Abstract
An estimated two billion people worldwide have been infected with hepatitis B virus (HBV). Despite the high infectivity of HBV in vivo, a lack of easily infectable in vitro culture systems hinders studies of HBV. Overexpression of the sodium taurocholate co-transporting polypeptide (NTCP) bile acid transporter in hepatoma cells improved infection efficiency. We report here a hepatoma cell culture system that does not require dimethyl sulfoxide (DMSO) for HBV infection. We overexpressed NTCP in Huh7.5 cells and allowed these cells to differentiate in a medium supplemented with human serum (HS) instead of fetal bovine serum (FBS). We show that human serum culture enhanced HBV infection in Huh7.5-NTCP cells, e.g., in HS cultures, HBV pgRNA levels were increased by as much as 200-fold in comparison with FBS cultures and 19-fold in comparison with FBS+DMSO cultures. Human serum culture increased levels of hepatocyte differentiation markers, such as albumin secretion, in Huh7.5-NTCP cells to similar levels found in primary human hepatocytes. N-glycosylation of NTCP induced by culture in human serum may contribute to viral entry. Our study demonstrates an in vitro HBV infection of Huh7.5-NTCP cells without the use of potentially toxic DMSO.
Highlights
Hepatitis B virus (HBV) represents an enormous public health burden with an estimated two billion individuals worldwide having been infected with the virus, resulting in250–300 million people chronically carrying the infection [1,2,3,4,5]
The resulting Huh.7.5-NTCP cell line had more than a 3500-fold increase in NTCP mRNA when compared with the parental Huh.7.5 cell line using real-time quantitative polymerase chain reaction (RT-qPCR) (Figure 1A)
We tested the permissiveness of Huh7.5-NTCP cells in human serum culture to hepatitis B virus (HBV)
Summary
Hepatitis B virus (HBV) represents an enormous public health burden with an estimated two billion individuals worldwide having been infected with the virus, resulting in250–300 million people chronically carrying the infection [1,2,3,4,5]. Other hepatoma cell lines cannot be infected with HBV, but can be transfected with HBV expression plasmids and, having bypassed cell entry, proceed with HBV infection from the genome transcription step [27,28]. Some cell lines, such as HepAD38 and HepG2.2.15, have integrated HBV genomes, which recapitulate infection from the point of genome transcription to the release of infectious virus [29,30]. These systems permit investigations into post-transcriptional stages of infection
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