{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,30]],"date-time":"2026-09-30T07:36:54Z","timestamp":1790753814602,"version":"4.1.0"},"reference-count":35,"publisher":"Public Library of Science (PLoS)","issue":"4","license":[{"start":{"date-parts":[[2022,4,1]],"date-time":"2022-04-01T00:00:00Z","timestamp":1648771200000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/100005564","name":"Gilead Sciences","doi-asserted-by":"publisher","id":[{"id":"10.13039\/100005564","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":["www.plosone.org"],"crossmark-restriction":false},"short-container-title":["PLoS ONE"],"abstract":"<jats:p>Nucleos(t)ide analogs are standard-of-care for the treatment of chronic hepatitis B and can effectively reduce hepatitis B virus (HBV) replication but rarely leads to cure. Nucleos(t)ide analogs do not directly eliminate the viral episome, therefore treatment cessation typically leads to rapid viral rebound. While treatment is effective, HBV DNA is still detectable (although not quantifiable) in the periphery of the majority of nucleos(t)ide analog treated HBV patients, even after prolonged treatment. Addressing whether the detectable HBV DNA represents infectious virus is a key unknown and has important implications for the development of a curative treatment for HBV. The minimum HBV genome equivalents required to establish infection in human liver chimeric mice was determined by titration of HBV patient sera and the infectivity in chimeric mice of serum from patients (n = 7) suppressed to the limit of detection on nucleos(t)ide analog therapy was evaluated. A minimum of 5 HBV genome equivalents were required to establish infection in the chimeric mice, confirming this model has sufficient sensitivity to determine whether serum from virally suppressed patients contains infectious virus. Strikingly, serum from 75% (n = 3 out of 4) of nucleos(t)ide-treated HBV patients with DNA that was detectable, but below the lower limit of quantitation, also established infection in the chimeric mice. These results demonstrate that infectious virus is still present in some HBV patients on suppressive nucleos(t)ide therapy. This residual virus may support viral persistence via continuous infection and explain the ongoing risk for HBV-related complications despite long-term suppression on therapy. Thus, additional treatment intensification may facilitate HBV cure.<\/jats:p>","DOI":"10.1371\/journal.pone.0262516","type":"journal-article","created":{"date-parts":[[2022,4,1]],"date-time":"2022-04-01T13:35:34Z","timestamp":1648820134000},"page":"e0262516","update-policy":"https:\/\/doi.org\/10.1371\/journal.pone.corrections_policy","source":"Crossref","is-referenced-by-count":19,"title":["Ongoing viral replication and production of infectious virus in patients with chronic hepatitis B virus suppressed below the limit of quantitation on long-term nucleos(t)ide therapy"],"prefix":"10.1371","volume":"17","author":[{"ORCID":"https:\/\/orcid.org\/0000-0003-3856-0815","authenticated-orcid":true,"given":"Dara L","family":"Burdette","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Scott","family":"Lazerwith","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jenny","family":"Yang","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Henry L. Y.","family":"Chan","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"William E.","family":"Delaney IV","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Simon P.","family":"Fletcher","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Tomas","family":"Cihlar","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Becket","family":"Feierbach","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"340","published-online":{"date-parts":[[2022,4,1]]},"reference":[{"key":"pone.0262516.ref001","author":"World Health Organization (WHO)","journal-title":"Hepatitis B. Introduction"},{"issue":"4","key":"pone.0262516.ref002","doi-asserted-by":"crossref","first-page":"607","DOI":"10.1016\/j.cld.2016.06.006","article-title":"Epidemiology