Residues critical for duck hepatitis B virus neutralization are involved in host cell interaction

To date, no detailed analysis of the neutralization properties of duck hepa titis a virus (DHBV) has been reported, and it is not clear whether any of the known neutralization epitopes correspond to the viral receptor binding site or to sequences involved in the cell entry pathway. We demonstrate her e that antibodies directed against two overlapping peptides (amino acids 83 to 97 and 93 to 107), covering the sequences of most DHBV pre-S neutralizi ng epitopes, both inhibit virus binding to primary duck hepatocytes and neu tralize virus infectivity. An extensive mutagenesis of the motif (WTP90)-W- 88, which is the shortest sequence of the epitope recognized by the virus-n eutralizing monoclonal antibody (MAb) 900 was performed in order to define the amino acids involved in these interactions. Single point mutations with in this epitope affected neither virus replication nor infectivity but abol ished virus neutralization by MAb 900 completely. Interestingly, mutants wi th two and three consecutive residue replacements (SIP and SIH) within this epitope retained replication competence but were no longer infectious. The loss of infectivity of SIH and SIP mutant particles was associated with si gnificantly reduced binding to primary duck hepatocytes and could be rescue d by trans complementation with wild-type pre-S protein. Taken together, th ese results indicate that each amino acid of the DHBV pre-S sequence (WTP90 )-W-88 is critical for recognition by the neutralizing MAb 900 and that rep lacement of the first two or all three residues strongly reduces virus inte raction with hepatocytes and abrogates infectivity. These data imply that t he motif (WTP90)-W-88 contains key residues which are critical for interact ion with both the neutralizing MAb and the host cell.