Isolation and Identification of Inter-Species Enterovirus Recombinant Genomes

Figures

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  Figure 1. Schematic representation of donor replicon RNA templates. The genetic structure is shown for EV71/rep, E7/rep, PV1/rep, PV3/rep, and EV70/rep. Numbers below refer to the nucleotide number within VP1 at which the virus sequence restarts, relative to its position in the full-length genome. For EV70/rep, we also denoted the last nucleotide of the VP4 fragment present upstream of the luciferase gene.

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  Figure 2. Representations of 2C-CRE stem-loop structures and mutations. The wild-type structure is shown for the 2C CRE of EV-A71, E7, PV3, and EV-D68. Nucleotides in boxes denote those mutated to knockout the CRE function, with replacement nucleotides shown with arrows.

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  Figure 3. Reverse-transcription PCR analysis of CRE-REP assay samples. Example gel electrophoresis analysis of total RNA isolated and amplified by RT-PCR from CRE-REP assays. In this example, we show results from both 33 °C (A) and 37 °C (B) following co-transfection of the EV68/CRE acceptor template with either a species B, C, or D donor template. Lanes are numbered 1–8 for reference. Numbers in brackets below gel images are the expected sizes for a precise recombinant of each assay template pairing.

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  Figure 4. Recombinant genomes isolated from EV68/EV70 CRE-REP assay: (A) parallel coordinates visualisation of EV68/EV70 recombinant genomes. The location of each recombination junction was mapped respectively to each parental genome. Each line represents a unique recombinant; (B) schematic representation of each identified recombinant. Recombinant name is given top left and the size of each sequence duplication is top right. The relevant part of the recombinant sequence chromatogram is shown below, with the acceptor and donor-derived regions indicated based on the determined junction position. Nucleotides underlined in recombinant sequence represent regions of ambiguity where both parental sequences match. Although labelled as acceptor, it cannot be determined from which template these particular nucleotides were derived.

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  Figure 5. Recombinants isolated from inter-species CRE-REP assays. Schematic representation of each identified recombinant. Labelling and presentation are as described in Figure 4B.

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  Figure 6. Replication of inter-species recombinant EV68/PV1-1: (A) schematic of inter-species recombinant genome EV68/PV1-1. Open arrowheads mark positions of primers for cDNA synthesis and closed arrowheads mark positions of primers for PCR; (B) PCR assay for negative- and positive-strand RNA. Left-hand panels denote amplification of recombination window with primers specific to the recombinant (EV68(F) and PV1(R)), and right-hand panels denote amplification of EV-D68 controls with EV-D68 specific primers (EV68(F) and EV68(R)). Amplification from positive-strand (upper panels) and negative-strand (lower panels) RNA is shown. Arrows indicate expected product sizes.