{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2025,10,11]],"date-time":"2025-10-11T01:39:17Z","timestamp":1760146757159,"version":"build-2065373602"},"reference-count":26,"publisher":"MDPI AG","issue":"12","license":[{"start":{"date-parts":[[2024,12,7]],"date-time":"2024-12-07T00:00:00Z","timestamp":1733529600000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"Korea National Institute of Health","award":["2022-NG-003-02"],"award-info":[{"award-number":["2022-NG-003-02"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Viruses"],"abstract":"<jats:p>Hepatitis B virus (HBV) can cause chronic infections, significantly increasing the risk of death from cirrhosis and hepatocellular carcinoma (HCC). A key player in chronic HBV infection is covalently closed circular DNA (cccDNA), a stable episomal form of viral DNA that acts as a persistent reservoir in infected hepatocytes and drives continuous viral replication. Despite the development of several animal models, few adequately replicate cccDNA formation and maintenance, limiting our understanding of its dynamics and the evaluation of potential therapeutic interventions targeting cccDNA. In this study, we aimed to develop a mouse model to investigate cccDNA formation and maintenance. We infected C57BL\/6 mice with recombinant adeno-associated virus (rAAV) carrying a 1.3-overlength HBV genome (genotype C) and collected liver tissue at various time points to assess cccDNA levels and viral replication. Our results demonstrated the successful establishment of a chronic hepatitis B mouse model using rAAV-HBV1.3, which supported persistent HBV infection with sustained cccDNA expression in hepatocytes. Serum levels of HBsAg and HBeAg were elevated for up to 12 weeks, while alanine transaminase (ALT) levels remained within the normal range, indicating limited liver damage during this period. We confirmed HBV DNA expression in hepatocytes, and importantly, cccDNA was detected using qPCR after Plasmid-Safe ATP-Dependent DNase treatment, which selectively removes non-cccDNA forms. Additionally, Southern blot analysis confirmed the presence of cccDNA isolated using the Hirt extraction method. This established model provides a valuable platform for studying the long-term maintenance of cccDNA in chronic HBV infection and offers an important tool for testing novel therapeutic strategies aimed at targeting cccDNA.<\/jats:p>","DOI":"10.3390\/v16121890","type":"journal-article","created":{"date-parts":[[2024,12,9]],"date-time":"2024-12-09T06:16:14Z","timestamp":1733724974000},"page":"1890","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Chronic Hepatitis B Genotype C Mouse Model with Persistent Covalently Closed Circular DNA"],"prefix":"10.3390","volume":"16","author":[{"ORCID":"https:\/\/orcid.org\/0009-0001-0787-5155","authenticated-orcid":false,"given":"Deok-Hwa","family":"Seo","sequence":"first","affiliation":[{"name":"The Catholic University Liver Research Center, College of Medicine, The Catholic University of Korea, Seoul 06591, Republic of Korea"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Wonhee","family":"Hur","sequence":"additional","affiliation":[{"name":"Division of Chronic Viral Diseases, Center for Emerging Virus Research, National Institute of Health (NIH), Cheongju 28159, Republic of Korea"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Juhee","family":"Won","sequence":"additional","affiliation":[{"name":"Department of Precision Medicine, School of Medicine, Sungkyunkwan University, Suwon 16419, Republic of Korea"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-1456-1450","authenticated-orcid":false,"given":"Ji-Won","family":"Han","sequence":"additional","affiliation":[{"name":"Division of Gastroenterology and Hepatology, Department of Internal Medicine, College of Medicine, Seoul St. Mary\u2019s Hospital, The Catholic University of Korea, Seoul 06591, Republic of Korea"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-4476-4868","authenticated-orcid":false,"given":"Seung-Kew","family":"Yoon","sequence":"additional","affiliation":[{"name":"Division of Gastroenterology and Hepatology, Department of Internal Medicine, College of Medicine, Seoul St. Mary\u2019s Hospital, The Catholic University of Korea, Seoul 06591, Republic of Korea"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Songmee","family":"Bae","sequence":"additional","affiliation":[{"name":"Division of Chronic Viral Diseases, Center for Emerging Virus Research, National Institute of Health (NIH), Cheongju 28159, Republic of