An occult hepatitis B-derived hepatoma cell line carrying persistent nuclear viral DNA and permissive for exogenous hepatitis B virus infection
Authors: Chau-Ting Yeh
Rong-Nan Chien
Chen-Chun Lin
Chih-Lang Lin
Po-Yuan Ke
Shi-Ming Lin
Journal: PLoS ONE (2013)
Discipline: Multidisciplinary
Keywords: Occult hepatitis B virus; HBV DNA; hepatoma cell lines; chronic hepatitis C virus; virological basis
Affiliations: Liver Research Unit, Chang Gung Memorial Hospital and Chang Gung University College of Medicine, Keelung, Taiwan.

Abstract:  Occult hepatitis B virus (HBV) infection is defined as persistence of HBV DNA in liver tissues, with or without detectability of HBV DNA in the serum, in individuals with negative serum HBV surface antigen (HBsAg). Despite accumulating evidence suggesting its important clinical roles, the molecular and virological basis of occult hepatitis B remains unclear. In an attempt to establish new hepatoma cell lines, we achieved a new cell line derived from a hepatoma patient with chronic hepatitis C virus (HCV) and occult HBV infection. Characterization of this cell line revealed previously unrecognized properties. Two novel human hepatoma cell lines were established. Hep-Y1 was derived from a male hepatoma patient negative for HCV and HBV infection. Hep-Y2 was derived from a female hepatoma patient suffering from chronic HCV and occult HBV infection. Morphological, cytogenetic and functional studies were performed. Permissiveness to HBV infection was assessed. Both cell lines showed typical hepatocyte-like morphology under phase-contrast and electron microscopy and expressed alpha-fetoprotein, albumin, transferrin, and aldolase B. Cytogenetic analysis revealed extensive chromosomal anomalies. An extrachromosomal form of HBV DNA persisted in the nuclear fraction of Hep-Y2 cells, while no HBsAg was detected in the medium. After treated with 2% dimethyl sulfoxide, both cell lines were permissive for exogenous HBV infection with transient elevation of the replication intermediates in the cytosol with detectable viral antigens by immunoflurescence analysis. In conclusions, we established two new hepatoma cell lines including one from occult HBV infection (Hep-Y2). Both cell lines were permissive for HBV infection. Additionally, Hep-Y2 cells carried persistent extrachromosomal HBV DNA in the nuclei. This cell line could serve as a useful tool to establish the molecular and virological basis of occult HBV infection. 

Retracted: Yes

PubPeer comments:
 Figure 5. * Green arrows: Several cuts appear to be visible in panels A and B. * Red boxes: In panel 5A, lanes 11 and 12 look similar to lanes 17 and 18, and to 19 and 20 * Blue boxes: In panel 5B, lanes C and 1 look similar to lanes 4 and 5 ![file] , 
 These problems have already been raised by a PLOS one editor a few months after publication. The cut and paste figure was done when adding data requested by a reviewer. All original figures were then provided to the editors for confirmation. , 
 #2 Thank you for your reply. I find it difficult to understand why the reviewers or editors of PLoS ONE asked for the repeated sections outlined in the red and blue boxes to be inserted into this figure. Would you be able to share that correspondence to help clarify this request? Also could you provide the original uncropped scans here. , 
 In the pre-revised manuscript, we did not provide sufficient day-to-day data (because they were negative). So the reviewer ask for these data even they were negative results. We performed the experiments again and add in the data. Please note that the added data (from the lower panel) were negative results (no positive band). We were unable to identify which film area was used in Figure 5A. ![file] , 
 For Figure 5B, we also attach one set of the original film. Note that we cannot identify the exact negative results area.![file] , 
 Retraction, January 28, 2021. From the Retraction Notice:  Following the publication of this article [1], concerns were raised regarding results presented in Fig 5. Specifically: * Vertical irregularities suggestive of image splicing have been detected between lanes 10u201311, 17u201318, 18u201319, and 20-P in Fig 5A and between lanes 3u20134, 5u20136, 10u201311, 12u201313, 15u201316, and 17u201318 in Fig 5B. The authors explained that the panels were spliced after additional data points were added to the original figure at the revieweru2019s request. * In Fig 5A, the results shown in lanes 11u201312 appear to partially overlap with those shown in lanes 17u201318 and 19u201320 despite representing different experimental conditions. * In Fig 5B, the results shown in lanes C-1 appear to partially overlap with those shown in lanes 3u20135 despite representing different experimental conditions. * The authors have provided underlying blots for the panels presented in Fig 5A and Fig 5B, and although these blots confirm the image splicing, they do not clarify the similarity within the panels discussed above. Furthermore, the underlying data provided for lanes 9, 10, 18 and 19 of Fig 5A and lanes C-7, 11, 12, 16, and 17 of Fig 5B did not match the results presented in the published panels. In light of the concerns affecting multiple figure panels that question the integrity of these data, the PLOS ONE Editors retract this article. I initially reported this paper to the journal in July 2014. ,