Determination of infectious hepatitis B virus particles by an end-point dilution assay identifies a novel class of inhibitors

Abstract

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 (TCID₅₀) 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 TCID₅₀/mL, converted to plaque-forming units (PFUs), and generated the specific infectivity (ratio of PFU/GEq). As a result, a TCID₅₀/mL of 7.22 × 10⁶ and 2.16 × 10⁶, 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 TCID₅₀/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 TCID₅₀ 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.

Keywords: Hepatitis B virus; Immunofluorescence analysis; Infection assay; Infectious virus particle; Multiplicity of infection; Novel antivirals; Quantitative PCR; Specific infectivity; Tissue culture infectious dose 50.