Details

Autor(en) / Beteiligte

• Cavallone, Daniela
• Moriconi, Francesco
• Colombatto, Piero
• Oliveri, Filippo
• Bonino, Ferruccio
• Brunetto, Maurizia Rossana

Titel

Optimization of in vitro HBV replication and HBsAg production in HuH7 cell line

Ist Teil von

• Journal of virological methods, 2013-04, Vol.189 (1), p.110-117

Ort / Verlag

Netherlands: Elsevier B.V

Erscheinungsjahr

2013

Link zum Volltext

Quelle

MEDLINE

Beschreibungen/Notizen

• ► In vitro cell culture. ► HBV replication cycle. ► In vitro transfection system. ► HBV surface antigen. 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.