{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"institution":[{"name":"medRxiv"}],"indexed":{"date-parts":[[2026,1,16]],"date-time":"2026-01-16T13:00:48Z","timestamp":1768568448420,"version":"3.49.0"},"posted":{"date-parts":[[2025,1,23]]},"group-title":"Infectious Diseases (except HIV\/AIDS)","reference-count":21,"publisher":"openRxiv","license":[{"start":{"date-parts":[[2025,1,23]],"date-time":"2025-01-23T00:00:00Z","timestamp":1737590400000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/www.medrxiv.org\/about\/FAQ#license"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"accepted":{"date-parts":[[2025,1,23]]},"abstract":"<jats:title>Abstract<\/jats:title>\n                <jats:sec>\n                  <jats:title>Background and Aims<\/jats:title>\n                  <jats:p>Hepatitis B virus (HBV) generates a double-stranded linear DNA (dslDNA) byproduct during replication. This dslDNA can undergo intermolecular and intramolecular nonhomologous end-joining (NHEJ) recombination, resulting in viral integration and dslDNA derived covalently closed circular DNAs (dsl-cccDNAs), respectively. The insertions and deletions (INDELs) at the end-joining site around the direct repeat (DR) 1 motif have been used to differentiate dsl-cccDNA from the authentic cccDNA. The frequency and characteristics of dsl-cccDNA in chronic hepatitis B (CHB) patients remain unclear.<\/jats:p>\n                <\/jats:sec>\n                <jats:sec>\n                  <jats:title>Methods<\/jats:title>\n                  <jats:p>HBV-targeted next-generation sequencing (NGS) was used to identify 32 dsl-cccDNA positive candidates, 22 HBeAg(+) and 10 HBeAg(-), from 56 liver biopsies of antiviral treatment-naive CHB patients for dsl-cccDNA confirmation and characterization by cccDNA-PCR NGS. INDELs within the DR2-1 region (nt 1600\u20131840) of the cccDNA were analyzed.<\/jats:p>\n                <\/jats:sec>\n                <jats:sec>\n                  <jats:title>Results<\/jats:title>\n                  <jats:p>\n                    Various clonally expanded, heterogenous \u223c22-nt deletions in the X-gene were detected in all 32 samples, which are likely quasi-species from the authentic cccDNA. We, therefore, defined dsl-cccDNA only by the presence of INDELs clustered at the DR1 surrounding region (nt 1800\u20131840). The percentage of dsl-cccDNA in total cccDNA was higher among HBeAg(+) compared to HBeAg(-) samples [11.32 (3.24\u201326.94)%\n                    <jats:italic>vs.<\/jats:italic>\n                    7.72 (2.16\u201328.23)%, p=0.01]. The diversity of dsl-cccDNA species correlated with cccDNA levels (log-transformed; r=0.82, p&lt;0.001), HBeAg(+) CHB (p&lt;0.001), and serum HBV DNA (p&lt;0.001).\n                  <\/jats:p>\n                <\/jats:sec>\n                <jats:sec>\n                  <jats:title>Conclusions<\/jats:title>\n                  <jats:p>dsl-cccDNA is more prevalent and heterogenous among the HBeAg(+) CHB subjects, which is likely due to the active viral cccDNA transcription and reverse transcription at the HBeAg(+) phase. The existence of replication-defective dsl-cccDNA may facilitate immune evasion and HBV integration, and complicate HBV pathogenesis. The potential impact of dsl-cccDNA in HBV therapeutic response deserves further assessment.<\/jats:p>\n                <\/jats:sec>","DOI":"10.1101\/2025.01.20.25320854","type":"posted-content","created":{"date-parts":[[2025,1,23]],"date-time":"2025-01-23T19:15:12Z","timestamp":1737659712000},"source":"Crossref","is-referenced-by-count":0,"title":["Detection and characterization of Hepatitis B virus double-stranded linear DNA-derived covalently closed circular DNA in chronic hepatitis B patients"],"prefix":"10.64898","author":[{"given":"Hsin-Ni","family":"Liu","sequence":"first","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Elena","family":"Kim","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Zhili","family":"Wang","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"ThiThuyTu","family":"Nguyen","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Fwu-Shan","family":"Shieh","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Yuanjie","family":"Liu","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Marc G.","family":"Ghany","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Raymond T.","family":"Chung","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Richard K.","family":"Sterling","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Selena Y","family":"Lin","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Haitao","family":"Guo","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Daryl T-Y","family":"Lau","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Ying-Hsiu","family":"Su","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"54368","reference":[{"key":"2025012510501098000_2025.01.20.25320854v1.1","doi-asserted-by":"publisher","DOI":"10.1016\/j.cld.2007.08.002"},{"key":"2025012510501098000_2025.01.20.25320854v1.2","doi-asserted-by":"crossref","unstructured":"Seeger C , Mason WS . 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