TABLE 2.

Quantitation of replicating wild-type DHBV contained in rDHBV stocks produced by cotransfection of helper plasmid pMD4 or pMD5 or with an LMH packaging cell line

Plasmid or cell line	Total no. of rDHBV in inoculum (v.p.)a	No. of cells infectedb	No. of foci of infected cellsc	WT DHBV/rDHBV ratiod
Helper plasmids
pMD4 with:
pCD16-GFPe	108	106	222 ± 52.5	5 × 10−6
pCD16-GFP-2e	108	106	205.0 ± 27.6	4 × 10−6
pMD5 with:
pCD16-GFPe	108	106	27.5 ± 12.0	7 × 10−7
pCD16-GFP-2e	108	106	5 ± 2.6	3 × 10−7
Packaging cell line	5 × 108	5 × 106	0 ± 0	<2 × 10−8

^aTriplicate infection of 10⁶ PDHs infected with the indicated virus stock. The number of rDHBV is given as DNA-containing enveloped viral particles.

^bNumber of hepatocytes infected in each experiment.

^cThe number of foci of cells infected with wild-type virus was determined at day 8 p.i. by immunofluorescence staining for the DHBV L protein, which is not expressed by the recombinant viruses. The means and standard deviations of three independent experiments are shown.

^dThe number of replicating wild-type (WT) DHBV in the respective rDHBV stock was calculated from the number of foci of infected cells. The detection limit of the assay shown was determined to be 2 × 10⁻⁸ by using serum-derived wild-type DHBV. In none of the infection experiments with rDHBV generated with the help of the packaging cell line was a focus of infected cells detected.

^eTransfer plasmid used for rDHBV production.