Aurélie Schnuriger1,2, Patrick Soussan3,4, Jules Sotty1, Pierre Bablon1, Bouchra Lekbaby1, Jérémy Augustin1,5, Morgane Girier-Dufournier1, Lucas Langlois1, Céline Dorival6, Fabrice Carrat6, Stanislas Pol7, Hélène Fontaine7, Nazim Sarica8, Christine Neuveut8, Chantal Housset1, Dina Kremdsorf1. 1. Sorbonne Université, Institut National de la Santé et de la Recherche Médicale (INSERM), Centre de Recherche de Saint Antoine (CRSA), Paris, France. 2. Assistance Publique-Hôpitaux de Paris (AP-HP), Sorbonne Université, Département de Virologie, GHU Paris-Est, Paris, France. 3. Sorbonne Université, Institut National de la Santé et de la Recherche Médicale (INSERM), Centre de Recherche de Saint Antoine (CRSA), Paris, France. patrick.soussan@inserm.fr. 4. Assistance Publique-Hôpitaux de Paris (AP-HP), Sorbonne Université, Département de Virologie, GHU Paris-Est, Paris, France. patrick.soussan@inserm.fr. 5. Université Paris-Est Créteil, Département de Pathologie, Hôpital Henri Mondor, Créteil, France. 6. Sorbonne Université, INSERM, Institut Pierre Louis d'Epidémiologie et de Santé Publique, Département de santé publique, Hôpital Saint-Antoine, AP-HP, Paris, France. 7. Université de Paris, AP-HP, Département d'hépatologie, Hôpital Cochin, Paris, France. 8. Institut de Génétique Humaine, Université de Montpellier, Laboratoire de Virologie Moléculaire CNRS-UMR9002, Montpellier, France.
Abstract
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), spliced1RNA (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 Log10 copies/ml; p < 0.001) characterized C2 compared to C1 patients. Among the different HBVnaf, pgRNA was more prevalent in C1 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 HBVnaf diversity, our data highlight an impact of this diversity on the dynamics of viral cycle. CLINICAL TRIAL REGISTRATION: Patients were included from a prospective multicenter French national cohort (ANRS CO22 HEPATHER, NCT01953458).
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), spliced1RNA (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 Log10 copies/ml; p < 0.001) characterized C2 compared to C1 patients. Among the different HBVnaf, pgRNA was more prevalent in C1 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 HBVnaf diversity, our data highlight an impact of this diversity on the dynamics of viral cycle. CLINICAL TRIAL REGISTRATION: Patients were included from a prospective multicenter French national cohort (ANRS CO22 HEPATHER, NCT01953458).
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