Optimised Conditions for the Production of Hepatitis B Virus from Cell Culture

Abstract

In chronically infected patients, hepatitis B virus (HBV) particles reach numbers as large as >10⁹ genome equivalents (GE)/ml of serum. However, expression of infectious HBV particles in cell culture only yields 10⁵–10⁶ GE/ml, which is insufficient for many studies. HBV transcription and possibly replication is dependent on hepatocyte-specific differentiation. Thus, we tested several cell culture parameters that have been reported to enhance the expression of hepatocyte-specific markers, such as growth on different extracellular matrices, different cell culture media, low concentrations of fetal calf serum (FCS) and the addition of dimethyl sulfoxide (DMSO) to the medium. Lower concentrations of FCS, growth on collagen and inclusion of DMSO in the medium only moderately enhanced HBV production in vitro when applied individually. However, combinations of these parameters optimised cell culture conditions and reproducibly increased the release of HBV particles about 100-fold to titres >10⁸ GE/ml of culture medium.

Stable HepG2- and Huh7-based human hepatoma cell lines for efficient regulated expression of infectious hepatitis B virus

“…DMSO further boosted HBV expression whereas no similar enhancement was seen with HepG2.2.15 cells. Though others reported a 2-fold stimulation by DMSO [19] this is in line with an only minor effect on HepG2.2.15 cells. While surface protein expression was, as expected, largely unaffected, Dox regulation of HBV replication was preserved in all cell lines tested (Table 1).…”

“…One such cell line, HepG2.2.15 [18], is widely used in antiviral research. Derived from HepG2 cells by transfection of a complex four-copy HBV construct, it produces modest levels [19] of virus. More expedient would be stable cell lines in which HBV expression is regulatable, preferentially up to higher levels.…”

Molecular Mechanism of p73-Mediated Regulation of Hepatitis B Virus Core Promoter/Enhancer II: Implications for Hepatocarcinogenesis

“…22 HBsAg and HBeAg are translated from preS/S mRNA and precore mRNA individually. As HBsAg and HBeAg expression does not always correlate with the amount of replicating virus, 23 we measured particle-associated HBV DNA from the supernatant by quantitative realtime PCR. For replication studies, we transfected a linear monomer spanning from nucleotide 1820 to 1819, which was shown to lead to virus production.…”

Hepatitis B Virus Activates Deoxynucleotide Synthesis in Nondividing Hepatocytes by Targeting the R2 Gene

“…4 The Michaelis constant (K m ) of the viral polymerase at a dNTP concentration of 0.4 lM is only 62%, whereas a 4 lM concentration gives 93.3% activity. 5 Furthermore, one hepatocyte produces 50-300 hepatitis B virions per day, 6 and because the HBV genome is approximately 3.2 kilobases, between 3 Â 10 5 to 2 Â 10 6 dNTPs are consumed per day in this process. Considering cell volume as 500 fL, 7 a resting cell contains approximately 1.2 Â 10 5 dNTP molecules.…”