In vivo selection of duck hepatitis B virus pre-S variants which escape from neutralization

To better understand the role of specific residues within the duck hepatitis B virus (DHBV) pre-S protein in neutralization and infectivity, we have selected and identified pre-S variants which escape neutralization. A highly neutralizing monoclonal antibody (Mab 900) which recognizes an epitope 83IPQPQWTP90 localized previously on the DHBV pre-S protein, within a region suspected to mediate the virus interaction with hepatocytes, was used as immune pressure. After only two in vivo neutralization rounds with Mab 900, five different pre-S mutant genomes were identified, which harbored point mutations affecting only proline residues located at position 90 within this epitope (83IPQPQWTP90) and/or at a distance at position 5. We have shown that a single (P5L) or double proline (P5L + P90H) substitution affect neither virus replication capacity nor in vivo infectivity. However, the P5 mutation reduces mutant recognition by Mab 900 twofold, while the substitution of both prolines 5 and 90 almost completely abolishes mutant P5L + P90H reactivity with this Mab and leads to a decrease of neutralization. Therefore we describe here an experimental system which allows rapid in vivo selection and identification of DHBV pre-S variants and provide evidence that residues within and at a distance from the neutralization epitope are important in DHBV neutralization but do not affect its replication capacity and infectivity.