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Production and characterization of high-titer serum-free cell culture grown hepatitis C virus particles of genotype 1-6.

Virology (2014-06-15)
Christian K Mathiesen, Tanja B Jensen, Jannick Prentoe, Henrik Krarup, Alfredo Nicosia, Mansun Law, Jens Bukh, Judith M Gottwein
ABSTRACT

Recently, cell culture systems producing hepatitis C virus particles (HCVcc) were developed. Establishment of serum-free culture conditions is expected to facilitate development of a whole-virus inactivated HCV vaccine. We describe generation of genotype 1-6 serum-free HCVcc (sf-HCVcc) from Huh7.5 hepatoma cells cultured in adenovirus expression medium. Compared to HCVcc, sf-HCVcc showed 0.6-2.1 log10 higher infectivity titers (4.7-6.2 log10 Focus Forming Units/mL), possibly due to increased release and specific infectivity of sf-HCVcc. In contrast to HCVcc, sf-HCVcc had a homogeneous single-peak density profile. Entry of sf-HCVcc depended on HCV co-receptors CD81, LDLr, and SR-BI, and clathrin-mediated endocytosis. HCVcc and sf-HCVcc were neutralized similarly by chronic-phase patient sera and by human monoclonal antibodies targeting conformational epitopes. Thus, we developed serum-free culture systems producing high-titer single-density sf-HCVcc, showing similar biological properties as HCVcc. This methodology has the potential to advance HCV vaccine development and to facilitate biophysical studies of HCV.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
丙酮, ACS reagent, ≥99.5%
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丙酮, puriss. p.a., ACS reagent, reag. ISO, reag. Ph. Eur., ≥99.5% (GC)
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丙酮, Laboratory Reagent, ≥99.5%
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丙酮, Pharmaceutical Secondary Standard; Certified Reference Material
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丙酮, suitable for HPLC, ≥99.9%
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丙酮, United States Pharmacopeia (USP) Reference Standard
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丙酮, puriss., meets analytical specification of Ph. Eur., BP, NF, ≥99% (GC)
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氯丙嗪 盐酸盐, VETRANAL®, analytical standard
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