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Evolutionary pattern of pandemic influenza )] TJ ET BT 26.250 639.438 Td /F1 9.8 Tf [(\(H1N1\) 2009 virus in the late phases of the 2009 pandemic.. PLOS Currents Influenza. 2010 Mar 4 . Edition 1. doi: )] TJ ET BT 26.250 627.533 Td /F1 9.8 Tf [(10.1371/currents.RRN1149.)] TJ ET q 15.000 -179.563 577.500 804.715 re W n 0.271 0.267 0.267 rg BT 26.250 598.430 Td /F4 12.0 Tf [(Abstract)] TJ ET BT 26.250 578.476 Td /F1 9.8 Tf [(Influenza A\(H1N1\)v has spread rapidly in all parts of the globe in 2009 as a true pandemic, although fortunately a clinically mild )] TJ ET BT 26.250 566.571 Td /F1 9.8 Tf [(one. The relevant evolutionary steps for the new virus to adapt to human populations occurred very early during the pandemic, )] TJ ET BT 26.250 554.667 Td /F1 9.8 Tf [(before the end of April. Of the several resulting clades or clusters, clade 7 appeared later and proved more successful, )] TJ ET BT 26.250 542.762 Td /F1 9.8 Tf [(substituting all other early clades before the bulk of the worldwide infections occurred.)] TJ ET BT 26.250 506.159 Td /F4 12.0 Tf [(Funding Statement)] TJ ET BT 26.250 486.205 Td /F1 9.8 Tf [(This work has been partly supported by grants from the Italian Ministry of Health \(Ricerca Corrente e Finalizzata\).)] TJ ET BT 26.250 457.103 Td /F4 12.0 Tf [(Introduction)] TJ ET BT 26.250 437.148 Td /F1 9.8 Tf [(2009 influenza A\(H1N1\)v pandemic virus has emerged following a recent reassortment event between swine strains )] TJ ET 0.267 0.267 0.267 rg BT 529.116 437.148 Td /F1 9.8 Tf [([1][2])] TJ ET 0.271 0.267 0.267 rg BT 550.800 437.148 Td /F1 9.8 Tf [(. Its )] TJ ET BT 26.250 425.244 Td /F1 9.8 Tf [(jump in the human population has been tentatively dated back to the beginning of the year)] TJ ET 0.267 0.267 0.267 rg BT 414.856 425.244 Td /F1 9.8 Tf [( [3][4])] TJ ET 0.271 0.267 0.267 rg BT 439.250 425.244 Td /F1 9.8 Tf [(, and very early in its history the )] TJ ET BT 26.250 413.339 Td /F1 9.8 Tf [(new virus could be differentiated in clades or, as later defined, clusters. )] TJ ET 0.267 0.267 0.267 rg BT 335.140 413.339 Td /F1 9.8 Tf [([5])] TJ ET 0.271 0.267 0.267 rg BT 345.982 413.339 Td /F1 9.8 Tf [( The significance of these findings is not clear, both in )] TJ ET BT 26.250 401.434 Td /F1 9.8 Tf [(terms of a possible evolutionary pathway of the pandemic virus and in terms of pathogenicity. The early data showed that clade )] TJ ET BT 26.250 389.529 Td /F1 9.8 Tf [(7 \(as in ref. 4, or cluster 2 in ref. 5\) appeared in New York a few weeks after clades 1 and 2 were isolated in Mexico and )] TJ ET BT 26.250 377.625 Td /F1 9.8 Tf [(California, and originated late in March, but all clades were reported to co-circulate in all continents thereafter. After September, )] TJ ET BT 26.250 365.720 Td /F1 9.8 Tf [(a second more intense peak has involved most temperate countries in the Northern Hemisphere. However, viral sequence )] TJ ET BT 26.250 353.815 Td /F1 9.8 Tf [(information on this second outbreak is relatively scant, and no clear trend in viral evolution has been outlined yet. In