of Hepatitis B Virus Infection and Impact of Vaccination on Disease","volume":"20","author":"NP Nelson","year":"2016","journal-title":"Clinics in liver disease"},{"issue":"2","key":"pone.0262516.ref003","doi-asserted-by":"crossref","first-page":"297","DOI":"10.1053\/j.gastro.2018.10.048","article-title":"What Is Needed to Eliminate Hepatitis B Virus and Hepatitis C Virus as Global Health Threats","volume":"156","author":"JW Ward","year":"2019","journal-title":"Gastroenterology"},{"issue":"12","key":"pone.0262516.ref004","doi-asserted-by":"crossref","first-page":"1943","DOI":"10.1136\/gutjnl-2013-306409","article-title":"Patients with chronic hepatitis B treated with oral antiviral therapy retain a higher risk for HCC compared with patients with inactive stage disease","volume":"63","author":"JY Cho","year":"2014","journal-title":"Gut"},{"issue":"1","key":"pone.0262516.ref005","doi-asserted-by":"crossref","first-page":"98","DOI":"10.1002\/hep.26180","article-title":"Long-term entecavir treatment reduces hepatocellular carcinoma incidence in patients with hepatitis B virus infection","volume":"58","author":"T Hosaka","year":"2013","journal-title":"Hepatology"},{"issue":"1","key":"pone.0262516.ref006","doi-asserted-by":"crossref","first-page":"399","DOI":"10.1002\/hep.25937","article-title":"Prevention of hepatitis B virus-related hepatocellular carcinoma with antiviral therapy","volume":"57","author":"CL Lai","year":"2013","journal-title":"Hepatology"},{"issue":"1","key":"pone.0262516.ref007","doi-asserted-by":"crossref","first-page":"143","DOI":"10.1053\/j.gastro.2014.03.048","article-title":"Association of nucleos(t)ide analogue therapy with reduced risk of hepatocellular carcinoma in patients with chronic hepatitis B: a nationwide cohort study","volume":"147","author":"CY Wu","year":"2014","journal-title":"Gastroenterology"},{"issue":"5","key":"pone.0262516.ref008","doi-asserted-by":"crossref","first-page":"1457","DOI":"10.1007\/s10620-014-3486-7","article-title":"Seven-year efficacy and safety of treatment with tenofovir disoproxil fumarate for chronic hepatitis B virus infection","volume":"60","author":"M Buti","year":"2015","journal-title":"Digestive diseases and sciences"},{"issue":"4","key":"pone.0262516.ref009","doi-asserted-by":"crossref","first-page":"341","DOI":"10.1016\/S1473-3099(11)70314-0","article-title":"Selection of chronic hepatitis B therapy with high barrier to resistance","volume":"12","author":"R Gish","year":"2012","journal-title":"The Lancet Infectious diseases"},{"issue":"5","key":"pone.0262516.ref010","doi-asserted-by":"crossref","first-page":"1593","DOI":"10.1053\/j.gastro.2009.08.063","article-title":"Hepatitis B virus resistance to nucleos(t)ide analogues","volume":"137","author":"F Zoulim","year":"2009","journal-title":"Gastroenterology"},{"issue":"2","key":"pone.0262516.ref011","doi-asserted-by":"crossref","first-page":"335","DOI":"10.1002\/hep.28916","article-title":"Low-level viremia and the increased risk of hepatocellular carcinoma in patients receiving entecavir treatment","volume":"66","author":"JH Kim","year":"2017","journal-title":"Hepatology"},{"issue":"5","key":"pone.0262516.ref012","doi-asserted-by":"crossref","first-page":"1240","DOI":"10.1053\/j.gastro.2014.01.044","article-title":"Effects of Tenofovir Disoproxil Fumarate in Hepatitis B e Antigen-Positive Patients With Normal Levels of Alanine Aminotransferase and High Levels of Hepatitis B Virus DNA","volume":"146","author":"HL Chan","year":"2014","journal-title":"Gastroenterology"},{"issue":"4","key":"pone.0262516.ref013","doi-asserted-by":"crossref","first-page":"683","DOI":"10.1016\/j.jhep.2016.05.014","article-title":"Decay of ccc-DNA marks persistence of intrahepatic viral DNA synthesis under tenofovir in HIV-HBV co-infected patients","volume":"65","author":"A Boyd","year":"2016","journal-title":"J