The HA tree confirms the clade shift during the summer. To be noted that a separate subclade of clade 7 consists of 12 sequences with the D222E substitution, which has been found at higher frequency in Italy, in Turkey and in Sweden, and whose biological meaning is still unknown. In contrast, a phylogenetic tree comparing NA sequences (full-length with a few exceptions), shows co-clustering of most clade 7 sequences with those from other clades until June, followed by progressive divergence of the late clade 7 Italian sequences from early New York sequences. By the end of June, clade 7 sequences from Rome isolates were clustering in three distinct subclades of clade 7. One of these was apparently the most successful as it was associated with the big autumn wave of infections. NA sequences from viruses bearing the HA G222E cluster together (including those from USA and Sweden), indicating that this mutation did not appear by converging evolution of different strains but rather may represent a signature of an authentic subclade within clade 7 sequences.

To establish whether the significant clade shift was due to the local epidemic rather than a global phenomenon, and to identify the time course and the local trends of this evolution in different parts of the world, the total set of HA sequences downloadable from GISAID database (December 2009) was divided by country (from countries with a reasonable number of sequences available over a period of at least three months) or geographical area and by month, and analyzed at the signature nucleotide 658 in the HA sequence. There are a few limits to such a database analysis: 1) the geographical origin of the isolates does not necessarily indicate the actual origin of the infection, rather merely the origin of the infected person, especially for early European and Asian isolates; 2) the collection times have been submitted with a variable degree of precision and some might be unreliable; 3) the majority of isolates have been sequenced in the first months of the epidemic, while very few sequences of later isolates have been published. Despite these limits, the pattern of cluster substitution appears quite clearly everywhere. Fig. 2 shows the increasing proportion of clade 7 sequences in different countries from April to December. These patterns are completely superimposable if other signature nucleotides [4][5] of clade 7/cluster2 virus are analyzed (not shown): NS position 367, MA positions 492 and 600, PB2 position 2163, confirming the stability of the clade and the absence of late reassortment events for these segments.

The time frame of this phenomenon was different depending on the country. In the Southern hemisphere (Oceania and South America) clade 7 was already predominant by the end of April, reflecting perhaps the faster spread of the epidemic in the Southern (winter) part of the globe. In Singapore, China and Scandinavian countries the shift occurred a few weeks later, while in the majority of northern hemisphere countries it occurred mostly between June and July. The apparent fall of clade 7 sequences from April to May 2008 in the USA, UK and Canada, might be considered an artefact due to the overrepresentation of sequences from the New York clade 7 outbreak in April, which ignited the boom of scientific and public interest. The somewhat erratic behaviour of South American sequences (mostly from Brazil, Chile and Argentina) can be attributed to the fact that the aggregation of data from different countries with such a huge North-South extension and with different timing in the epidemic peaks (necessary because of the low number of available sequences) might not be reliably representative of the whole area epidemic. The selection of clade 7 virus in Italian isolates (data from our lab, n= 66, aggregated to those published by other labs, n= 133 ) appears slightly anticipated compared to other Northern Hemisphere countries such as Mexico, USA, Japan, Spain. In USA and in Japan a few sequences other than clade 7 were collected apparently as late as October/November.

While the dynamics of the epidemics suggests a possible selective advantage of clade 7 virus over other early clades, the evolutionary trends within clade 7 need further investigation. One interesting analysis consists in the quantification of the selective pressure acting on the single genetic segments of the virus. For this purpose, 2 groups of clade 7 sequences (for each genomic segment) were selected, respectively collected in May (mostly from the initial New York outbreak) and in November (from all parts of the world, after the October peak). May sequences were randomly selected to match the much smaller number of November sequences. The selective pressure acting on each segment was computed as the ratio between the rate of non synonymous substitutions per non synonymous site and the rate of synonymous substitutions per synonymous sites (Ka/Ks) using the Nei and Gojobori substitution model ([6] with the Jukes-Cantor correction) implemented in the MEGA 4.0 package [7]. The Ka/Ks values suggest that a strong purifying selection was active on all segments, in agreement with previous findings for this and other influenza viruses [1][8]. In particular, purifying selection was extreme (<0.1) on NP, MP, PA and PB1, moderate (>0.2) on NS and HA. To identify if this evolutionary pattern changed during the course of the pandemic, and to identify the period of the maximum positive selective pressure on the virus, the ratio of average pairwise Ka and Ks was computed separately within the May and the November groups of sequences (again for each segment), while the evolutionary step during the period May-November (that encompassed the greatest number of infections for most countries) was analyzed as the ratio of average pairwise Ka and Ks between each May and each November sequence (Fig. 3a).

Two different patterns can be observed: for PB1, PA, and NA segments Ka/Ks remained very low and constant for the whole period; by contrast, for NS, HA , PB2, NP and MP, Ka/Ks, although highly variable among segments, showed a common decreasing pattern. The dramatic reduction from May to November suggests that the strongest “positive” selective pressure acted before May on these segments. By contrast, the genetic distance values (Fig. 3b) for the same sets of sequences, indicate a progressive increase over time. Taken together, these findings suggest that the October-November peak of infections, which occurred in the Northern Hemisphere, did not impose any significant positive selective pressure on clade 7 virus, but only a random genetic drift in strict purifying selection conditions.