Applied Molecular Virology Laboratory, Discovery Biology Division, Institut Pasteur Korea, 696 Sampyeong-dong, Bundang-gu, Seongnam-si, Gyeonggi-do, South Korea.
Applied Molecular Virology Laboratory, Discovery Biology Division, Institut Pasteur Korea, 696 Sampyeong-dong, Bundang-gu, Seongnam-si, Gyeonggi-do, South Korea; Division of Bio-Medical Science and Technology, University of Science and Technology, 217, Gajeong-ro, Yuseong-gu, Daejeon, South Korea.
Antiviral Res. 2021 Dec;196:105195. doi: 10.1016/j.antiviral.2021.105195. Epub 2021 Nov 1.
The quantification of infectious virus particles is fundamental to perform in vitro virology studies. To determine the number of hepatitis B virus (HBV) genome-containing particles in vitro, the genome equivalents (GEq) are measured using quantitative PCR (qPCR). However, in addition to infectious virions, HBV DNA-containing, non-infectious HBV particles are also produced in vitro, which can lead to an over-estimation of the number of infectious HBV particles when analyzed by qPCR. Here, we establish an end-point dilution assay that can precisely determine the number of infectious HBV particles. The cell-based HBV infection assay uses a 384-well plate format and enables the calculation of the 50% tissue culture infective dose (TCID) in a semi-automated manner. Cell culture-derived HBV (HBVcc), produced by either stable HBV-replicating cells (HepAD38) or HBV-infected HepG2-NTCP cells, as well as patient-derived HBV sera were serially diluted and used to infect naïve target cells. Applying the end-point dilution assay, we infected HepG2-NTCP cells with PEG precipitated HBV derived from HepAD38-and HepG2-NTCPsec+ cell supernatants, calculated the TCID/mL, converted to plaque-forming units (PFUs), and generated the specific infectivity (ratio of PFU/GEq). As a result, a TCID/mL of 7.22 × 10 and 2.16 × 10, and the specific infectivity of 1/13,816 and 1/8798 were calculated for HepAD38 and HepG2-NTCPsec+ cell supernatants, respectively. The specific infectivity further increased by approximately 2-fold after removal of non-infectious "naked" particles by immunoprecipitation. Purification of HepAD38 cell supernatants by heparin columns increased the TCID/mL and specific infectivity by 18- and 15-fold, respectively. Interestingly, non-purified patient-derived HBV sera from two individuals had a specific infectivity of 1/88 and 1/3609. After converting TCID to multiplicity of infection (MOI) values, we inoculated HepG2-NTCP cells with HBVcc based on GEq or MOI values and demonstrated that MOI-based infection leads to more reproducible infection rates. Furthermore, the assay was validated using serially diluted lamivudine, an HBV replication inhibitor, inhibiting HBV DNA secretion and infectious viral progeny by approx. 56- and 470-fold, respectively. Interestingly, we identified dexmedetomidine (DMM), an alpha-2 adrenergic agonist, inhibiting the secretion of infectious viral progeny by approx. 6-fold, without interfering in the secretion of HBV DNA. Taken together, we developed an assay that is suitable for the standard quantification of infectious HBV particles. We identified DMM as a novel inhibitor that exclusively interferes with the secretion of infectious HBV particles without affecting the secretion of HBV genomes. This end-point dilution assay enables the precise determination of the number of infectious HBV particles, assessment of the specific infectivity and stability of HBV particles, and identification of novel classes of HBV inhibitors.
乙型肝炎病毒（HBV）体外感染性病毒粒子的定量分析是进行病毒学研究的基础。为了确定体外 HBV 基因组含量的病毒粒子数量，使用定量 PCR（qPCR）测量基因组当量（GEq）。然而，除了有感染性的病毒粒子外，HBV DNA 还会产生无感染性的 HBV 粒子，这可能导致 qPCR 分析时感染性 HBV 粒子数量的高估。在此，我们建立了一种终点稀释测定法，可以精确确定感染性 HBV 粒子的数量。基于细胞的 HBV 感染测定法使用 384 孔板格式，能够半自动地计算 50%组织培养感染剂量（TCID）。通过稳定复制 HBV 的细胞（HepAD38）或 HBV 感染的 HepG2-NTCP 细胞产生的细胞培养衍生的 HBV（HBVcc），以及来自患者的 HBV 血清被连续稀释并用于感染未感染的靶细胞。应用终点稀释测定法，我们用 PEG 沉淀的源自 HepAD38 和 HepG2-NTCPsec+细胞上清液的 HBV 感染 HepG2-NTCP 细胞，计算 TCID/mL，转换为噬菌斑形成单位（PFU），并生成特异性感染力（PFU/GEq 的比值）。结果，HepAD38 和 HepG2-NTCPsec+细胞上清液的 TCID/mL 分别为 7.22×10 和 2.16×10，特异性感染力分别为 1/13816 和 1/8798。免疫沉淀去除无感染性的“裸”颗粒后，特异性感染力进一步增加约 2 倍。用肝素柱纯化 HepAD38 细胞上清液可分别使 TCID/mL 和特异性感染力提高 18 倍和 15 倍。有趣的是，来自两名个体的未经纯化的患者源性 HBV 血清的特异性感染力分别为 1/88 和 1/3609。将 TCID 转换为感染复数（MOI）值后，我们基于 GEq 或 MOI 值用 HBVcc 感染 HepG2-NTCP 细胞，结果表明基于 MOI 的感染可导致更可重复的感染率。此外，我们使用连续稀释的拉米夫定（一种 HBV 复制抑制剂）验证了该测定法，该抑制剂可分别将 HBV DNA 分泌和感染性病毒产物抑制约 56 倍和 470 倍。有趣的是，我们发现右美托咪定（DMM），一种 α-2 肾上腺素能激动剂，可将感染性病毒产物的分泌抑制约 6 倍，而不干扰 HBV DNA 的分泌。总之，我们开发了一种适用于感染性 HBV 粒子定量分析的测定法。我们发现 DMM 是一种新型抑制剂，它专门干扰感染性 HBV 粒子的分泌，而不影响 HBV 基因组的分泌。这种终点稀释测定法能够精确确定感染性 HBV 粒子的数量，评估 HBV 粒子的特异性感染力和稳定性，并鉴定新型 HBV 抑制剂。
