The current outbreak of pandemic influenza A(H1N1) that was first detected in Mexico is a reminder that IFV is an evolving pathogen capable of significant reassortment events resulting in the generation of novel strains. The “Spanish flu” pandemic of 1918–1919 infected approximately 20% of the world's population and killed 20–50 million people, making it the most devastating disease in all recorded history 
. The initial epidemiology of the pandemic influenza A(H1N1) virus suggests a lower case fatality rate compared to the “Spanish flu”, however concerns remain with regard to the potential for secondary waves of infection, as occurred with the “Spanish flu”, resulting in higher mortality compared with the initial wave of infection 
Amidst a ~99% frequency of oseltamivir resistance in 2008–2009 seasonal A/H1N1 influenza isolates the novel 2009 pandemic influenza A(H1N1) was fortuitously found to retain oseltamivir sensitivity. However, this virus acquired the adamantane resistance-conferring gene via a reassortment event 
. Co-circulation of viruses with different drug resistance profiles in humans poses a threat of emergence of viruses resistant to both oseltamivir and adamantanes 
. Simultaneous infection of a single host with both strains could lead to gene reassortment and ultimate production of a virus with both oseltamivir-resistance and the characteristics of the novel influenza A(H1N1) virus. The process of swapping gene segments between two viral strains (segment reassortment, genetic drift) and has been widely reported to be a major mechanism of viral evolution 
. Because the pandemic influenza A(H1N1) virus, or any other emerging strain of influenza, could potentially gain the oseltamivir-resistance mutation (H274Y) it is critical to continue to develop anti-influenza compounds with alternative mechanisms of action.
DAS181 is a sialidase fusion protein currently in development for use as a broad-spectrum inhibitor of influenza virus and parainfluenza virus infection. DAS181 acts by reducing viral binding to the respiratory epithelium, a novel mechanism of action for anti-influenza drugs, and as such it complements existing anti-influenza approaches. Furthermore, since DAS181 targets the host cell, the potential for generating viral resistance may be less than with traditional influenza inhibitors, which target the virus itself (M2 inhibitors, neuraminidase inhibitors). Here we demonstrate for the first time that DAS181 inhibits replication of several pandemic influenza A(H1N1) viruses in MDCK cells, HAE culture, ex vivo human bronchi tissue and in a murine model. The effective inhibitory DAS181 concentration for the pandemic influenza A(H1N1) viruses was similar to that with seasonal IFV, suggesting that the pandemic influenza A(H1N1) viruses binds to sialic acid residues comparably recognized by DAS181. This finding highlights the potential broad spectrum activity of DAS181 against current and future IFV strains.
A common hallmark of novel IFV strains is altered sialic acid (Sia) recognition. All influenza A viruses bind to terminal Sia acid residues expressed on the cellular receptors. However, the efficiency of this interaction depends on several factors such as the structure of adjacent oligosaccharides. When avian or swine viruses cross the inter-species barrier and infect humans they may interact differently with the ‘human’ Sia-containing receptors, as evidenced by HPAI H5N1 and 1918 “Spanish flu” viruses isolated from clinical material 
. While the exact Sia residues recognized by these emerging strains (pandemic influenza A(H1N1) or HPAI) may vary, DAS181 exhibits broad spectrum sialic acid removal and therefore may be effective against current and future influenza viruses with altered HA-Sia binding specificity.
The data obtained using MDCK cell culture should be interpreted with caution because this commonly used cell line may not accurately represent the human respiratory tract in terms of cell types and expression pattern of various sialic acids 
. Furthermore, the mouse infection data is limited by the fact that the mouse and human respiratory systems present different viral binding patterns 
, and the novel 2009 A(H1N1) virus is not a mouse-adapted strain. As such the dynamics of 2009 A(H1N1) infection shown in these models may not accurately represent infection in humans. It should be noted that in the absence of daily ketamine anesthesia, A/Mexico/4108/2009 does not result in substantial morbidity or mortality in the mouse model. However, the significant protective efficacy of DAS181 under these experimental conditions is remarkable and the further evaluation of DAS181 utilizing additional A(H1N1) viruses is warranted. In contrast, the DAS181 effectiveness shown here in HAE culture and ex vivo
bronchi model, two of the most representative models of the human respiratory tract, correlates with the MDCK and mouse data and suggests that DAS181 may indeed be effective against pandemic influenza A(H1N1) infections although further clinical confirmation is required. Additionally, it will be valuable to evaluate the affect of treatment timing relative to infection; in the studies herein treatment was initiated within 6 hours of infection. We have previously observed that NAIs, oseltamivir and zanamivir, do not inhibit DAS181 activity in vitro
or in vivo
(unpublished data). In cell culture, DAS181 exhibits a strongly synergistic relationship with the NAIs against a panel of laboratory IFV strains (unpublished data), although this has not been examined in the context of 2009 A(H1N1) infection.
Because of the potential for emerging IFV strains to attain oseltamivir resistance via reassortment with the current seasonal H1N1 strain we also tested DAS181 activity against an oseltamivir-resistant IFV. The most common mutation conferring oseltamivir-resistance involves H274Y (H275Y in N1 numbering) substitution in the neuraminidase of viruses of N1 antigenic subtype (H1N1 and H5N1). This mutation was originally identified in isolates from oseltamivir-treated individuals infected with H1N1 virus 
, and more recently, in patients infected with HPAI of H5N1 background, a troubling observation given the pandemic potential and extremely virulent nature of this IFV strain 
. Perhaps more alarming was the finding that seasonal H1N1 viruses, carrying the oseltamivir resistance-conferring H274 mutation, emerged simultaneously in several countries in 2007–2008, including countries where oseltamivir is not prescribed 
. The frequency of IFV isolates with this mutation reached ~99.5% in the US in 2008–2009 
. Here we show that influenza A/Hawaii/21/2007, one of the first oseltamivir-resistant viruses from the 2007–2008 season, is highly sensitive to DAS181 in MDCK cells. This in vitro
finding indicates that DAS181 may be active against currently circulating oseltamivir-resistant IFV and further suggests that if novel strains, such as the pandemic influenza A(H1N1) viruses, attain the H274Y mutation, DAS181 may be active against these new isolates as well. Additional testing of DAS181 sensitivity of several NAI-resistant viruses is ongoing.
While the 2009 pandemic IFV A(H1N1) and the oseltamivir-resistant seasonal IFV of 2007–2009 currently exhibit low case fatality rates, these patterns are potentially unstable. Given the IFV' propensity for rapid evolution, the constant threat of an emerging highly virulent drug-resistant strain is concerning and supports the need to monitor evolution of the 2009 A(H1N1) IFV strains and simultaneously develop specific vaccines and novel antivirals.
The safety and efficacy of DAS181 as a novel anti-IFV agent is currently being evaluated in clinical trials. Based on its broad spectrum preclinical in vitro and in vivo activity against seasonal and potentially pandemic IFV, as well as against the 2009 pandemic IFV A(H1N1) and oseltamivir-resistant IFV, DAS181 represents a potentially valuable treatment option for emerging and drug-resistant IFV.