Novel hepatitis D virus (HDV) RNA mutants carrying large fragment deletions were identified in the serum samples of two hepatitis B/D patients receiving antiviral therapy. Sequence analysis revealed that the deleted regions encompassed both ribozyme domains. The mutant persisted in the serum samples for at least 2 and 10 months, respectively in the two patients, raising the question of whether such mutants could replicate in the absence of ribozyme domains. Thirty anti-HDV antibody-positive serum samples derived from 17 patients receiving antiviral therapy were submitted for RT-PCR detection of HDV RNA deletion mutants. Large fragment HDV RNA deletions were found in 4 patients. Of them, two had liver biopsy samples available. Northern blot analysis revealed high molecular weight HDV RNA replication intermediates, genomic and anti-genomic senses, in the liver tissues. Transfection of an in vitro transcribed HDV RNA deletion mutant (1.28 kb) into Huh7 and HepG2 cells also resulted in generation of high molecular weight HDV RNA species in the hepatoma cells (>6.5 kb) with secretion of a 6.5 kb HDV RNA species into the medium. In conclusion, we discovered novel large fragment deletion mutants of HDV RNA in hepatitis B/D patients receiving antiviral therapy. Such mutants did not contain ribozyme domains but could replicate in the liver cells to generate high molecular weight but not unit-length HDV RNA. (C) 2014 Elsevier Inc. All rights reserved.