IMPORTANCEDespite the high impact of influenza pandemics on human health, some mechanisms underlying the emergence of pandemic influenza viruses still are poorly understood. Thus, it was unclear why both H2N2/1957 and H3N2/1968 reassortant pandemic viruses contained, in addition to the avian HA, the PB1 gene segment of the avian parent. Here, we addressed this long-standing question by modeling the emergence of the H3N2/1968 virus from its putative human and avian precursors. We show that the avian PB1 segment increased activity of the viral polymerase and facilitated viral replication. Our results suggest that in addition to the acquisition of antigenically novel HA (i.e., antigenic shift), enhanced viral polymerase activity is required for the emergence of pandemic influenza viruses from their seasonal human precursors. Influenza A viruses are single-stranded negative-sense RNA viruses. Their genome consists of 8 RNA segments, each of which encodes 1 to 3 proteins (1). Pandemic influenza has been occurring at irregular intervals for at least 500 years, causing significant, occasionally catastrophic mortality in the human population (2, 3). Genetic data available for the last four pandemic viruses (H1N1/1918, H2N2/1957, H3N2/1968, and H1N1/2009) indicate that they all contained an antigenically novel hemagglutinin (HA) which was derived from animal influenza A viruses (reviewed in references 2-5). The origin of the other seven gene segments of the pandemic viruses varied (3,4,6). The source of the non-HA gene segments of the H1N1/1918 virus is not clear due to the lack of sequence data for coexistent avian and mammalian influenza A viruses. The precursor of the H1N1/2009 virus emerged and circulated in pigs; this virus was transmitted and adapted to humans as a whole. Two other pandemic viruses emerged via gene mixing (reassortment) between animal viruses and contemporary human viruses and contained either 5 gene segments (H2N2/1957) or 6 gene segments (H3N2/1968) of the human virus parent.Avian influenza viruses do not replicate efficiently in humans. It is believed that exchange via reassortment of nonadapted gene segments from an avian virus by segments from a human-adapted parent might allow the emerging pandemic virus to overcome host range restriction (2,7,8). Surprisingly, both 1957 and 1968 pandemic viruses contained the avian PB1 segment (gene segment 2) (9). This segment encodes three proteins, polymerase basic protein 1 (PB1), PB1-F2, and PB1-N40, which are translated from the start codons 1, 4, and 5, respectively (1, 10). PB1 is the core subunit of the trimeric viral RNA-dependent RNA polymerase complex (PB1, PB2, and PA), which is responsible for transcription and replication of the viral genome (1, 11). PB1-F2 and PB1-N40 are not essential for virus replication, but they can affect replication efficiency and contribute to pathogenicity (10, 12).

The major natural hosts of influenza A viruses are wild aquatic birds. Occasionally, viruses are transmitted to mammalian and other avian species, including humans. Due to the high mutation rate and reassortment of the viral genome, the viruses may undergo adaptation to humans and then give rise to a pandemic.