Rolling circle amplification: A high fidelity and efficient alternative to plasmid preparation for the rescue of infectious clones

Abstract

Alphaviruses (genus Alphavirus; family Togaviridae) are a medically relevant family of viruses that include chikungunya virus and Mayaro virus. Infectious cDNA clones of these viruses are necessary molecular tools to understand viral biology. Traditionally, rescuing virus from an infectious cDNA clone requires propagating plasmids in bacteria, which can result in mutations in the viral genome due to bacterial toxicity or recombination and requires specialized equipment and knowledge to propagate the bacteria. Here, we present an alternative- rolling circle amplification (RCA), an in vitro technology. We demonstrate that the viral yield of transfected RCA product is comparable to midiprepped plasmid, albeit with a slight delay in kinetics. RCA, however, is cheaper and less time-consuming. Further, sequential RCA did not introduce mutations into the viral genome, subverting the need for glycerol stocks and retransformation. These results indicate that RCA is a viable alternative to traditional plasmid-based approaches to viral rescue.

Keywords: Alphaviruses; Bacteria-free; Infectious clone; Molecular virology; Rolling circle amplification; Viral rescue.

Fig. 1

Comparison of plasmid- and RCA-based workflows for viral rescue. The plasmid-based system involves the transformation of a plasmid into bacteria. The bacteria are then selected and propagated using antibiotic enriched media, and the plasmid is purified from the bacteria and transfected into the cell type of interest. The red exclamation points indicate points during the workflow where mutations and error can be introduced or enhanced. The RCA-based system involves amplification of the plasmid using random hexamers to produce the hyperbranched product. This product can then be directly transfected into cells.

Fig. 2

Comparison of viral titers produced by either plasmid or RCA in various cell lines. The kinetics of plasmid and RCA techniques to produce virus were assessed in Vero, BHK21 Clone 13, and HEK293T cells. Cells were transfected in triplicate with either 500 ng of Evomics SuperPhi RCA or plasmid DNA in triplicate. The experiment was done in two independent biological replicates. The supernatant was collected each day post-transfection until cells reached 90% CPE for plaque assay. Error bars represent the standard deviation from the mean. Statistical analysis was performed using two-way ANOVA with ad hoc Sidak’s correction for multiple comparisons (ns P > 0.05, **P ≤ 0.01, ***P ≤ 0.001).

Fig. 3

Assessing the effects of RCA input on the resulting viral titer. The effect of RCA input on viral production kinetics was examined using Vero cells. Cells were transfected in triplicate with 100 ng, 250 ng, 500 ng, or 1000 ng of Evomics SuperPhi RCA or 500 ng of the plasmid. The supernatant was collected at one and 2-days post-transfection for plaque assay. Error bars represent the standard deviation from the mean. Statistical analysis was performed using two-way ANOVA with ad hoc Dunnett’s correction for multiple comparisons.

Fig. 4

Assessing the effects of RCA kits on resulting viral titer. The effect of RCA kits on viral production kinetics was examined using Vero cells. Cells were transfected with 250 ng or 500 ng of either Evomics SuperPhi or GE GenomiPhi RCA or 500 ng of plasmid DNA. The supernatant was collected at one and 2-days post-transfection for plaque assay. Error bars represent the standard deviation from the mean. Statistical analysis was performed using two-way ANOVA with ad hoc Dunnett’s correction for multiple comparisons against a plasmid control.

Fig. 5

Assessing the effects of repetitive RCA on resulting viral titer. We examined the impact of sequential RCA on the ability to rescue virus in Vero cells. We used the Evomics SuperPhi RCA kit and a sample of midiprepped DNA as a template to generate RCA products (Passage 0). We then used the RCA product as the template for subsequent RCA reactions (Passage 1–3). We transfected the cells using 500 ng of RCA product or 500 ng of plasmid DNA in triplicate. The supernatant was collected 2-days post-transfection for plaque assay. Error bars represent the standard deviation from the mean. Statistical analysis was performed using one-way ANOVA with ad hoc Dunnett’s correction for multiple comparisons against the plasmid control (ns P > 0.05, **P ≤ 0.01, ***P ≤ 0.001).