TABLE 1.

Analysis of liver tissue from each of the group 1 to 4 DHBV-infected ducks

Group (treatment)DuckgDaya% of DHBsAg-positive liver cellsbcccDNA copy no. perc:Total DHBV DNAdFatty changesePresence of amyloidf
Liver cellDHBsAg-positive cell
1 (ETV plus vaccine)159160 (M)18>835645NT
42>83101234NT
91394107NT
181237306
2441921111
161162 (M)18>836728NT
42>83131615NT
9133395NT
181152143
287287256++++
298>834567++
165166 (F)18>834552NT
42>83101220NT
91325167NT
18183404+
24461176
163164 (F)18>835616NT
42>83787NT
9137383NT
181264154++
167168 (M)18>835626NT
42>839116NT
91234177NT
181461585
287122229+
320>831012116+
2 (ETV plus vector)169170 (M)18>836729NT
42>83141713NT
91394104NT
181148587
2871033011++
320>831316233
39172 (F)18>8381017NT
42>83141710NT
91435126NT
181147496
244261415+++
175176 (M)18>83121433NT
42>8381013NT
91264157NT
181331035
24422878
177178 (M)18>83172044NT
42>83141720NT
913771915NT
18132713++
287>83111315+
298>8356107++
173174 (M)18>83182254NT
42>83161913NT
911931612NT
181164614
28753569+
320>836775+++
3 (water plus vaccine)17999 (M)18>831012180NT
42>83111393NT
91>831822156NT
181>831518118
244>831214192++−/PICK>
Liver cellDHBsAg-positive cell
183184 (M)18>83151889NT
42>83911274NT
91>831518125+NT
181>831922229
287>837842++
298>834543++++
181182 (F)18>831316100NT
42>831113209NT
91>831417126NT
181>831012183++
244>8345195+
379718>83101295NT
42>831012320NT
91>83151891NT
181>832530292
185186 (M)18>831316236NT
42>831619393NT
91>831619122NT
181>831619353++
287>83394696+
320>83141790++
4 (water plus vector)195196 (M)18>831012115NT
42>831619448NT
91>832024124NT
181>833239372
287>833239104++
320>831720128+
193194 (M)18>831316119NT
42>831417575NT
91>831923136NT
181>832733317+
244>8367154+++
100200 (F)18>831619198NT
42>831720364NT
91>831619130+NT
181>832024528+++
244>8378548+++
197198 (M)18>831518131NT
42>832429666NT
91>831923105NT
181>832935396++
287>83182268+++
298>8378119++++
  • a All ducks underwent surgical liver biopsy on days 18, 42, 91, and 181. Two of the five ducks from each of groups 1 to 4 were autopsied on day 243 (the day before drug was withdrawn), and the remaining ducks in groups 1 to 4 were biopsied on day 287 and then autopsied on either day 298 or day 320.

  • b The percentage of DHBsAg-positive liver cells was determined from cell counts by using a microscope eyepiece grid at a magnification of ×400. DHBsAg-positive cells were counted in 5 to 10 representative grids and expressed as a percentage of total liver cell nuclei in the same area. The group 3 and 4 ducks had detectable DHBsAg in all hepatocytes at each time point. Hepatocytes comprise ∼83% of total liver cells (13).

  • c Levels of cccDNA are expressed as the average copy number per liver cell based on liver weight as previously described (14) and as the calculated average copy number per DHBsAg-positive liver cell.

  • d Levels of total DHBV DNA are expressed as the average copy number per liver cell based on liver weight as previously described (14).

  • e Fatty changes were detected in formalin-fixed liver tissue stained by hematoxylin and eosin. −, not tested; +, mild; ++, moderate; +++, marked.

  • f Amyloid was detected in sections of formalin-fixed liver tissue using Congo red staining. NT, not tested. Liver samples collected from day 181 onwards only were tested for the presence of amyloid.

  • g The sex of each duck was determined to be female (F) or male (M). Ducks 163164 and 165166 from group 1, 39172 from group 2, 181182 and 185186 from group 3, and 100200 from group 4 contained high levels of visible lipid from day 140 (23 weeks of age) that resulted in 5 to 10-fold increases in the OD values obtained in the DHBsAg ELISA, so all samples from these ducks were excluded from the analysis of DHBsAg. Ducks 163164 (group 1) and 3797 (group 3) were euthanatized due to poor health on day 200. No tissues were available from these ducks after the liver biopsy samples collected on day 181.