiology, Kozielska 4, 01-163 Warsaw, Poland"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-8150-4187","authenticated-orcid":false,"given":"Piotr","family":"B\u0105ska","sequence":"additional","affiliation":[{"name":"Division of Pharmacology and Toxicology, Department of Preclinical Sciences, Institute of Veterinary Medicine, Warsaw University of Life Sciences-SGGW, 02-786 Warsaw, Poland"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Aleksandra","family":"Mielnikow","sequence":"additional","affiliation":[{"name":"Division of Microbiology, Department of Preclinical Sciences, Institute of Veterinary Medicine, Warsaw University of Life Sciences, 02-786 Warsaw, Poland"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-5346-3760","authenticated-orcid":false,"given":"Michalina","family":"Bartak","sequence":"additional","affiliation":[{"name":"Division of Microbiology, Department of Preclinical Sciences, Institute of Veterinary Medicine, Warsaw University of Life Sciences, 02-786 Warsaw, Poland"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Marcin W.","family":"Ba\u0144bura","sequence":"additional","affiliation":[{"name":"Division of Microbiology, Department of Preclinical Sciences, Institute of Veterinary Medicine, Warsaw University of Life Sciences, 02-786 Warsaw, Poland"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-5273-3930","authenticated-orcid":false,"given":"Joanna","family":"Cymerys","sequence":"additional","affiliation":[{"name":"Division of Microbiology, Department of Preclinical Sciences, Institute of Veterinary Medicine, Warsaw University of Life Sciences, 02-786 Warsaw, Poland"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1968","published-online":{"date-parts":[[2024,2,9]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"586","DOI":"10.1055\/s-0036-1584923","article-title":"Adenovirus: Epidemiology, Global Spread of Novel Serotypes, and Advances in Treatment and Prevention","volume":"37","author":"Lynch","year":"2016","journal-title":"Semin. Respir. Crit. Care Med."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"441","DOI":"10.1128\/CMR.00116-13","article-title":"Adenovirus infections in immunocompetent and immunocompromised patients","volume":"27","author":"Lion","year":"2014","journal-title":"Clin. Microbiol. Rev."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"300","DOI":"10.1016\/j.jcv.2013.03.017","article-title":"Adenovirus infection associated with central nervous system dysfunction in children","volume":"57","author":"Huang","year":"2013","journal-title":"J. Clin. Virol."},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"235","DOI":"10.1080\/13550280600846633","article-title":"Acute meningoencephalitis caused by adenovirus serotype 26","volume":"12","author":"Dubberke","year":"2006","journal-title":"J. Neurovirol."},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"2361","DOI":"10.1128\/JCM.00080-11","article-title":"Adenoviral infection presenting as an isolated central nervous system disease without detectable viremia in two children after stem cell transplantation","volume":"49","author":"Frange","year":"2011","journal-title":"J. Clin. Microbiol."},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"830","DOI":"10.1086\/314041","article-title":"Isolation and characterization of adenovirus 5 from the brain of an infant with fatal cerebral edema","volume":"31","author":"Chatterjee","year":"2000","journal-title":"Clin. Infect. Dis."},{"key":"ref_7","doi-asserted-by":"crossref","unstructured":"Nesti\u0107, D., Bo\u017einovi\u0107, K., Pehar, I., Wallace, R., Parker, A.L., and Majhen, D. (2021). The Revolving Door of Adenovirus Cell Entry: Not All Pathways Are Equal. Pharmaceutics, 13.","DOI":"10.3390\/pharmaceutics13101585"},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"572","DOI":"10.5604\/17322693.1203721","article-title":"Mechanisms of endocytosis utilized by viruses during infection","volume":"70","author":"Cymerys","year":"2016","journal-title":"Post. High. Med. Do\u015bw."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"591","DOI":"10.1016\/j.virol.2015.03.006","article-title":"Adenovirus membrane penetration: Tickling the tail of a sleeping dragon","volume":"479\u2013480","author":"Wiethoff","year":"2015","journal-title":"Virology"},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"152","DOI":"10.1002\/jgm.553","article-title":"Adenovirus endocytosis","volume":"6","author":"Meier","year":"2004","journal-title":"J. Gene. Med."