The duck hepatitis B virus (DHBV) pregenomic RNA is a bicistronic mRNA encoding the core and polymerase proteins. Thirteen AUGs (C2 to C14) and 10 stop codons (S1 to S10) are located between the C1 AUG for the core protein and the P1 AUG that initiates polymerase translation. We previously found that the translation of the DHBV polymerase is initiated by ribosomal shunting. Here, we assessed the biosynthetic events after shunting. Translation of the polymerase open reading frame was found to initiate at the C13, C14, and P1 AUGs. Initiation at the C13 AUG occurred through ribosomal shunting because translation from this codon was cap dependent but was insensitive to blocking ribosomal scanning internally in the message. C13 and C14 are in frame with P1, and translation from these upstream start codons led to the production of larger isoforms of P. We named these isoforms "pre-P" by analogy to the pre-C and pre-S regions of the core and surface antigen open reading frames. Pre-P was produced in DHBV16 and AusDHBV-infected duck liver and was predicted to exist in 80% of avian hepadnavirus strains. Pre-P was not encapsidated into DHBV core particles, and the viable strain DHBV3 cannot make pre-P, so it is not essential for viral replication. Surprisingly, we found that pre-P is an N-linked glycoprotein that is secreted into the medium of cultured cells. These data indicate that DHBV produces an additional protein that has not been previously reported. Identifying the role of pre-P may improve our understanding of the biology of DHBV infection.Hepadnaviruses are small DNA-containing viruses that replicate by reverse transcription (39). Hepadnaviruses have been found in birds, rodents, and primates (22,38). Human hepatitis B virus (HBV) chronically infects over 350 million people worldwide and is a major cause of liver disease and liver cancer (23). Duck hepatitis B virus (DHBV) is a common model for HBV (25,46 The organization of the 3,021-nucleotide (nt)-long DHBV genome ( Fig. 1) is very compact. All nucleotides are within at least one of three open reading frames (ORFs), and the expression of multiple proteins from one ORF via initiation at multiple in-frame AUG codons is common. The first ORF encodes the core protein (C) and e antigen (e-Ag) (37), with the e-Ag being encoded as an N-terminal extension of the C ORF. The second ORF encodes the envelope proteins L and S, and like the organization of C and e-Ag, the L protein is an N-terminal extension of the S protein. The third ORF encodes the polymerase/reverse transcriptase protein (P). In mammalian viruses, a fourth ORF encodes the X protein, a multifunctional regulatory protein (2, 3). DHBV lacks an apparent X ORF, but a potential cryptic X-like ORF has been reported (6). In vivo experiments revealed no functional role for this protein in short-term infection (26).The products of all of the hepadnaviral ORFs possess regulatory functions in addition to their structural and enzymatic roles. The S ORF encodes the viral surface glycoproteins, but these proteins ar...