Italy most )] TJ ET BT 26.250 341.910 Td /F1 9.8 Tf [(clades were circulating in the first months of the pandemic, when the great majority of the infections were imported by travellers )] TJ ET BT 26.250 330.006 Td /F1 9.8 Tf [(\(mostly from North and South America\) who had become infected abroad. As in most European countries, a second, more )] TJ ET BT 26.250 318.101 Td /F1 9.8 Tf [(intense wave of infections occurred in Italy during the period October-November 2009. Unlike the first epidemic peak of )] TJ ET BT 26.250 306.196 Td /F1 9.8 Tf [(imported infections, this peak was powered by the rapid local spread of the virus \(which had been circulating at low intensity )] TJ ET BT 26.250 294.291 Td /F1 9.8 Tf [(during the whole summer\) in children and adolescents \(and their contacts\) due to the opening of schools, kindergartens and )] TJ ET BT 26.250 282.387 Td /F1 9.8 Tf [(other communities after the summer holidays.)] TJ ET BT 26.250 245.784 Td /F4 12.0 Tf [(Results)] TJ ET BT 26.250 225.830 Td /F1 9.8 Tf [(We examined the nucleotide sequences \(amplified from nasal swabs\) of the hemagglutinin \(HA, bases 440-828 of the coding )] TJ ET BT 26.250 213.925 Td /F1 9.8 Tf [(sequence\) and neuraminidase \(NA\) genes \(variable length\) from respectively 19 and 23 influenza A\(H1N1\)v strains isolated in )] TJ ET BT 26.250 202.020 Td /F1 9.8 Tf [(the city of Rome, in the period May-August 2009. At position 658 \(from the start codon\) of HA the frequency of T was 63% )] TJ ET BT 26.250 190.116 Td /F1 9.8 Tf [(\(12/19\), while the frequency of A \(signature of clade 7/cluster2 virus\) was 37%. These percentages were similar to those )] TJ ET BT 26.250 178.211 Td /F1 9.8 Tf [(deduced from 589 HA sequences isolated globally \(mostly in Mexico and in the United States\) before August )] TJ ET 0.267 0.267 0.267 rg BT 497.175 178.211 Td /F1 9.8 Tf [([5])] TJ ET 0.271 0.267 0.267 rg BT 508.017 178.211 Td /F1 9.8 Tf [(. Viral strains )] TJ ET BT 26.250 166.306 Td /F1 9.8 Tf [(isolated after September can be considered genuinely representative of the local evolution of the pandemic. The complete or )] TJ ET BT 26.250 154.401 Td /F1 9.8 Tf [(partial HA and NA nucleotide sequences of an additional 43 influenza A\(H1N1\)v isolates were obtained. The frequency of the )] TJ ET BT 26.250 142.497 Td /F1 9.8 Tf [(signature HA nucleotide 658 variants in those later isolates was 0% and 100% respectively for T and A, documenting the )] TJ ET BT 26.250 130.592 Td /F1 9.8 Tf [(disappearance of other clades in favour of clade 7. Among these sequences, no mutations considered to be biologically )] TJ ET BT 26.250 118.687 Td /F1 9.8 Tf [(significant were detected, including the D222G/N \(position 239 from the start codon in the H1N1 2009 pandemic virus\) in HA )] TJ ET BT 26.250 106.782 Td /F1 9.8 Tf [(and oseltamivir resistance mutations in NA, despite most patients had been subjected to treatment. Fig.1a and b shows )] TJ ET BT 26.250 94.878 Td /F1 9.8 Tf [(Neighbour-Joining phylogenetic trees \(with bootstrap test\) of HA and NA sequences \(respectively\) isolated at INMI