Résumé
Background and aim: Besides the prototype of HBV infectious particle containing a 3.2 kb partially double-stranded DNA (wtDNA), additional circulating virus-like particles containing pregenomic RNA ( pgRNA), spliced-RNA (spRNA) or spliced-derived DNA (defDNA) genomes have been described. Independently, detection of these circulating viral genomes may contribute to the clinical management of HBV infection and/or as a predictive marker of therapy response. Here, the diversity of circulating viral genomes was investigated in chronic infected patients then, its impact on viral lifecycle was explored.Method: 335 chronic HBV infected patients treated (n = 158) or not (n = 177) were selected from the Hepather French Cohort. Pangenomic qPCRs were set up to quantify the four forms of circulating viral genomes from the same nucleic extract. Principal component analysis and hierarchical clustering were performed to characterize the circulating HBV genome diversity. Viral infection of differentiated HepG2-NTCP cells using blood samples from untreated patients was achieved to study its impacts on viral replication.Results: Hierarchical clustering of circulating viral genome diversity in bloodstream divided untreated patients in 2 clusters. The cluster 1 (C1) was defined by the predominance (>50% of circulating genomes) of wtDNA while the cluster 2 (C2) contained various amount of the four viral genomes with a majority of pgRNA, wtDNA and defDNA in 77%, 18% and 5% of cases, respectively. Genomic diversity in C2 was associated with a higher level of viral load (7.1 ± 7.3 Log10 copies/ml of wtDNA), compared with C1 (6.6 ± 7.1 Log10 copies/ml of wtDNA; p < 0.001). The greatest proportion of circulating pgRNA forms was preferentially observed in high replicative patients in C1 and unexpectedly, in low replicative patients in C2. Analysis of treated patients reinforced the contribution of HBV replication in the genome diversity occurrence. In vitro infection with untreated patient samples from both clusters showed that viral diversity in inoculum modulated the intracellular viral cycle. Before infection, inoculum enrichment with pgRNA particles demonstrated their ability to interfere on viral cycle.Conclusion: Our data provide evidences for the main role of viral replication in circulating HBV genomes diversity. Novel biomarker of HBV infection, circulating pgRNA genomes may also contribute to modulate the viral cycle in an unconventional process.