Korea"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-5266-072X","authenticated-orcid":false,"given":"Kyun-Hwan","family":"Kim","sequence":"additional","affiliation":[{"name":"Department of Precision Medicine, School of Medicine, Sungkyunkwan University, Suwon 16419, Republic of Korea"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-5780-9607","authenticated-orcid":false,"given":"Pil-Soo","family":"Sung","sequence":"additional","affiliation":[{"name":"The Catholic University Liver Research Center, College of Medicine, The Catholic University of Korea, Seoul 06591, Republic of Korea"},{"name":"Division of Gastroenterology and Hepatology, Department of Internal Medicine, College of Medicine, Seoul St. Mary\u2019s Hospital, The Catholic University of Korea, Seoul 06591, Republic of Korea"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1968","published-online":{"date-parts":[[2024,12,7]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"104925","DOI":"10.1016\/j.antiviral.2020.104925","article-title":"Hepatitis B Virus Biology and Life Cycle","volume":"182","author":"Tsukuda","year":"2020","journal-title":"Antivir. Res."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"201","DOI":"10.1016\/j.jcmgh.2023.03.011","article-title":"Hepatitis B Virus Targets Lipid Transport Pathways to Infect Hepatocytes","volume":"16","author":"Esser","year":"2023","journal-title":"Cell Mol. Gastroenterol. Hepatol."},{"key":"ref_3","doi-asserted-by":"crossref","unstructured":"Sung, P.S., Park, D.J., Kim, J.H., Han, J.W., Lee, E.B., Lee, G.W., Nam, H.C., Jang, J.W., Bae, S.H., and Choi, J.Y. (2019). Ex Vivo Detection and Characterization of Hepatitis B Virus-Specific CD8(+) T Cells in Patients Considered Immune Tolerant. Front. Immunol., 10.","DOI":"10.3389\/fimmu.2019.01319"},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"478","DOI":"10.1097\/MCG.0000000000001695","article-title":"An Evidence-Based Practical Guide to Vaccination for Hepatitis B Virus","volume":"56","author":"Jacobson","year":"2022","journal-title":"J. Clin. Gastroenterol."},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"605","DOI":"10.3350\/cmh.2022.0342","article-title":"Development of Hepatocellular Carcinoma in Treated and Untreated Patients with Chronic Hepatitis B Virus Infection","volume":"29","author":"Lin","year":"2023","journal-title":"Clin. Mol. Hepatol."},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"1249","DOI":"10.1016\/j.jhep.2021.11.024","article-title":"How to Achieve Functional Cure of Hbv: Stopping Nucs, Adding Interferon or New Drug Development?","volume":"76","author":"Wong","year":"2022","journal-title":"J. Hepatol."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"232","DOI":"10.5009\/gnl18546","article-title":"Nucleos(T)Ide Analogues for Reducing Hepatocellular Carcinoma in Chronic Hepatitis B Patients: A Systematic Review and Meta-Analysis","volume":"14","author":"Wang","year":"2020","journal-title":"Gut Liver"},{"key":"ref_8","doi-asserted-by":"crossref","unstructured":"Li, Q., Sun, B., Zhuo, Y., Jiang, Z., Li, R., Lin, C., Jin, Y., Gao, Y., and Wang, D. (2022). Interferon and Interferon-Stimulated Genes in Hbv Treatment. Front. Immunol., 13.","DOI":"10.3389\/fimmu.2022.1034968"},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"539","DOI":"10.3350\/cmh.2024.0060","article-title":"Novel Role of MHC Class II Transactivator in Hepatitis B Virus Replication and Viral Counteraction","volume":"30","author":"Dezhbord","year":"2024","journal-title":"Clin. Mol. Hepatol."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"1591","DOI":"10.1038\/s41467-021-21850-9","article-title":"Hepatitis B Virus Cccdna Is Formed through Distinct Repair Processes of Each Strand","volume":"12","author":"Wei","year":"2021","journal-title":"Nat. Commun."},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"71","DOI":"10.1038\/cmi.2013.43","article-title":"A Mouse Model for Hbv Immunotolerance and Immunotherapy","volume":"11","author":"Yang","year":"2014","journal-title":"Cell Mol. Immunol."},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"85","DOI":"10.5625\/lar.2018.34.3.85","article-title":"Mouse Models for Hepatitis B Virus Research","volume":"34","author":"Hwang","year":"2018","journal-title":"Lab. Anim. Res."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"1001","DOI":"10.1016\/j.jcmgh.2021.11.011","article-title":"A Novel Mouse Model Harboring Hepatitis B Virus Covalently Closed Circular DNA","volume":"13","author":"Xu","year":"2022","journal-title":"Cell Mol. Gastroenterol. Hepatol."