Replication deficient hepadnaviral mutants are widely used to study the hepadnaviral life cycle. Due to the production procedure employing cotransfection of plasmids, it is possible that recombinant virus stocks get contaminated with replicating wildtype virus disturbing the investigations. To test our recombinant virus stocks for wildtype virus, we used DHBV which - in contrast to HBV - infects, replicates, and spreads efficiently in primary cell cultures. This allowed us to detect very low amounts of wildtype virus.
Replication deficient recombinant hepadnaviruses were produced in a chicken hepatoma cell line (LMH) by cotransfecting a transfer plasmid, containing a replication deficient vector genome in which instead of the viral S-gen a GFP reporter gen was inserted, and a helper plasmid, providing all viral proteins from an encapsidation deficient genome.
Infection of PDH with recombinant virus particles at a multipicity of infection of 30 resulted in GFP expression by single cells. Investigating the same culture by immunofluorescence for the expression of the viral L-protein, nearly all cells stained positive, pointing to a contamination with replication competent wildtype virus, which was estimated to account for 0,5% of the total viruses. Because homologous recombination is known to occur in LMH cells, we checked by constructing a helper plasmid containing the preS-region of the DHBV-subtyp 3 whether this is a source for arising of wildtype virus.
To avoid recombination, a LMH packaging cell line stably transfected with a helper plasmid was created. A clone was selected in which by Southern blot analysis only monomers of the DHBV-helper genome and no replicative intermediates were detected and which released no replication competent virus. Using this cellline, recombinant viruses at titers of 108 DNA containing enveloped viral particles per ml cell culture medium were produced. At the moment we are investigating, whether this recombinant virus is completely free of wildtype virus contamination.
Although the observed wildtype virus contamination is low, this contamination may mask the infection behavior of viral recombinants exspecially when wildtype virus outgrows with time.