NA H274Y mutation has been identified as the major mutation that confers H1N1 influenza A virus oseltamivir resistance (14
). While H274Y has been thought to damage viral fitness, clinical isolations in recent years suggest that permissive mutations exist such that the drop in the fitness effect from H274Y can be minimized (16
). Phylogenetic analysis which relies on existing clinical isolations is often used to discover beneficial mutations (16
). However, the searching space is often limited by the defined number of clinical isolates being sequenced. In vitro
selection or serial passaging could be employed for discovering beneficial mutations in virus (21
), but it is limited by the stochasticity and frequency of spontaneous mutation. In addition, the selection process restricts the sensitivity to mutants that offer a minor beneficial effect, due to the highly competitive environment of selection. The high-throughput screening method presented in this study provides an alternative approach to screen for beneficial mutations in an unbiased fashion.
A few recent studies have demonstrated the concept of high-throughput surveillance of a mutant library such as functional mapping for the human WW domain using a phage display system (35
) and genome functional element screening using random transposon insertion (36
). This is the first time, to the best of our knowledge, that this approach has been applied with single-nucleotide resolution to a whole gene. By surveying the mutant pool in early rounds of selection with 454 sequencing, we were able to avoid an out-competing effect exerted by the optimum mutation and discover suboptimum mutations, such as F239Y and L250P. By increasing the scale of the sequencing sampling with newer sequencing technologies, which would provide greater sequencing depth, it is possible to build a single-nucleotide profile of the whole genome and predict the dynamics of viral mutation space under different conditions. The technique employed in this study is widely applicable to different fields where reverse-genetics systems exist such as human immunodeficiency virus (38
), hepatitis C virus (39
), etc. It also has the potential to be adapted to screen for mutations in cellular genes.
The screen performed here identified a novel mutation, E214D, which can fully restore the H274Y viral replication efficiency. In contrast to the seasonal H1N1 strain, aspartic acid at position 214 is present in every clinical isolate of the swine-origin 2009 H1N1 strain. It could provide an explanation for the phenomenon that H274Y imposed a less deleterious effect on swine-origin influenza virus than on seasonal influenza virus (32
). The novel compensatory mutations presented in this study enhance the forecasting power for the emergence of oseltamivir-resistant influenza virus. Influenza virus harboring a compensatory mutation is likely to acquire the oseltamivir-resistant mutation NA H274Y under conditions of oseltamivir treatment and to be less responsive to the treatment without any compromise in fitness. Therefore, to prevent future spread and minimize the social cost, precautions should be taken before treating an influenza virus-infected patient with oseltamivir when most isolates in an area are acquiring the compensatory mutations.
In summary, we present a new approach allowing the identification of beneficial mutations in a comprehensive fashion with a high sensitivity for detection of mutations with suboptimal potency. Besides identifying a novel compensatory mutation, E214D, which can fully restore the viral replication fitness of the H274Y mutant, our data also revealed mutations that could offer a smaller degree of beneficial effect, such as F239Y and L250P. The results of this study not only demonstrate the usage of high-throughput functional profiling to study a gene at single-nucleotide resolution but also broaden the understanding of NA protein evolution of oseltamivir-resistant influenza virus by offering a tool for quasispecies dynamic prediction.