Sorbonne Université, Institut National de la Santé et de la Recherche Médicale (INSERM), Centre de Recherche de Saint Antoine (CRSA), Paris, France.
Université Paris-Est Créteil, Département de Pathologie, Hôpital Henri Mondor, Créteil, France.
Hepatol Int. 2022 Dec;16(6):1259-1272. doi: 10.1007/s12072-022-10389-6. Epub 2022 Aug 4.
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.
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 (HBV). In vitro infection assays were performed to address the impact of HBV.
Hierarchical clustering individualized two clusters of HBV 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 copies/ml; p < 0.001) characterized C2 compared to C1 patients. Among the different HBV, pgRNA was more prevalent in C1 patients with high vs low HBV viral load (22.1% ± 2.5% vs 4.1% ± 1.8% of HBV, 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) HBV secretion was independent of the viral strain; (2) the viral cycle efficiency differed according to the proportion of HBV in the inoculum, independently of cccDNA formation. Inoculum enrichment before infection suggests that pgRNA-containing particles drive this impact on viral replication.
Besides the critical role of HBV replication in circulating HBV diversity, our data highlight an impact of this diversity on the dynamics of viral cycle.
Patients were included from a prospective multicenter French national cohort (ANRS CO22 HEPATHER, NCT01953458).
除了含有全长双链 DNA（flDNA）的典型乙型肝炎病毒（HBV）感染性颗粒外，还描述了其他循环病毒样颗粒，这些颗粒携带前基因组 RNA（pgRNA）、剪接 1 RNA（sp1RNA）或剪接衍生 DNA（defDNA）形式。我们旨在确定这些四种循环形式在患者中的水平，并评估它们对病毒生命周期的影响。
对包括在 HEPATHER 队列中的 162 名未经治疗的慢性 HBV 患者进行了研究。建立了 pan 基因组 qPCR 来定量 HBV 四种循环核酸（HBV）。进行体外感染实验以研究 HBV 的影响。
对患者的 HBV 多样性进行分层聚类，将其分为两个聚类：（1）聚类 1（C1）表现为 flDNA 优势；（2）聚类 2（C2）表现为不同形式的不同比例。与 C1 患者相比，C2 患者表现为 HBeAg 阳性慢性肝炎期和更高的病毒载量（7.0±6.4 与 6.6±6.2 Log 拷贝/ml；p<0.001）。在不同的 HBV 中，pgRNA 在高病毒载量（22.1%±2.5%与 4.1%±1.8%的 HBV，p<0.0001）与低病毒载量（22.1%±2.5%与 4.1%±1.8%的 HBV，p<0.0001）的 C1 患者中更为常见，但在 C2 患者中仍然高度普遍，而与复制水平无关。在感染实验中使用的 C2 患者样本表明：（1）HBV 分泌独立于病毒株；（2）病毒周期效率根据接种物中 HBV 的比例而不同，与 cccDNA 形成无关。感染前接种物的富集表明，含有 pgRNA 的颗粒对病毒复制产生了这种影响。
除了 HBV 复制对循环 HBV 多样性的关键作用外，我们的数据还强调了这种多样性对病毒周期动力学的影响。
患者来自法国前瞻性多中心国家队列（ANRS CO22 HEPATHER，NCT01953458）。
