Development of an improved polykaryon-based influenza virus rescue system

Abstract

Background: Virus rescue from transfected cells is an extremely useful technique that allows defined viral clones to be engineered for the purpose of rational vaccine design or fundamental reverse genetics studies. However, it is often hindered by low primary rescue success rates or yields, especially with field-derived viral strains.

Approach: We investigated the possibility of enhancing influenza virus rescue by eliciting cell fusion to increase the chances of having all necessary plasmids expressed within the same polykaryon. To this end we used the Maedi-Visna Virus envelope protein which has potent fusion activity in cells from a wide range of different species.

Results: Co-transfecting cells with the eight plasmids necessary to rescue influenza virus plus a plasmid expressing the Maedi-Visna Virus envelope protein resulted in increased rescue efficiency. In addition, partial complements of the 8-plasmid rescue system could be transfected into two separate populations of cells, which upon fusion led to live virus rescue.

Conclusion: The simple modification described here has the potential to improve the efficiency of the virus rescue process and expand the potential applications for reverse genetic studies.

Figure 1

Principle of the fusion approach applied to the influenza A 8-plasmid rescue system. The eight virus rescue plasmids are transfected in 293 T cells together with the Env expressing plamsid. Our hypothesis is that individual 293 T cells tend to be transfected with partial complements of the viral rescue plasmids. Upon fusion, all eight necessary viral rescue plasmids can be expressed in one syncytium, allowing rescue of complete virions.

Figure 2

Yield of rescued PR8 virus obtained using different plasmid proportions. The total amount of plasmid transfected per well was set to 0.8 μg in a 24-well plate format for the whole experiment. A proportion of this amount was dedicated to the viral rescue plasmids, while the rest of the amount was dedicated either to the Env-expressing plasmid (black symbols) or the B1 inert plasmid (grey symbols), referred to as "other plasmids" in the chart. Each point shows the average yield from three replicate transfections and error bars indicate standard deviation. Negative results (0 p.f.u./mL) are plotted as 1 p.f.u./mL on the logarithmic scale. **, p < 0.01 at the 0.2 μg level.

Figure 3

Effect of using the MVV Env on recovered virus yield of a field-derived LPAI strain. This figure shows the effect of substituting 25% of the viral genes containing plasmids for the MVV envelope gene-containing plasmid on the rescue efficiency of a low pathogenicity avian influenza strain derived from a field sample. The total amount of transfected DNA is 4 μg per well in 6 well plates and 0.8 μg per well in 24 well plates. The average of three replicates is shown and error bars indicate standard deviation. Asterisks indicate levels of statistical significance as follows: *, p < 0.05; **, p < 0.01. wp, well plate.

Figure 4

Effect of adding Env-expressing or inert plasmid beyond 0.8 μg total DNA on primary rescue yields. This figure shows the effect of adding increasing amounts of Env-expressing or inert B1 plasmid to a fixed amount of 0.6 μg of PR8 rescue plasmids on primary rescue efficiency in a 24-well plate format. Each point shows the average yield from three replicate transfections and error bars indicate standard deviation.