HVR1 quasispecies analysis from a long-term culture of hepatitis C virus in Hep G2 derived cells grown in a haemodialysis cartridge.
Studies on the in vitro hepatitis C virus (HCV) infection are hampered by the lack of an appropriate system to culture permissive cells to be continuously infected with HCV. Trypsinization required for cell passage can lead to possible temporary loss of permissiveness for infection, whereas refreshment of the medium can result in loss of infectious particles necessary for perpetuation of the infection; it is therefore very difficult to maintain a continuous HCV infection in cell cultures. A new infection method was designed and evaluated in order to prevent these unfavourable circumstances. A cell line derived from the human hepatoblastoma cell line Hep G2 was grown in the extracapillary space of a haemodialysis cartridge, in the presence of a HCV-positive inoculum, while the culture medium was recirculated through the intracapillary space, supplying the cells with nutrients and oxygen. HCV RNA could continuously be detected in the cells up to 77 days of culture. Sequence analysis of the HCV hypervariable region 1 (HVR1) revealed that 56% and 75%, respectively, of the clones obtained from the cells at day 20 and 40 after start of the infection were different from the clones obtained from the original inoculum and that certain nucleotide positions in this region were more susceptible to mutations, leading to an alteration in amino acid sequence. As none of these sequences were present in the clones from the inoculum, it is suggested that new HCV quasispecies have emerged as a result of viral replication in the hepatocytes in vitro. This system seems a valuable tool for the in vitro evaluation of antiviral drugs.
Department of Liver and Pancreatic Diseases, University Hospital Gasthuisberg, Herestraat 49, B-3000 Leuven, Belgium.
C
C
Clarysse
L
L
Lin
T
T
Crabbé
J F
JF
van Pelt
N
N
Cammack
S H
SH
Yap
D000595:Amino Acid Sequence; D001483:Base Sequence; D018929:Cell Culture Techniques / Q000379:methods; D002460:Cell Line; D003470:Culture Media; D016174:Hepacivirus / Q000737:chemistry / Q000235:genetics*; D006801:Humans; D008969:Molecular Sequence Data; D012367:RNA, Viral / Q000032:analysis; D020539:Sequence Analysis, Protein; D013997:Time Factors; D014764:Viral Proteins / Q000737:chemistry / Q000235:genetics*; D014779:Virus Replication
D003470:Culture Media; C400562:HVR1 protein, Hepatitis C virus; D012367:RNA, Viral; D014764:Viral Proteins