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. 2016 Nov 7;6:36483. doi: 10.1038/srep36483

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Copyright © 2016, The Author(s)

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Huh7.5 cells were transfected with mcHBV-GLuc cccDNA and mcHBV-GLuc YMHA cccDNA (a mutant with defective reverse transcriptase activity). The viruses in the culture supernatant were concentrated with 6% PEG8000, and fractioned with idixanol gradient centrifugation. The fractions from 6 to 17 were analyszed with PCR primers covering the Intron. (a) A 317bp product was detected using the input mcHBV cccDNA as template, and a smaller 167bp product was detected from virion-associated HBV genome. (b) HBV viron-associated HBV genome was detected in fractions 13 to 17 in the transfection with mcHBV-GLuc cccDNA (arrow and box), but was undetectable in the transfection with the mcHBV-GLuc YMHA cccDNA mutant DNA. Con 1: PCR product with intron sequence using mcHBV cccDNA plasmid as template. Con 2: PCR product using HBV-GLuc plasmid without intron sequence as template. M: DNA ladder, 300, 200 and 100 bp are shown.