Hepatol"},{"issue":"3","key":"pone.0262516.ref014","doi-asserted-by":"crossref","first-page":"273","DOI":"10.1016\/j.bpg.2017.04.014","article-title":"Animal models of HBV infection","volume":"31","author":"M Dandri","year":"2017","journal-title":"Best practice & research Clinical gastroenterology"},{"issue":"5","key":"pone.0262516.ref015","doi-asserted-by":"crossref","first-page":"1610","DOI":"10.1002\/hep.26428","article-title":"Discovery of naturally occurring transmissible chronic hepatitis B virus infection among Macaca fascicularis from Mauritius Island","volume":"58","author":"T Dupinay","year":"2013","journal-title":"Hepatology"},{"issue":"1","key":"pone.0262516.ref016","first-page":"1","article-title":"Hepatitis B virus infection of tupaia hepatocytes in vitro and in vivo","volume":"24","author":"E Walter","year":"1996","journal-title":"Hepatology"},{"issue":"1","key":"pone.0262516.ref017","doi-asserted-by":"crossref","first-page":"234","DOI":"10.1053\/j.gastro.2005.03.090","article-title":"Mapping of the hepatitis B virus attachment site by use of infection-inhibiting preS1 lipopeptides and tupaia hepatocytes","volume":"129","author":"D Glebe","year":"2005","journal-title":"Gastroenterology"},{"issue":"19","key":"pone.0262516.ref018","doi-asserted-by":"crossref","first-page":"9652","DOI":"10.1128\/JVI.00867-09","article-title":"The size of the viral inoculum contributes to the outcome of hepatitis B virus infection","volume":"83","author":"S Asabe","year":"2009","journal-title":"Journal of virology"},{"issue":"5","key":"pone.0262516.ref019","doi-asserted-by":"crossref","first-page":"1046","DOI":"10.1002\/hep.20892","article-title":"Infection of human hepatocyte chimeric mouse with genetically engineered hepatitis B virus","volume":"42","author":"M Tsuge","year":"2005","journal-title":"Hepatology"},{"issue":"6","key":"pone.0262516.ref020","doi-asserted-by":"crossref","DOI":"10.3390\/v9060156","article-title":"The Role of cccDNA in HBV Maintenance","volume":"9","author":"L Allweiss","year":"2017","journal-title":"Viruses"},{"issue":"3","key":"pone.0262516.ref021","doi-asserted-by":"crossref","first-page":"542","DOI":"10.1136\/gutjnl-2016-312162","article-title":"Proliferation of primary human hepatocytes and prevention of hepatitis B virus reinfection efficiently deplete nuclear cccDNA in vivo","volume":"67","author":"L Allweiss","year":"2018","journal-title":"Gut"},{"issue":"3","key":"pone.0262516.ref022","doi-asserted-by":"crossref","first-page":"685","DOI":"10.1002\/hep.24758","article-title":"Humanized chimeric uPA mouse model for the study of hepatitis B and D virus interactions and preclinical drug evaluation","volume":"55","author":"M Lutgehetmann","year":"2012","journal-title":"Hepatology"},{"issue":"2","key":"pone.0262516.ref023","doi-asserted-by":"crossref","first-page":"243","DOI":"10.1007\/s12072-015-9614-4","article-title":"Long-term clinical outcomes in cirrhotic chronic hepatitis B patients treated with tenofovir disoproxil fumarate for up to 5 years","volume":"9","author":"M Buti","year":"2015","journal-title":"Hepatology international"},{"issue":"3","key":"pone.0262516.ref024","doi-asserted-by":"crossref","first-page":"901","DOI":"10.1016\/S0002-9440(10)63352-4","article-title":"Near completely humanized liver in mice shows human-type metabolic responses to drugs","volume":"165","author":"C Tateno","year":"2004","journal-title":"The American journal of pathology"},{"issue":"2","key":"pone.0262516.ref025","doi-asserted-by":"crossref","first-page":"473","DOI":"10.1002\/hep.29891","article-title":"Acute hepatitis B virus infection in humanized chimeric mice has multiphasic viral kinetics","volume":"68","author":"Y Ishida","year":"2018","journal-title":"Hepatology"},{"issue":"2","key":"pone.0262516.ref026","doi-asserted-by":"crossref","first-page":"233","DOI":"10.1177\/0192623314544378","article-title":"Chimeric mice with hepatocyte-humanized liver as an appropriate model to study human peroxisome proliferator-activated receptor-\u03b1","volume":"43","author":"C