Human infection with an avian influenza virus persists. To prepare for a potential outbreak of avian influenza, we constructed a candidate vaccine virus (CVV) containing hemagglutinin (HA) and neuraminidase (NA) genes of a H5N1 virus and evaluated its antigenic stability after serial passaging in embryonated chicken eggs. The passaged CVV harbored the four amino acid mutations (R136K in PB2; E31K in PA; A172T in HA; and R80Q in M2) without changing its antigenicity, compared with the parental CVV. Notably, the passaged CVV exhibited much greater replication property both in eggs and in Madin-Darby canine kidney and Vero cells. Of the four mutations, the PA E31K showed the greatest effect on the replication property of reverse genetically-rescued viruses. In a further luciferase reporter, mini-replicon assay, the PA mutation appeared to affect the replication property by increasing viral polymerase activity. When applied to different avian influenza CVVs (H7N9 and H9N2 subtypes), the PA E31K mutation resulted in the increases of viral replication in the Vero cell again. Taken all together, our results suggest the PA E31K mutation as a single, substantial growth determinant of avian influenza CVVs and for the establishment of a high-yield avian influenza vaccine backbone.
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