from April to )] TJ ET BT 26.250 82.973 Td /F1 9.8 Tf [(December 2009 \(with indicated the month of collection\) and compared to representative sequences of the initial pandemic from )] TJ ET BT 26.250 71.068 Td /F1 9.8 Tf [(North America in April.)] TJ ET 0.965 0.965 0.965 rg 26.250 -179.563 555.000 240.750 re f 0.267 0.267 0.267 rg 0.267 0.267 0.267 RG 26.250 61.187 m 581.250 61.187 l 581.250 60.437 l 26.250 60.437 l f q 168.750 0 0 225.000 35.250 -173.563 cm /I3 Do Q q 35.250 -179.563 537.000 0.000 re W n Q Q q 15.000 684.354 577.500 53.646 re W n 0.267 0.267 0.267 rg BT 15.000 718.042 Td /F2 21.0 Tf [(Evolutionary pattern of pandemic influenza \(H1N1\) 2009 virus )] TJ ET BT 15.000 693.094 Td /F2 21.0 Tf [(in the late phases of the 2009 pandemic.)] TJ ET Q 0.271 0.267 0.267 rg BT 15.000 675.088 Td /F3 9.8 Tf [(March 4, 2010)] TJ ET BT 77.556 675.088 Td /F3 9.8 Tf [()] TJ ET 0.267 0.267 0.267 rg BT 82.431 675.088 Td /F3 9.8 Tf [(Influenza)] TJ ET BT 26.250 663.247 Td /F1 9.8 Tf [(Maria Beatrice Valli)] TJ ET 0.271 0.267 0.267 rg BT 110.227 663.247 Td /F1 9.8 Tf [(, )] TJ ET 0.267 0.267 0.267 rg BT 115.648 663.247 Td /F1 9.8 Tf [(Silvia Meschi)] TJ ET 0.271 0.267 0.267 rg BT 172.529 663.247 Td /F1 9.8 Tf [(, )] TJ ET 0.267 0.267 0.267 rg BT 177.950 663.247 Td /F1 9.8 Tf [(Marina Selleri)] TJ ET 0.271 0.267 0.267 rg BT 237.542 663.247 Td /F1 9.8 Tf [(, )] TJ ET 0.267 0.267 0.267 rg BT 242.963 663.247 Td /F1 9.8 Tf [(Paola Zaccaro)] TJ ET 0.271 0.267 0.267 rg BT 305.821 663.247 Td /F1 9.8 Tf [(, )] TJ ET 0.267 0.267 0.267 rg BT 311.242 663.247 Td /F1 9.8 Tf [(Giuseppe Ippolito)] TJ ET 0.271 0.267 0.267 rg BT 387.117 663.247 Td /F1 9.8 Tf [(, )] TJ ET 0.267 0.267 0.267 rg BT 392.538 663.247 Td /F1 9.8 Tf [(Maria Rosaria Capobianchi)] TJ ET 0.271 0.267 0.267 rg BT 510.113 663.247 Td /F1 9.8 Tf [(, )] TJ ET 0.267 0.267 0.267 rg BT 515.534 663.247 Td /F1 9.8 Tf [(Stefano Menzo)] TJ ET 0.271 0.267 0.267 rg BT 26.250 651.342 Td /F1 9.8 Tf [(Valli MB, Meschi S, Selleri M, Zaccaro P, Ippolito G, Capobianchi MR, Menzo S. Evolutionary pattern of pandemic influenza )] TJ ET BT 26.250 639.438 Td /F1 9.8 Tf [(\(H1N1\) 2009 virus in the late phases of the 2009 pandemic.. PLOS Currents Influenza. 2010 Mar 4 . Edition 1. doi: )] TJ ET BT 26.250 627.533 Td /F1 9.8 Tf [(10.1371/currents.RRN1149.)] TJ ET q 15.000 -179.563 577.500 804.715 re W n 0.271 0.267 0.267 rg BT 26.250 598.430 Td /F4 12.0 Tf [(Abstract)] TJ ET BT 26.250 578.476 Td /F1 9.8 Tf [(Influenza A\(H1N1\)v has spread rapidly in all parts of the globe in 2009 as a true pandemic, although fortunately a clinically mild )] TJ ET BT 26.250 566.571 Td /F1 9.8 Tf [(one. The relevant evolutionary steps for the new virus to adapt to human populations occurred very early during the pandemic, )] TJ ET BT 26.250 554.667 Td /F1 9.8 Tf [(before the end of April. Of the several resulting clades or clusters, clade 7 appeared later and proved more successful, )] TJ ET BT 26.250 542.762 Td /F1 9.8 Tf [(substituting all other early clades before the bulk of the worldwide infections occurred.)] TJ ET BT 26.250 506.159 Td /F4 12.0 