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

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

<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 -

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

Characterisation of 7 occult HBV ID DNA loadAnti-HBc Anti-HBeAnti-HBs IU/ml Neg Pos Neg Neg Pos Pos Neg Neg Pos Neg Neg Neg Neg

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

Preliminary data on occult HBV infection ID Viral anti-HBsAg S seq BCP/PC Complete load capture sequence genome

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 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

Main sequence features in 4 OBI IDBCP Pre-core Pre-S1/Pre-S2‘a’ SC WT +WT S140L WT + C 2930 del. S140L 1504WT WT T131A 3461WTWT - WT

Substitutions in the ‘a’ loops of HBV genotype E

Anti-HBs response to HBV vaccine of 2 asymptomatic donors with anti-HBc only Days after primary vaccination Anti-HBs (S/CO) Primary injection2d injection

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

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 substitutions/site Partial S region (490 bp) Neighbour-Joining Tree A D B C G E H F