Avian PB2 with 627E were introduced into North American SIVs more than 10 years ago, and this avian signature (i.e., 627E) remains stable rather than changing to the mammalian signature (i.e., 627K). In this study, we found that the PB2 protein with aa 627K is a key determinant for efficient viral replication in MDCK cells and for virulence in mice. These findings confirm previous results showing that PB2 627K is essential for virulence of the 1997 H5N1 and other highly pathogenic H5N1 avian influenza viruses in mice (Hatta et al., 2001
; Hatta et al., 2007
; Liu et al., 2005
The virus containing the avian-origin PB2 with 627E was able to infect mice and pigs, indicating that other important aa mutations can compensate for the absence of 627K in the PB2 protein as reported previously (Li et al., 2009
). Our sequence comparison of PB2s from the Tx/98 and 1930 viruses with those from mouse-adapted viruses revealed that both Tx/98 and 1930 PB2s contain the same aa (i.e., 333T, 482K, 701D, and 714S), which are completely different from the conserved PB2 aa signatures (i.e., 333I, 482A, 701N, and 714R) normally required in mouse-adapted viruses (Naffakh et al., 2008
), suggesting that other mutations can also confer adaptation of the avian-origin PB2 to mice and other mammalian species. Normally, the avian-origin PB2 contains E at aa position 627 and alanine (A) at aa position 271 whereas the swine-origin PB2 contains K and threonine (T) at those respective positions (Bussey et al., 2010
; Naffakh et al., 2008
). The avian PB2 271A residue plays a key role in enhanced polymerase activity and viral growth of influenza A viruses in mammalian host cells and likely contributes to virus spread and cross-species transmission of pandemic H1N1 viruses (Bussey et al., 2010
). Therefore, PB2 271A might compensate for the presence of 627E in the 1930-Tx/98PB2-627E virus and in currently circulating SIVs. The avian-origin PB2 in the currently circulating SIVs and in the pandemic H1N1 viruses contains a conserved serine (S) at position 590 and a conserved arginine (R) at position 591, called the SR polymorphism, which increase polymerase activity and viral replication in human and porcine cells if present with PB2 627E (Mehle and Doudna, 2009
). This SR polymorphism is considered to be an adaptive strategy of the avian influenza virus polymerase for replication in humans (Mehle and Doudna, 2009
). Because the Tx/98 avian-origin PB2 contains this SR polymorphism, it could contribute to both mammalian adaptation and disease-causing capabilities of the 1930-Tx/98PB2-627E virus.
In pig challenge experiments, the 1930 viruses with PB2 627K (i.e., 1930-Tx/98PB2-627K and 1930-PB2-627K) were able to replicate in pig lungs and induce severe macroscopic and histopathologic lung lesions whether in the avian-origin or swine PB2 background. Additionally, when the classical 1930 virus contains an avian-origin PB2, either 627K or 627E allow for pathogenicity in the pig model. This result is different from our discovery in the mouse model and in a previous study that 627K in the H5N1 avian PB2 polymerase is crucial for replication in pigs (Manzoor et al., 2009
). Our comparisons of the histopathologic lung lesions and virus titers caused by each virus revealed that the 627E residue of PB2 decreases the pathogenicity of the classical 1930 virus in pigs when it contains the swine-origin PB2 but not when it contains the avian-origin PB2.
Thus, the importance of the aa at position 627 in PB2 appears to differ among different virus strains and in different animal species. These results correlate with previous observations in which substituting the E at position 627 in PB2 with K does not change virus replication and virulence of the 2009 pandemic H1N1 virus in mice (Herfst et al., 2010
; Zhu et al., 2010
) or the pathogenicity and transmission of the virus in ferrets (Herfst et al., 2010
). Furthermore, although the majority of the lethal H5N1 viruses for ferrets and mice possess PB2 627K, some H5N1 viruses with PB2 627E are lethal in ferrets (Katz et al., 2000
; Maines et al., 2005
; Salomon et al., 2006
). Whether PB2 is derived from swine or avian influenza viruses, 627K of PB2 is crucial for virulence of the classical H1N1 SIV in the mouse model. In the pig model, PB2 627E decreases pathogenicity of the classical 1930 SIV when it contains the swine-origin PB2, but not when it possesses the avian-origin PB2. Our study suggests that the pathogenicity of SIVs with different PB2 genes and mutation of codon 627 in mice does not correlate with the pathogenicity of the same SIVs in the natural host, the pig.