Diversity of the nucleic acid forms of circulating HBV in chronically infected patients and its impact on viral cycle

Background Besides the prototypical hepatitis B virus (HBV) infectious particle, which contains a full-length double-stranded DNA (flDNA), additional circulating virus-like particles, which carry pregenomic RNA (pgRNA), spliced1 RNA (sp1RNA) or spliced-derived DNA (defDNA) forms have been described. We aimed to determine the level of these four circulating forms in patients and to evaluate their impact on viral lifecycle. Methods Chronic HBV untreated patients (n = 162), included in the HEPATHER cohort, were investigated. Pangenomic qPCRs were set up to quantify the four circulating forms of HBV nucleic acids (HBVnaf), In vitro infection assays were performed to address the impact of HBVnaf. Results Hierarchical clustering individualized two clusters of HBVnaf diversity among patients: (1) cluster 1 (C1) showing a predominance of flDNA; (2) cluster 2 (C2) showing various proportions of the different forms. HBeAg-positive chronic hepatitis phase and higher viral load (7.0 +/- 6.4 vs 6.6 +/- 6.2 Log(10) copies/ml; p < 0.001) characterized C2 compared to C1 patients. Among the different HBVnaf, pgRNA was more prevalent in Cl patients with high vs low HBV viral load (22.1%+/- 2.5% vs 4.1%+/- 1.8% of HBVnaf, p < 0.0001) but remained highly prevalent in C2 patients, whatever the level of replication. C2 patients samples used in infection assays showed that: (1) HBVnaf secretion was independent of the viral strain; (2) the viral cycle efficiency differed according to the proportion of HBVnaf in the inoculum, independently of cccDNA formation. Inoculum enrichment before infection suggests that pgRNA-containing particles drive this impact on viral replication. Conclusion Besides the critical role of HBV replication in circulating HBV llaf diversity, our data highlight an impact of this diversity on the dynamics of viral cycle. [GRAPHICS] .

Automated summary

This study aimed to analyze the level of circulating forms of hepatitis B virus (HBV) in patients and evaluate their impact on the viral lifecycle. The results showed differences in the levels of different circulating forms among patients and their correlation with disease severity and viral replication activity. Furthermore, particles containing pregenomic RNA were found to play a crucial role in viral replication. These findings have important implications for further understanding the mechanisms of HBV infection and developing new treatment strategies.