FIG. 1.

(A to C) Serum DHBV DNA levels decline rapidly in ducks treated with lamivudine and a ddG prodrug. The drug treatment and test bleeds of all ducks began at 6 a.m., and antiviral drugs were administered by intramuscular injection every 12 h thereafter. The ducks were initially bled every 6 h, then every 8 h, and finally every 12 h as indicated by the symbols on the graphs. Serum virus levels were measured by dot blot hybridization to a ³²P-labeled DHBV probe. Radioactivity was measured with a Fuji phosphorimager, and viremia is reported in arbitrary units (PI units). The birds were housed in a room with a cycle of 10 h of light (commencing at 8 a.m.) followed by 14 h of darkness. (A) Duck 33 was treated with lamivudine and ddG prodrug, and its serum virus level was monitored over 3.5 days. (B) Duck 19 was treated identically to duck 33. (C) Four control ducks (⧫, duck 6; ▪, duck 26; ▴, duck 42; ✖, duck 979) were bled on the same schedule as ducks 33 and 19 but received no drug treatment. (D) Dot blot assay of viremia in ducks. Ten microliters of serum from each duck in the cccDNA half-life study was obtained at each of the indicated time points, applied to a Hybond-N membrane (Amersham), hybridized to a ³²P-labeled DHBV probe, and autoradiographed. Ducks 17, 48, and 77 were left untreated, and ducks 50, 65, 68, 992, and 998 were treated with lamivudine and a ddG prodrug. (E) DNA was isolated from serum samples from treated duck 992 at the indicated time points, and DHBV sequences were amplified by PCR. The amount of amplicon for each time point was estimated by measuring the intensity of its band on a stained agarose gel. The amounts of amplicon produced by the indicated serial dilutions of DNA isolated on day 0 are marked by horizontal lines. (F) Quantitative Southern blot of cccDNA present in liver DNA at the start of the half-life study. One microgram of DNA from duck liver tissue (Duck) or uninfected duck erythrocyte doped with known amounts of a plasmid carrying the DHBV genome (Copies DHBV × 10⁶) was digested with EcoRI endonuclease, separated on an agarose gel, transferred to a Hybond-N+ membrane, and probed with ³²P-labeled DHBV DNA. A Fuji phosphorimager was used to quantify the hybridized probe. The cccDNA copy numbers inferred from the Southern blot are shown and are compared to the copy numbers derived from the competitive PCR assay.