},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"14","DOI":"10.1016\/j.antiviral.2017.07.006","article-title":"Detection of the Hepatitis B Virus (Hbv) Covalently-Closed-Circular DNA (Cccdna) in Mice Transduced with a Recombinant Aav-Hbv Vector","volume":"145","author":"Lucifora","year":"2017","journal-title":"Antivir. Res."},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"24066","DOI":"10.1021\/acsomega.2c02216","article-title":"Hbv Cccdna\u2014A Culprit and Stumbling Block for the Hepatitis B Virus Infection: Its Presence in Hepatocytes Perplexed the Possible Mission for a Functional Cure","volume":"7","author":"Bhat","year":"2022","journal-title":"ACS Omega"},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"e163175","DOI":"10.1172\/JCI163175","article-title":"Functional Cure of Hepatitis B Requires Silencing Covalently Closed Circular and Integrated Hepatitis B Virus DNA","volume":"132","author":"Ghany","year":"2022","journal-title":"J. Clin. Investig."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"972","DOI":"10.1136\/gutjnl-2022-328380","article-title":"Quantification of the Hepatitis B Virus Cccdna: Evidence-Based Guidelines for Monitoring the Key Obstacle of Hbv Cure","volume":"72","author":"Allweiss","year":"2023","journal-title":"Gut"},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"e0046824","DOI":"10.1128\/jvi.00468-24","article-title":"Tripartite Motif-Containing Protein 21 Is Involved in Ifn-Gamma-Induced Suppression of Hepatitis B Virus by Regulating Hepatocyte Nuclear Factors","volume":"98","author":"Won","year":"2024","journal-title":"J. Virol."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"677","DOI":"10.1016\/j.jhep.2023.01.017","article-title":"Eliminating Cccdna to Cure Hepatitis B Virus Infection","volume":"78","author":"Zoulim","year":"2023","journal-title":"J. Hepatol."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"311","DOI":"10.1053\/j.gastro.2018.07.057","article-title":"Development of Direct-Acting Antiviral and Host-Targeting Agents for Treatment of Hepatitis B Virus Infection","volume":"156","author":"Xia","year":"2019","journal-title":"Gastroenterology"},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"1275","DOI":"10.1002\/hep.32245","article-title":"Hbv Covalently Closed Circular DNA Minichromosomes in Distinct Epigenetic Transcriptional States Differ in Their Vulnerability to Damage","volume":"75","author":"Wang","year":"2022","journal-title":"Hepatology"},{"key":"ref_22","doi-asserted-by":"crossref","unstructured":"Wettengel, J.M., and Burwitz, B.J. (2020). Innovative Hbv Animal Models Based on the Entry Receptor Ntcp. Viruses, 12.","DOI":"10.3390\/v12080828"},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"105140","DOI":"10.1016\/j.antiviral.2021.105140","article-title":"Intramolecular Recombination Enables the Formation of Hepatitis B Virus (Hbv) Cccdna in Mice after Hbv Genome Transfer Using Recombinant Aav Vectors","volume":"194","author":"Ko","year":"2021","journal-title":"Antivir. Res."},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"e27941","DOI":"10.1097\/MD.0000000000027941","article-title":"Distribution of Hepatitis B Virus Genotypes and Subgenotypes: A Meta-Analysis","volume":"100","author":"Liu","year":"2021","journal-title":"Medicine"},{"key":"ref_25","doi-asserted-by":"crossref","unstructured":"Du, Y., Broering, R., Li, X., Zhang, X., Liu, J., Yang, D., and Lu, M. (2021). In Vivo Mouse Models for Hepatitis B Virus Infection and Their Application. Front. Immunol., 12.","DOI":"10.3389\/fimmu.2021.766534"},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"103617","DOI":"10.1016\/j.drudis.2023.103617","article-title":"Targeting Hepatitis B Virus Cccdna Levels: Recent Progress in Seeking Small Molecule Drug Candidates","volume":"28","author":"Jin","year":"2023","journal-title":"Drug Discov. Today"}],"container-title":["Viruses"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1999-4915\/16\/12\/1890\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,10]],"date-time":"2025-10-10T16:49:26Z","timestamp":1760114966000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1999-4915\/16\/12\/1890"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2024,12,7]]},"references-count":26,"journal-issue":{"issue":"12","published-online":{"date-parts":[[2024,12]]}},"alternative-id":["v16121890"],"URL":"https:\/\/doi.org\/10.3390\/v16121890","relation":{},"ISSN":["1999-4915"],"issn-type":[{"type":"electronic","value":"1999-4915"}],"subject":[],"published":{"date-parts":[[2024,12,7]]}}}