},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"3509","DOI":"10.1242\/jcs.02476","article-title":"Coxsackievirus-adenovirus receptor (CAR) is essential for early embryonic cardiac development","volume":"118","author":"Dorner","year":"2005","journal-title":"J. Cell Sci."},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"956","DOI":"10.1038\/emboj.2008.38","article-title":"Subversion of CtBP1-controlled macropinocytosis by human adenovirus serotype 3","volume":"27","author":"Amstutz","year":"2008","journal-title":"EMBO J."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"9664","DOI":"10.1128\/JVI.73.11.9664-9668.1999","article-title":"Rab5 GTPase regulates adenovirus endocytosis","volume":"73","author":"Rauma","year":"1999","journal-title":"J. Virol."},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1006\/viro.2000.0468","article-title":"Cell receptors involved in adenovirus entry","volume":"274","author":"Nemerow","year":"2000","journal-title":"Virology"},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"237","DOI":"10.1126\/science.1094823","article-title":"How viruses enter animal cells","volume":"304","author":"Smith","year":"2004","journal-title":"Science"},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"309","DOI":"10.1016\/0092-8674(93)90231-E","article-title":"Integrins alpha v beta 3 and alpha v beta 5 promote adenovirus internalization but not virus attachment","volume":"73","author":"Wickham","year":"1993","journal-title":"Cell"},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"4454","DOI":"10.1128\/JVI.78.9.4454-4462.2004","article-title":"The human membrane cofactor CD46 is a receptor for species B adenovirus serotype 3","volume":"78","author":"Sirena","year":"2004","journal-title":"J. Virol."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"715","DOI":"10.1016\/0092-8674(92)90306-W","article-title":"The small GTPase rab5 functions as a regulatory factor in the early endocytic pathway","volume":"70","author":"Bucci","year":"1992","journal-title":"Cell"},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"377","DOI":"10.1016\/S0092-8674(00)81966-2","article-title":"Oligomeric complexes link Rab5 effectors with NSF and drive membrane fusion via interactions between EEA1 and syntaxin 13","volume":"98","author":"McBride","year":"1999","journal-title":"Cell"},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"765","DOI":"10.1007\/s13365-019-00762-x","article-title":"Disturbances of mitochondrial dynamics in cultured neurons infected with human herpesvirus type 1 and type 2","volume":"25","author":"Cymerys","year":"2019","journal-title":"J. Neurovirol."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"91","DOI":"10.1016\/j.tjem.2018.08.001","article-title":"User\u2019s guide to correlation coefficients","volume":"18","author":"Akoglu","year":"2018","journal-title":"Turk. J. Emerg. Med."},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"1763","DOI":"10.1213\/ANE.0000000000002864","article-title":"Correlation Coefficients: Appropriate Use and Interpretation","volume":"126","author":"Schober","year":"2018","journal-title":"Anesth. Analg."},{"key":"ref_23","first-page":"371","article-title":"Detection of human adenoviruses with real-time PCR assay using TaqMan fluorescent probes","volume":"59","author":"Rola","year":"2007","journal-title":"Med. Dosw. Mikrobiol."},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"28","DOI":"10.3389\/fnana.2020.00028","article-title":"Location of the Cell Adhesion Molecule \u201cCoxsackievirus and Adenovirus Receptor\u201d in the Adult Mouse Brain","volume":"14","author":"Wehbi","year":"2020","journal-title":"Front. Neuroanat."},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"2895","DOI":"10.1128\/JVI.74.6.2895-2899.2000","article-title":"Productive replication of human adenoviruses in mouse epidermal cells","volume":"74","author":"Ganly","year":"2000","journal-title":"J. Virol."