The best likelihood scores were as follows: for the pandemic H1N1 dataset ln - 9857.691488 (additional file 5
) and for the seasonal H1N1 dataset ln -13871.895684 (additional file 6
To detect positive selection we used the criterion of statistically significant bias of non-synonymous mutations (dN) relative to synonymous mutations (dS) at a codon [11
]. For the seasonal H1N1 lineage, we see evidence for positive natural selection for resistance to oseltamivir conferred by the genotype 275Y (dN-dS = 6.097; p = 0.035). The codon corresponding to amino acid position 275 is the sole region on the NA segment currently exhibiting significant dN-dS bias.
In the pandemic clade of H1N1, we see evidence for positive natural selection for resistance to oseltamivir as conferred by genotype 275Y (dN-dS = 7.69; p = 0.006). There is also significant dN-dS bias at codon 248 (dN-dS = 5.18; p = 0.031). Of the 225 reports of cases of resistance to oseltamivir among pandemic H1N1 [4
], a subset of 28 isolates has been sequenced and put in public databases as of January 4, 2010. Based on our NA phylogeny, the members of the H1N1 pandemic lineage containing the 275Y NA genotype include: isolates from patients infected with pandemic H1N1 and treated with oseltamivir in the United States (Washington State) [5
], two clades of isolates restricted to Japan (Yamaguchi plus Chiba; Shiga plus Niigata), a clade of isolates with a worldwide distribution (Japan, Denmark, plus the United States), and various unrelated isolates from China, Israel, Africa, the United States, Japan, and Europe. There are also reports of oseltamivir resistance in pandemic H1N1 isolates from Singapore but their NA has not yet been sequenced.
In the case of seasonal H1N1, some workers have dismissed the possibility of H1N1 responding to use of drugs based on low sales of oseltamivir in Norway between 2002 and 2007 [12
]. However, large numbers of doses of oseltamivir have been purchased by many entities around the world. These doses have been recently used widely in treatment and prophylaxis of mild cases [13
], especially in Japan [14
]. Moreover, any illegal trade and use of drugs around the world will be difficult to measure. There are reports of evolution of resistance and spread among patients as a result of the use of oseltamivir in prophylaxis [5
The mutations D79G, S247G, and S247N in NA also confer resistance to oseltamivir [8
]. For amino acid positions 79 and 247 there is very little variation in seasonal H1N1. For amino acid position 79, most of the seasonal clade contains the wild type, 79D. The pandemic clade largely contains 79S. For position 247 in the seasonal clade, most isolates contain the wild type, 247S. Two seasonal isolates from humans in Montserrat contains 247N. Regarding position 247 in the pandemic clade, all isolates thus far sequenced and put into public databases contain the wild type, 247S.
The mutations H126N or Q136K in NA confer resistance to zanamivir in H1N1 [8
]. For amino acid position 126 in NA, the seasonal clade contains the wild type, 126H. The pandemic clade contains largely 126P, which merits further study.
Due to the increasing importance of zanamivir, we provide a Keyhole Mark-up Language (KML) file in which we mapped isolates from Australia, Thailand, New Zealand, Brazil, Nicaragua, and Macau that contain the genotype 136K in NA for the seasonal clade (additional file 7
). The pandemic clade uniformly contains the wild type, 136Q in NA.
As either selection or reassortment can lead to strains of pandemic H1N1 that are resistant to drugs, we complement analyses of selection with a map of co-occurrence of oseltamivir-resistant seasonal H1N1 and pandemic H1N1 (Figure ). We found 53 regions in which these influenza strains co-circulate (Table ). A subset of six of these 53 regions contains pandemic H1N1 isolates with NA segments that carry the genotype 275Y in NA. This subset includes regions within Japan and the United States. To precisely illustrate the 53 areas of co-circulation of seasonal and pandemic H1N1 we distribute a KML file (see http://pointmap.osu.edu
or additional file 8
Figure 1 Screen shot of an interactive visualization of populations of oseltamivir-resistant seasonal H1N1 influenza and pandemic H1N1 influenza in co-circulation across the globe. The visualization is available via a web browser on our website POINTMAP http://pointmap.osu.edu (more ...)
Areas in which populations of pandemic H1N1 and oseltamivir-resistant seasonal H1N1 and co-circulate.