Tateno","year":"2015","journal-title":"Toxicologic pathology"},{"key":"pone.0262516.ref027","article-title":"Evidence for Ongoing Low-Level Viremia in Patients With Chronic Hepatitis B Receiving Long-Term Nucleos(t)ide Analog Therapy [Poster 1861]","author":"P Marcellin","journal-title":"The 65th Annual Meeting of the American Association for the Study of Liver Diseases: The Liver Meeting (AASLD); 2014 November 7\u201314; Boston MA United States"},{"issue":"19","key":"pone.0262516.ref028","doi-asserted-by":"crossref","DOI":"10.1172\/jci.insight.140584","article-title":"Single hepatocytes show persistence and transcriptional inactivity of hepatitis B","volume":"5","author":"A Balagopal","year":"2020","journal-title":"JCI Insight"},{"issue":"4","key":"pone.0262516.ref029","doi-asserted-by":"crossref","first-page":"794","DOI":"10.1016\/j.jhep.2020.11.003","article-title":"Transition to HBeAg-negative chronic hepatitis B virus infection is associated with reduced cccDNA transcriptional activity","volume":"74","author":"A Suslov","year":"2021","journal-title":"J Hepatol"},{"issue":"1","key":"pone.0262516.ref030","doi-asserted-by":"crossref","first-page":"21097","DOI":"10.1038\/s41598-020-78001-1","article-title":"Quantification and epigenetic evaluation of the residual pool of hepatitis B covalently closed circular DNA in long-term nucleoside analogue-treated patients","volume":"10","author":"F Lebosse","year":"2020","journal-title":"Sci Rep"},{"issue":"1","key":"pone.0262516.ref031","doi-asserted-by":"crossref","first-page":"65","DOI":"10.1001\/jama.295.1.65","article-title":"Risk of hepatocellular carcinoma across a biological gradient of serum hepatitis B virus DNA level","volume":"295","author":"CJ Chen","year":"2006","journal-title":"Jama"},{"issue":"8","key":"pone.0262516.ref032","doi-asserted-by":"crossref","first-page":"1797","DOI":"10.1111\/j.1572-0241.2006.00647.x","article-title":"Past HBV viral load as predictor of mortality and morbidity from HCC and chronic liver disease in a prospective study","volume":"101","author":"G Chen","year":"2006","journal-title":"The American journal of gastroenterology"},{"issue":"1","key":"pone.0262516.ref033","doi-asserted-by":"crossref","first-page":"41","DOI":"10.1002\/hep.31240","article-title":"Rapid Turnover of Hepatitis B Virus Covalently Closed Circular DNA Indicated by Monitoring Emergence and Reversion of Signature-Mutation in Treated Chronic Hepatitis B Patients","volume":"73","author":"Q Huang","year":"2021","journal-title":"Hepatology"},{"issue":"1","key":"pone.0262516.ref034","doi-asserted-by":"crossref","first-page":"veaa063","DOI":"10.1093\/ve\/veaa063","article-title":"Estimating hepatitis B virus cccDNA persistence in chronic infection","volume":"7","author":"KA Lythgoe","year":"2021","journal-title":"Virus Evol"},{"issue":"12","key":"pone.0262516.ref035","doi-asserted-by":"crossref","first-page":"e13250","DOI":"10.1111\/cmi.13250","article-title":"Synchronised infection identifies early rate-limiting steps in the hepatitis B virus life cycle","volume":"22","author":"A Chakraborty","year":"2020","journal-title":"Cell Microbiol"}],"container-title":["PLOS ONE"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/dx.plos.org\/10.1371\/journal.pone.0262516","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2022,4,1]],"date-time":"2022-04-01T13:36:02Z","timestamp":1648820162000},"score":1,"resource":{"primary":{"URL":"https:\/\/dx.plos.org\/10.1371\/journal.pone.0262516"}},"subtitle":[],"editor":[{"given":"Haitao","family":"Guo","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"editor"}]}],"short-title":[],"issued":{"date-parts":[[2022,4,1]]},"references-count":35,"journal-issue":{"issue":"4","published-online":{"date-parts":[[2022,4,1]]}},"URL":"https:\/\/doi.org\/10.1371\/journal.pone.0262516","relation":{},"ISSN":["1932-6203"],"issn-type":[{"value":"1932-6203","type":"electronic"}],"subject":[],"published":{"date-parts":[[2022,4,1]]}}}