Replication of Hepatitis B Virus in Culture Systems With Adult Human Hepatocytes

Abstract

We developed a technique for isolation and primary culture of adult human hepatocytes from surgical liver biopsy specimens by in situ perfusion and a shaking method. Cultured hepatocytes were maintained in monolayers for more than three weeks and showed morphological and functional characteristics in vivo. The cultured human hepatocytes were inoculated with hepatitis B virus (HBV). Hepatitis B surface antigen (HBsAg) in the medium was detected for about three weeks after inoculation, which was longer than that reported in previous studies. In one case of high attachment efficiency, hepatitis B e antigen (HBeAg) was detected in the medium five to eight days after inoculation. HBsAg and HBeAg were also detected in the extracts of inoculated human hepatocytes. Immunofluorescence study revealed HBsAg in 20-30% of hepatocytes and hepatitis B core antigen (HBcAg) in 2-3% of the cultured human hepatocytes four days after inoculation. Free HBV DNA was identified in the human hepatocytes for at least two weeks after inoculation, although single-stranded HBV DNA was not detected. These studies suggest that HBsAg was actively produced and that HBV replicated in a small number of inoculated adult human hepatocytes in primary culture. However, further improvement of culture systems is needed for active replication of HBV in vitro.

In Vitro transmission of duck hepatitis B virus to primary duck hepatocyte cultures

“…As a result, infection in 20% of cultured cells was demonstrated by the immunostaining for hepatitis B core antigen (17). The limited number of cells infected was almost the same between DHBV and hepatitis B virus in the culture system.…”

Endogenous antiviral microRNAs determine permissiveness for hepatitis B virus replication in cultured human fetal and adult hepatocytes

“…The AdHBV vector efficiently transduced replication‐competent HBV genomes in all cells used in this study. In this way, difficulties were avoided in HBV infection of cultured hepatocytes and cell lines, e.g., HepG2, HuH‐7, or HepaRG cells required transfection of HBV plasmids for viral replication [Shimizu et al, ; Sells et al, ; Tsurimoto et al, ; Gripon et al, , 2002; Ochiya et al, ; Sun and Nassal, ]. Previously, infection of human hepatocytes with HBV was not always successful, and even required repeated exposure to HBV inocula or continuous co‐culture with HBV‐producing cells for many days [Gripon et al, ; Ochiya et al, ].…”

“…Recently, several anti‐HBV miRNA were identified, e.g., hsa‐miR‐15b, ‐125‐5p, ‐125‐b, ‐151‐5p, ‐199a‐3p, ‐122, ‐1231, etc. [Shimizu et al, ; Sells et al, ; Tsurimoto et al, ; Gripon et al, , 2002; Ochiya et al, ; Sun and Nassal, ]. In chronic hepatitis B and HBV‐associated HCC, several miRNA were overexpressed in blood, e.g., hsa‐miR‐7, ‐ 196b, ‐433, ‐511 targeting polymerase or surface genes, hsa‐miR‐205 targeting X gene, and hsa‐miR‐345 targeting HBV preC gene [Li et al, ].…”

“…The ability to use hepatocytes as cell culture models for hepatitis B virus (HBV) for basic studies and antiviral therapeutics is important. However, work over past several decades established that in cultured hepatocytes, HBV either does not complete replication leading to virion production or replicates only transiently and that too at extremely low levels [Shimizu et al, ; Gripon et al, ; Ochiya et al, ]. This nonpermissiveness of cultured hepatocytes for establishment of HBV replication with productive viral infection and/or viral persistence has been puzzling because of the otherwise extensive hepatotropism of HBV and susceptibility of nonimmune people or correct animals to the rapid establishment of productive HBV infection.…”