1. Natural history of HBV infection (adapted from Torbenson Lancet 2003) Chronic infection Healthy carriage Recovery occult DNA Anti-HBe Anti-HBc Immune response LowHigh No Serol. marker Viral load Anti-HBs Time 10E3 10E9 Occult chronic infection

2. Differential diagnosis of occult HBV Marker RecoveredChronic Anti-HBc PositivePositive Anti-HBe + ++ Anti-HBs PositiveNegative Viral load (copies/ml) <10E3 <10E4 PC stop codon No Yes BCP mutations FewFrequent Secondary response Yes No to HBsAg

3. 0 5 10 15 20 25 30 35 40 45 <10<1E2<1E3<1E4<1E5<1E6<1E7<1E8<1E9 HBV DNA load (IU/ml) Number of samples Distribution of HBV DNA load in 126 HBsAg + and 12 HBsAg - samples from Ghana, West Africa 11% of DNA+ samples are HBsAg -

4. Investigation of 7 occult HBV infections from Ghanaian blood donors Rapid test HBsAg negative : 5107 EIA neg HBV DNA pos = 12 2d sample negative 4 Studied 7 insufficient volume 1 Presence of virion by Ab capture No signal 1no capture 3Capture + 3 Sequencing

5. Characterisation of 7 occult HBV ID DNA loadAnti-HBc Anti-HBeAnti-HBs IU/ml 1237 13 Neg 979 16 Pos Neg Neg 1240 26 Pos Pos Neg 1252 6 Neg 245 117 Pos Neg Neg 1504 17 Neg 3461 29 Neg

6. Capture of HBV particles from plasma using magnetic beads Q-PCR Virus in plasma incubated with mouse mAb against ‘a’ determinant overnight + Add magnetic beads with sheep anti-mouse IgG covalently bound Capture on magnetWashLyse captured particle and extract nucleic acid

7. Preliminary data on occult HBV infection ID Viral anti-HBsAg S seq BCP/PC Complete load capture sequence genome 1237 13 0 0 0 979 16 0 0 0 1240 26 0 0 0 1252 6 0 + 0 245 117 + + + + 1504 17 + 0 + 3461 29 + + + 0

8. 0.01 substitutions/site HBV whole genome Neighbour-Joining tree A A A A A B B B B B C C D D D E 245-21 245-12 H H H F F F F F G G G G G E E EE C C C D D 100 Sample 245 VL 117 IU/ml Complete sequence of 2 clones

9. Main sequence features in 4 OBI IDBCP Pre-core Pre-S1/Pre-S2‘a’ 1762-64 1896 SC 245-12WT +WT S140L 245-21WT + C 2930 del. S140L 1504WT WT - - 1252 - - - T131A 3461WTWT - WT

10. Substitutions in the ‘a’ loops of HBV genotype E

11. Anti-HBs response to HBV vaccine of 2 asymptomatic donors with anti-HBc only 0 10 20 30 40 50 Days after primary vaccination 543210543210 Anti-HBs (S/CO) Primary injection2d injection

12. Conclusions Molecular studies of occult HBV are difficult Most include enveloped virions Viral load is consistently <500 IU/ml Detection requires single donation screening Serologically most seem to be anti-HBc only BCP and PC wild type suggest recovered infection despite absence of detectable anti-HBs No evidence of ‘a’ determinant escape was found

13. 3461 245-21 245-12 E E E E E 1252 H H H F F F F F A A A A A D D DD D B B B B B C C C C C G G G G G 0.005 substitutions/site Partial S region (490 bp) Neighbour-Joining Tree 98 100 95 99 A D B C G E H F