Hepatitis delta virus sequencing (360-bp)

Hepatitis delta virus (HDV) replicates using host polymerases. Codon 196 of its unique ORF can be edited from UAG (encoding the small delta antigen) to UGG (encoding the large delta antigen) by the ADAR1 host enzyme. These antigens have different, essential roles in the HDV cycle. This study sought to determine the percentage of unedited and edited genomes, and evaluate HDV quasispecies complexity and RNA evolution rates in serum of chronic hepatitis delta (CHD) treatment-naïve patients, using next-generation sequencing methods. A cross-sectional study determined the prevalence of the editing codon in the overall quasispecies and evaluated the complexity of HDV. The longitudinal studies included sequential samples from 3 CHD patients (mean, 12 years’ follow-up), and determined the percentage of unedited and edited genomes, HDV quasispecies complexity and RNA evolution rates. In total, 185,741 sequences were analyzed. Unedited genomes predominated over edited genomes in most samples (8/12 cross-sectional samples and 28/29 sequential samples). Quasispecies complexity was similar in edited and unedited genomes, but there were occasional differences between the 2 populations. Serum HDV RNA levels did not correlate with genome percentages or quasispecies complexity. The HDV evolution rate was 1.2x10-3-9.5x10-3 substitutions/site/year and showed a negative correlation with the time elapsed between samples (p<0.05). In the HDV genome, 72.6% (SD 11.1) of nucleotide substitutions were C to U or U to C transitions. Our findings support the idea that the circulating HDV quasispecies is composed of 2 genomic populations whose proportion, complexity and evolution rate fluctuate over the time of infection.