},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"412","DOI":"10.1016\/S1525-0016(03)00199-0","article-title":"Novel immunocompetent murine tumor models for the assessment of replication-competent oncolytic adenovirus efficacy","volume":"8","author":"Hallden","year":"2003","journal-title":"Mol. Ther."},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"417","DOI":"10.4149\/av_2016_04_417","article-title":"Primary murine neurons as in vitro model for studying neuroinfections caused by human adenoviruses","volume":"60","author":"Cymerys","year":"2016","journal-title":"Acta Virol."},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"213","DOI":"10.1111\/j.1365-2818.2006.01706.x","article-title":"A guided tour into subcellular colocalization analysis in light microscopy","volume":"224","author":"Bolte","year":"2006","journal-title":"J. Microsc."},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"C723","DOI":"10.1152\/ajpcell.00462.2010","article-title":"A practical guide to evaluating colocalization in biological microscopy","volume":"300","author":"Dunn","year":"2011","journal-title":"Am. J. Physiol. Cell Physiol."},{"key":"ref_30","doi-asserted-by":"crossref","unstructured":"Moser, B., Hochreiter, B., Herbst, R., and Schmid, J.A. (2017). Fluorescence colocalization microscopy analysis can be improved by combining object-recognition with pixel-intensity-correlation. Biotechnol. J., 12.","DOI":"10.1002\/biot.201600332"},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"1320","DOI":"10.1126\/science.275.5304.1320","article-title":"Isolation of a common receptor for Coxsackie B viruses and adenoviruses 2 and 5","volume":"275","author":"Bergelson","year":"1997","journal-title":"Science"},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"12125","DOI":"10.1128\/JVI.79.19.12125-12131.2005","article-title":"Adenovirus receptors","volume":"79","author":"Zhang","year":"2005","journal-title":"J. Virol."},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"224","DOI":"10.1016\/j.ymthe.2004.10.007","article-title":"Efficient species C HAdV infectivity in plasmocytic cell lines using a clathrin independent lipid raft\/caveola endocytic route","volume":"11","author":"Colin","year":"2005","journal-title":"Mol. Ther. J. Am. Soc. Gene Ther."},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"34","DOI":"10.1016\/S0960-9822(98)70018-1","article-title":"A novel role for Rab5-GDI in ligand sequestration into clathrin-coated pits","volume":"8","author":"Newell","year":"1998","journal-title":"Curr. Biol."},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"376","DOI":"10.1038\/14075","article-title":"Rab5 regulates motility of early endosomes on microtubules","volume":"1","author":"Nielsen","year":"1999","journal-title":"Nat. Cell Biol."},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"2657","DOI":"10.1091\/mbc.11.8.2657","article-title":"EEA1, a Tethering Protein of the Early Sorting Endosome, Shows a Polarized Distribution in Hippocampal Neurons, Epithelial Cells, and Fibroblasts","volume":"11","author":"Wilson","year":"2000","journal-title":"Mol. Biol. Cell"},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"2265","DOI":"10.1111\/j.1600-0854.2008.00835.x","article-title":"Infectious adenovirus type 2 transport through early but not late endosomes","volume":"9","author":"Gastaldelli","year":"2008","journal-title":"Traffic"},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"737","DOI":"10.1517\/14712590902988451","article-title":"Adenoviral virotherapy for malignant brain tumors","volume":"9","author":"Nandi","year":"2009","journal-title":"Expert. Opin. Biol. Ther."}],"container-title":["Pathogens"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/2076-0817\/13\/2\/158\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,10]],"date-time":"2025-10-10T13:58:07Z","timestamp":1760104687000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/2076-0817\/13\/2\/158"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2024,2,9]]},"references-count":38,"journal-issue":{"issue":"2","published-online":{"date-parts":[[2024,2]]}},"alternative-id":["pathogens13020158"],"URL":"https:\/\/doi.org\/10.3390\/pathogens13020158","relation":{},"ISSN":["2076-0817"],"issn-type":[{"value":"2076-0817","type":"electronic"}],"subject":[],"published":{"date-parts":[[2024,2,9]]}}}