PMC

Development of a reverse genetics system for BoDV-1. (a) A protocol for BoDV-1 reverse genetics. A BoDV-1 antigenome plasmid encoding the full-length BoDV-1 antigenome and three helper plasmids expressing N, P and L were co-transfected into cultured cells. At 72 h post-transfection, the transfected cells were cocultured with Vero cells to spread the infection efficiently. The cells were passaged every 3 or 4 days until the rBoDV-1 infection spread to most Vero cells. (b) Configuration of the BoDV-1 antigenome plasmid. The antigenome plasmid constructed by Schneider et al. encodes a BoDV-1 full-length antigenome flanked by a hammerhead ribozyme (Rz) and a hepatitis delta virus ribozyme (δ) between the T7 promoter (T7) and the T7 terminator (φ) []. The antigenome plasmid constructed by Martin et al. encodes a BoDV-1 full-length antigenome flanked by a hammerhead ribozyme (Rz) and a hepatitis delta virus ribozyme (δ) between the CAG promoter and polyadenylation signal (PSA) []. The antigenome plasmid constructed by Ackermann et al. was developed from the plasmid constructed by Martin et al. by inserting a nonsynonymous mutation, R66K, and two nonsynonymous mutations, L1116R and N1398D, into the P and L genes of the BoDV-1 antigenome [].

HDV genome, antigenome and mRNA are differentially affected by HDAg over a wide range of HDAg expression. (A) Schematic showing locations of RT and PCR primers used for detection of HDV antigenome RNA and mRNA. (B and C) Huh-7 cells were transfected with in vitro-synthesized HDAg-encoding mRNA-b for 4 h and then, after 20 h, with either circular HDAg(–) genome HDV RNA or circular HDAg(–) antigenome for 4 h. Cellular RNAs were harvested 24 h after the start of the circular RNA transfection and analyzed by strand-specific qRT-PCR. (B) Cells were pretransfected with various amounts (0.1 to 30 ng) of mRNA-b (horizontal axis), followed by 25 ng of circular HDAg(–) HDV genome or antigenome RNA. Orange, HDV antigenome RNA; purple, HDV mRNA; gray, HDV genome RNA. The values obtained for 30 ng of transfected mRNA are not shown to better illustrate the differences between the antigenome RNA and mRNA at low HDAg levels. The dotted line is a linear least-squares fit to the antigenome RNA data. Averaged background levels were as follows: genome, 5%; antigenome, 6%; mRNA, 2%. (C) Cells were pretransfected with either 1 ng (solid lines) or 7.75 ng (dotted lines) of mRNA-b, followed by various amounts of circular HDAg(–) HDV genome RNA (horizontal axis). The calculated amounts of RNA per cell assume 25 ng of total RNA per cell and 50% transfection efficiency.

Analyses on next-generation sequencing of rSRMV-eGFP progenies. Schematic representation of rSRMV-eGFP antigenome (A). All elements proportionally match their actual lengths in viral antigenome. ORF: open reading frame; UTR: untranslated region. Depth and coverage of next-generation sequencing across the rSRMV-eGFP antigenome at P10, P20, P30, P40, and P50 (B). All elements proportionally match their actual lengths in viral antigenome.

Next-generation sequencing of P50 rCDV-VP2. Schematic representation of rCDV-VP2 antigenome (A). All elements proportionally match their actual lengths in the viral antigenome. UTR, untranslated region. Depth and coverage of NGS across the rCDV-VP2 antigenome (B). All genetic elements proportionally match their actual lengths in the viral antigenome. Absolute (C) and relative (D) sequencing depths for identified sites with SNM. *Mutation rate of 100% at a given site. Profile of SNMs in rCDV-VP2 antigenome at P50 (E). SNMs are marked with arrows. The sole nonsense mutation is marked with a red arrow. Numbers in brackets indicate point mutation rates. #Missense mutation. Amino acid mutations (arrow-marked) in seven proteins (F). The sole nonsense mutation causes early termination of VP2 translation, marked with “Q8Stop” in red.