title: Optimization of in vitro HBV replication and HBsAg production in HuH7 cell line.
abstract: The Gunther's vector-free method (GM), using PCR-amplified full length HBV-DNA (fl-HBV-DNA), is currently the best in vitro HBV replication system despite the low intracellular HBV-DNA production. The replication efficiency and HBsAg secretion of 12 isolates from HBsAg/HBeAg positive sera by GM, Monomer-Linear-Sticky-Ends-DNA (MLSE) and Monomer-Circular-Closed (MCC) were compared in HuH7 cells. Eight of twelve genomes (67%) were replication competent by GM; however direct sequencing (DS) showed that more than 80% of input DNA was undigested in spite of SapI treatment. Replication Intermediates (RI) were detected earlier (24 vs. 48h) and in higher amounts (2.51±0.32 and 6.43±0.43 fold) by MCC than GM or MLSE. By MCC 10 of 12 genomes (83%) were replication competent and 7 produced high RI levels. RI and HBsAg kinetics correlated positively in MCC (R=0.696, p=0.017 overall; R=0.928, p=0.008), but not in GM (R=-0.437, p=0.179 overall; R=-0.395, p=0.439) in genotype D isolates. In conclusion, HBV-DNA circularization prior transfection improves in vitro viral replication and replication competent HBsAg production, mimicking better the in vivo conditions.
authors: Hepatology Unit and Liver Physiopathology Laboratory, University Hospital of Pisa, Pisa 56124, Italy.
authors: Daniela
authors: D
authors: Cavallone
authors: Francesco
authors: F
authors: Moriconi
authors: Piero
authors: P
authors: Colombatto
authors: Filippo
authors: F
authors: Oliveri
authors: Ferruccio
authors: F
authors: Bonino
authors: Maurizia Rossana
authors: MR
authors: Brunetto
mesh_terms: D000293:Adolescent; D000328:Adult; D000368:Aged; D001483:Base Sequence; D045744:Cell Line, Tumor; D004261:DNA Replication; D004270:DNA, Circular / Q000235:genetics*; D004279:DNA, Viral / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D005260:Female; D016679:Genome, Viral / Q000235:genetics; D005838:Genotype; D006514:Hepatitis B Surface Antigens / Q000096:biosynthesis*; D006513:Hepatitis B e Antigens / Q000378:metabolism; D006515:Hepatitis B virus / Q000235:genetics* / Q000378:metabolism / Q000502:physiology*; D006801:Humans; D008297:Male; D008875:Middle Aged; D017422:Sequence Analysis, DNA; D014779:Virus Replication / Q000235:genetics*; D055815:Young Adult
chemical_list: D004270:DNA, Circular; D004279:DNA, Viral; D006514:Hepatitis B Surface Antigens; D006513:Hepatitis B e Antigens