Human RNA polymerase I-driven reverse genetics for influenza a virus in canine cells

Abstract

We have established a human RNA polymerase I (pol I)-driven influenza virus reverse genetics (RG) system in the Madin-Darby canine kidney 33016-PF cell line, which is approved for influenza vaccine manufacture. RNA pol I polymerases are generally active only in cells of species closely related to the species of origin of the polymerases. Nevertheless, we show that a nonendogenous RNA pol I promoter drives efficient rescue of influenza A viruses in a canine cell line. Application of this system allows efficient generation of virus strains and presents an alternative approach for influenza vaccine production.

FIG. 1.

(A) Comparison of human and canine RNA pol I promoter activity in human and canine cells using a luciferase reporter assay. The influenza polymerase complex was provided by transfected plasmids (+ polymerase) or by influenza virus infection (+ infection). (B) Comparison of transfection efficiency in human and canine cells using a CMV promoter-driven luciferase reporter plasmid. The mean results and standard deviations of triplicate samples from three independent experiments are presented. RLU, relative light units.

FIG. 2.

Multiplex PCR amplification with species-specific primer pairs. (A) Detection of cytochrome b and cytochrome c oxidase I (cox I) genes from 293T cells (lanes 1 and 5), MDCK 33016-PF cells (lanes 2 and 6), MDCK ATCC cells (lanes 3 and 7), and a sorted subpopulation of MDCK 33016-PF cells that display high human pol I promoter activity (lanes 4 and 8). Expected sizes are 391 bp (human) and 172 bp (canine) for cytochrome b and 229 bp (human) and 153 bp (canine) for cox I. (B) Sensitivity to human cell contamination of canine cells. Human 293T DNA was added to canine MDCK ATCC DNA at ratios of 1:99, 5:95, 10:90, 20:80, and 100:0 (lanes 9 to 13, respectively) before performing multiplex PCR with the cox I primer pairs. “M” indicates the molecular weight marker (1-kb Plus DNA Ladder; Invitrogen). PCR products were separated on a 2% agarose gel. In each panel, a representative gel is presented from at least two independent experiments.

FIG. 3.

Rescue of influenza virus by human pol I promoter-based reverse genetics in human and canine cells. (A) Rescue of A/Puerto Rico/8/34 virus in the presence or absence of TMPRSS2 with the postrescue addition of feeder cells. (B) Rescue of A/Puerto Rico/8/34 virus in the presence or absence of TMPRSS2 helper plasmid without any addition of feeder cells. Titers of the indicated viruses from each cell type are expressed as focus-forming units (FFU) per milliliter. The mean results and standard deviations from at least three independent experiments are shown.

FIG. 4.

Rescue of A/Puerto Rico/8/34 influenza virus by human or canine pol I-driven reverse genetics in MDCK 33016-PF cells. Titers are expressed as focus-forming units (FFU) per milliliter. The mean results and standard deviations from three independent experiments are shown.