Investigation of Influenza Virus Polymerase Activity in Pig Cells

Abstract

bReassortant influenza viruses with combinations of avian, human, and/or swine genomic segments have been detected frequently in pigs. As a consequence, pigs have been accused of being a "mixing vessel" for influenza viruses. This implies that pig cells support transcription and replication of avian influenza viruses, in contrast to human cells, in which most avian influenza virus polymerases display limited activity. Although influenza virus polymerase activity has been studied in human and avian cells for many years by use of a minigenome assay, similar investigations in pig cells have not been reported. We developed the first minigenome assay for pig cells and compared the activities of polymerases of avian or human influenza virus origin in pig, human, and avian cells. We also investigated in pig cells the consequences of some known mammalian host range determinants that enhance influenza virus polymerase activity in human cells, such as PB2 mutations E627K, D701N, G590S/Q591R, and T271A. The two typical avian influenza virus polymerases used in this study were poorly active in pig cells, similar to what is seen in human cells, and mutations that adapt the avian influenza virus polymerase for human cells also increased activity in pig cells. In contrast, a different pattern was observed in avian cells. Finally, highly pathogenic avian influenza virus H5N1 polymerase activity was tested because this subtype has been reported to replicate only poorly in pigs. H5N1 polymerase was active in swine cells, suggesting that other barriers restrict these viruses from becoming endemic in pigs.

The Effect of the PB2 Mutation 627K on Highly Pathogenic H5N1 Avian Influenza Virus Is Dependent on the Virus Lineage

“…Interestingly, viruses from the 2009 H1N1 pandemic had PB2 627E but carried other PB2 mutations, 590-91SR and 271A, that appeared to compensate for PB2 627E and adapt the viruses for efficient replication in mammals (19)(20)(21). Avian species-derived influenza virus polymerases bearing PB2 627E are restricted in mammalian cells, including cells of human, murine, primate, and porcine origin (22)(23)(24)(25)(26). Despite recent developments, the exact mechanism for this host-specific restriction by PB2 627 remains unclear.…”

“…PB1, PB2, PA, and NP expression plasmids were transfected together with the minigenome firefly reporter plasmid pChicken-PolI-Firefly into avian DF-1 cells or together with pHumanPolI-Firefly into human 293T cells as previously described (26). Cells were incubated at 37, 39, or 41°C.…”

“…As an example of a historical pre-Asianlineage H5N1 HPAIV bearing the typical avian virus signature PB2 627E, we used A/turkey/England/50-92/1991 (50-92) virus, reportedly restricted to Galliformes (43,44 (26,46). The 50-92, Ty/05, and pH1N1 PB1, PB2, PA, and NP genes were subcloned into the pCAGGS expression plasmid as described previously (25,26).…”

“…Viruses were generated by reverse genetics using a 12-plasmid system as described previously (26). Briefly, all 12 plasmids were transfected into 293T cells in 12-well plates using the Fugene HD transfection reagent (Roche Diagnostics).…”

“…The 50-92, Ty/05, and pH1N1 PB1, PB2, PA, and NP genes were subcloned into the pCAGGS expression plasmid as described previously (25,26). Mutagenesis of PB2 genes was performed by overlapping PCR as described previously (25,26). Polymerase assays utilized the minigenome firefly reporter plasmid pChickenPolI-Firefly or pHuman-PolI-Firefly as described previously (25).…”