Presence of replicating virus in recombinant hepadnavirus stocks results from recombination and can be eliminated by the use of a packaging cell line.
Mutant hepatitis B viruses are useful tools to study the viral life cycle and viral pathogenesis. Furthermore, recombinant hepatitis B viruses are candidate vectors for liver-directed gene therapy. Because wild-type viruses present in recombinant or mutant virus stocks may falsify experimental results and are detrimental for a viral vector, we investigated whether and to what extent wild-type virus is present in recombinant virus stocks and where it originates from. We took advantage of the duck model of hepatitis B virus infection which allows very sensitive detection of replication-competent viruses by infection of primary duck hepatocytes or of ducklings in vivo. Recombinant hepatitis B virus stocks contained significant amounts of wild-type viruses, which were most probably generated by homologous recombination between plasmids containing homologous viral sequences. In addition, replication-competent viral genomes were reconstituted from plasmids which contained replication-deficient but redundant viral sequences. Using a stable cell line for packaging of deficient viral genomes, no wild-type virus was detected, neither by infection of primary hepatocytes nor in vivo.
Klöcker
Uta
U
Department of Virology,University of Heidelberg, D-69120 Heidelberg, Germany.
Oberwinkler
Heike
H
Kürschner
Timo
T
Protzer
Ulrike
U
D000818:Animals; D002478:Cells, Cultured; D004372:Ducks; D018347:Hepadnaviridae Infections / Q000821:virology; D015611:Hepatitis B Virus, Duck / Q000235:genetics* / Q000472:pathogenicity / Q000502:physiology*; D006524:Hepatitis, Viral, Animal / Q000821:virology; D022781:Hepatocytes / Q000821:virology; D010957:Plasmids; D011995:Recombination, Genetic; D014407:Tumor Cells, Cultured; D019065:Virus Assembly; D014779:Virus Replication