The PB 2E 627 K Mutation Attenuates Viruses Containing the 2009 H 1 N 1 Influenza Pandemic Polymerase

The swine-origin H1N1 influenza A virus emerged in early 2009 and caused the first influenza pandemic in 41 years. The virus has spread efficiently to both the Northern and the Southern Hemispheres and has been associated with over 16,000 deaths. Given the virus’s recent zoonotic origin, there is concern that the virus could acquire signature mutations associated with the enhanced pathogenicity of previous pandemic viruses or H5N1 viruses with pandemic potential. We tested the hypothesis that mutations in the polymerase PB2 gene at residues 627 and 701 would enhance virulence but found that influenza viruses containing these mutations in the context of the pandemic virus polymerase complex are attenuated in cell culture and mice. IMPORTANCE Influenza A virus (IAV) evolution is characterized by host-specific lineages, and IAVs derived in whole or in part from animal reservoirs have caused pandemics in humans. Because IAVs are known to acquire host-adaptive genome mutations, and since the PB2 gene of the 2009 H1N1 virus is of recent avian derivation, there exists concern that the pathogenicity of the 2009 H1N1 influenza A pandemic virus could be potentiated by acquisition of the host-adaptive PB2-E627K or -D701N mutations, which have been shown to enhance the virulence of other influenza viruses. We present data from a mouse model of influenza infection showing that such mutations do not increase the virulence of viruses containing the 2009 H1N1 viral polymerase. Received 3 March 2010 Accepted 8 March 2010 Published 18 May 2010 Citation Jagger, B. W. , M. J. Memoli, Z. -M. Sheng, L. Qi, R. J. Hrabal, et al. 2010. The PB2-E627K mutation attenuates viruses containing the 2009 H1N1 influenza pandemic polymerase. mBio 1(1):e00067-10. doi:10.1128/mBio.00067-10. Editor W. Ian Lipkin, Columbia University Copyright © 2010 Jagger et al. This is an open-access article distributed under the terms of the Creative Commons Attribution-Noncommercial-Share Alike 3.0 Unported License, which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the author and source are credited. Address correspondence to Jeffery K. Taubenberger, taubenbergerj@niaid.nih. gov. Influenza A viruses (IAVs) cause significant human respiratory disease in the form of annual, epidemic recurrences and the sporadic emergence of novel viruses that may give rise to pandemics (1). In April 2009, a novel swine-origin H1N1 influenza A virus was identified from patients in Mexico and the United States (2), spread globally (3), and caused the first influenza pandemic since 1968 (4). Influenza pandemics vary greatly in their severity, as measured by the numbers of pneumonia and influenza deaths they cause (5). While the pandemic’s full impact cannot yet be assessed, as of 21 February 2010 there have been at least 16,226 deaths and millions of cases worldwide (6). The worst influenza pandemic on record was the 1918-1919 “Spanish” influenza, which killed approximately 50 million people globally (7). It has been hypothesized that the 1918 pandemic circulated in a less virulent form in an initial spring-summer wave and a more virulent form in a second major wave (8). There has been speculation that the 2009 pandemic could also develop enhanced virulence following further adaptation to the human host, but data in support of these hypotheses are limiting (9). The basis for enhanced pathogenicity of pandemic influenza viruses relates in part to antigenic novelty and the lack of protective immunity in all or parts of the population but also likely relates to inherent viral virulence factors that differ between virus strains. Pandemic viruses, derived at least in part from zoonotic infections, must also be sufficiently host adapted to efficiently replicate and transmit in humans (10). The molecular basis as to why the 1918 pandemic virus, especially in the fall and winter of 19181919 (11), had enhanced pathogenicity is polygenic and still not fully elucidated. Experimental animal models have shown that the genes encoding the surface proteins hemagglutinin (HA) and neuraminidase (NA) and the genes encoding the viral ribonucleoprotein polymerase (RNP) complex (consisting of the viral polymerase subunits PB2, PB1, PA, and nucleoprotein [NP]) contain still-undefined virulence factors (12–16). Similarly, the Asian lineage of highly pathogenic avian influenza (HPAI) virus H5N1 has caused very high mortality rates among those humans with clinically apparent infections (286 deaths out of 478 confirmed cases since 2003 [17]), but sustained human-to-human transmission has not been observed. Mutations in the genes encoding the influenza A viral RNP have been implicated in the adaptation of avian influenza viruses to humans (18, 19). The PB2 subunit (20), in particular, PB2 residue 627 (PB2-627), has been identified as an important determinant of host range restriction (21) and virulence in animal models (22, 23). Avian influenza viruses generally encode a glutamic acid at this site, while human isolates typically encode a lysine. Residue 701, residing in a region of PB2 implicated in nuclear localization (24, 25), has similarly been identified as a host-adaptive locus (26), RESEARCH ARTICLE April 2010 Volume 1 Issue 1 e00067-10 mbio.asm. org 1 with the D701N mutation increasing both replication in mice (27, 28) and transmission in guinea pigs (29). Independent examples of selection of the PB2-D701N mutation have also been observed, for example, in the nonpandemic avian origin European swine H1N1 viruses (30) as well as in some HPAI H5N1 viruses. Along with its established role in host adaptation, the influenza A RNP has also been noted as a key determinant of virulence. A virus containing the 1918 RNP on the background of a seasonal human H1N1 virus was found to increase viral replication and worsen histopathologic findings in the lower respiratory tract in ferrets (16). Likewise, a reassortant virus containing the polymerase genes (encoding PB2, PB1, and PA) of the highly pathogenic H5N1 isolate A/Vietnam/1203/2004 (referred to as VN1203), isolated from a fatal human infection, and the nonpolymerase genes of A/chicken/Vietnam/C58/04 (H5N1) (referred to as CH58), an HPAI isolate that is nonlethal in mice and ferrets, retained significant lethality in these animals. In contrast, a virus containing the polymerase genes of CH58 and the nonpolymerase genes of VN1203 was entirely nonlethal even though both viruses express an HA with a polybasic cleavage site insertion (31), the principal determinant of high pathogenicity in poultry infections (32). The 2009 pandemic H1N1 virus possesses a PB2 protein of relatively recent avian derivation, and this protein retains the avian consensus residues PB2-627E and -701D. There has been concern that the 2009 pandemic H1N1 virus could acquire human-adaptive changes at these residues (33), and two viral isolates with the PB2-E627K mutation have been reported (34). It is therefore critical to assess the likelihood that pandemic H1N1 viruses with such mutations would have increased virulence.