Tf [(Funding Statement)] TJ ET BT 26.250 486.205 Td /F1 9.8 Tf [(This work has been partly supported by grants from the Italian Ministry of Health \(Ricerca Corrente e Finalizzata\).)] TJ ET BT 26.250 457.103 Td /F4 12.0 Tf [(Introduction)] TJ ET BT 26.250 437.148 Td /F1 9.8 Tf [(2009 influenza A\(H1N1\)v pandemic virus has emerged following a recent reassortment event between swine strains )] TJ ET 0.267 0.267 0.267 rg BT 529.116 437.148 Td /F1 9.8 Tf [([1][2])] TJ ET 0.271 0.267 0.267 rg BT 550.800 437.148 Td /F1 9.8 Tf [(. Its )] TJ ET BT 26.250 425.244 Td /F1 9.8 Tf [(jump in the human population has been tentatively dated back to the beginning of the year)] TJ ET 0.267 0.267 0.267 rg BT 414.856 425.244 Td /F1 9.8 Tf [( [3][4])] TJ ET 0.271 0.267 0.267 rg BT 439.250 425.244 Td /F1 9.8 Tf [(, and very early in its history the )] TJ ET BT 26.250 413.339 Td /F1 9.8 Tf [(new virus could be differentiated in clades or, as later defined, clusters. )] TJ ET 0.267 0.267 0.267 rg BT 335.140 413.339 Td /F1 9.8 Tf [([5])] TJ ET 0.271 0.267 0.267 rg BT 345.982 413.339 Td /F1 9.8 Tf [( The significance of these findings is not clear, both in )] TJ ET BT 26.250 401.434 Td /F1 9.8 Tf [(terms of a possible evolutionary pathway of the pandemic virus and in terms of pathogenicity. The early data showed that clade )] TJ ET BT 26.250 389.529 Td /F1 9.8 Tf [(7 \(as in ref. 4, or cluster 2 in ref. 5\) appeared in New York a few weeks after clades 1 and 2 were isolated in Mexico and )] TJ ET BT 26.250 377.625 Td /F1 9.8 Tf [(California, and originated late in March, but all clades were reported to co-circulate in all continents thereafter. After September, )] TJ ET BT 26.250 365.720 Td /F1 9.8 Tf [(a second more intense peak has involved most temperate countries in the Northern Hemisphere. However, viral sequence )] TJ ET BT 26.250 353.815 Td /F1 9.8 Tf [(information on this second outbreak is relatively scant, and no clear trend in viral evolution has been outlined yet. In Italy most )] TJ ET BT 26.250 341.910 Td /F1 9.8 Tf [(clades were circulating in the first months of the pandemic, when the great majority of the infections were imported by travellers )] TJ ET BT 26.250 330.006 Td /F1 9.8 Tf [(\(mostly from North and South America\) who had become infected abroad. As in most European countries, a second, more )] TJ ET BT 26.250 318.101 Td /F1 9.8 Tf [(intense wave of infections occurred in Italy during the period October-November 2009. Unlike the first epidemic peak of )] TJ ET BT 26.250 306.196 Td /F1 9.8 Tf [(imported infections, this peak was powered by the rapid local spread of the virus \(which had been circulating at low intensity )] TJ ET BT 26.250 294.291 Td /F1 9.8 Tf [(during the whole summer\) in children and adolescents \(and their contacts\) due to the opening of schools, kindergartens and )] TJ ET BT 26.250 282.387 Td /F1 9.8 Tf [(other communities after the summer holidays.)] TJ ET BT 26.250 245.784 Td /F4 12.0 Tf [(Results)] TJ ET BT 26.250 225.830 Td /F1 9.8 Tf [(We examined the nucleotide sequences \(amplified from nasal swabs\) of the hemagglutinin \(HA, bases 440-828 of the coding )] TJ ET BT 26.250 213.925 Td /F1 9.8 Tf [(sequence\) and neuraminidase \(NA\) genes \(variable length\) from respectively 19 and 23 influenza A\(H1N1\)v strains isolated in )] TJ ET BT 26.250 202.020 Td /F1 9.8 Tf [(the city of Rome, in the period May-August 2009. At position 658 \(from the start codon\) of HA the frequency of T was 63% )] TJ ET BT 26.250 190.116 Td /F1 9.8 Tf [(\(12/19\), while the frequency of A \(signature of clade 7/cluster2 virus\) was 37%. These percentages were similar to those )] TJ ET BT 26.250 178.211 Td /F1 9.8 Tf [(deduced from 589 HA sequences isolated globally \(mostly in Mexico and in the United States\) before August )] TJ ET 0.267 0.267 0.267 rg BT 497.175 178.211 Td /F1 9.8 Tf [([5])] TJ ET 0.271 0.267 0.267 rg BT 508.017 178.211 Td /F1 9.8 Tf [(. Viral strains )] TJ ET BT 26.250 166.306 Td /F1 9.8 Tf [(isolated after September can be considered genuinely representative of the local evolution of the pandemic. The complete or )] TJ ET BT 26.250 154.401 Td /F1 9.8 Tf [(partial HA and NA nucleotide sequences of an additional 43 influenza A\(H1N1\)v isolates were obtained. The frequency of the )] TJ ET BT 26.250 142.497 Td /F1 9.8 Tf [(signature HA nucleotide 658 variants in those later isolates was 0% and 100% respectively for T and A, documenting the )] TJ ET BT 26.250 130.592 Td /F1 9.8 Tf [(disappearance of other clades in favour of clade 7. Among these sequences, no mutations considered to be biologically )] TJ ET BT 26.250 118.687 Td /F1 9.8 Tf [(significant were detected, including the D222G/N \(position 239 from the start codon in the H1N1 2009 pandemic virus\) in HA )] TJ ET BT 26.250 106.782 Td /F1 9.8 Tf [(and oseltamivir resistance mutations in NA, despite most patients had been subjected to treatment. Fig.1a and b shows )] TJ ET BT 26.250 94.878 Td /F1 9.8 Tf [(Neighbour-Joining phylogenetic trees \(with bootstrap test\) of HA and NA sequences \(respectively\) isolated at INMI from April to )] TJ ET BT 26.250 82.973 Td /F1 9.8 Tf [(December 2009 \(with indicated the month of collection\) and compared to representative sequences of the initial pandemic from )] TJ ET BT 26.250 71.068 Td /F1 9.8 Tf [(North America in April.)] TJ ET 0.965 0.965 0.965 rg 26.250 -179.563 555.000 240.750 re f 0.267 0.267 0.267 rg 0.267 0.267 0.267 RG 26.250 61.187 m 581.250 61.187 l 581.250 60.437 l 26.250 60.437 l f q 168.750 0 0 225.000 35.250 -173.563 cm /I3 Do Q q 35.250 -179.563 537.000 0.000 re W n Q Q q 15.000 684.354 577.500 53.646 re W n 0.267 0.267 0.267 rg BT 15.000 718.042 Td /F2 21.0 Tf [(Evolutionary pattern of pandemic influenza \(H1N1\) 2009 virus )] TJ ET BT 15.000 693.094 Td /F2 21.0 Tf [(in the late phases of the 2009 pandemic.)] TJ ET Q 0.271 0.267 0.267 rg BT 15.000 675.088 Td /F3 9.8 Tf [(March 4, 2010)] TJ ET BT 77.556 675.088 Td /F3 9.8 Tf [()] TJ ET 0.267 0.267 0.267 rg BT 82.431 675.088 Td /F3 9.8 Tf [(Influenza)] TJ ET BT 26.250 663.247 Td /F1 9.8 Tf [(Maria Beatrice Valli)] TJ ET 0.271 0.267 0.267 rg BT 110.227 663.247 Td /F1 9.8 Tf [(, )] TJ ET 0.267 0.267 0.267 rg BT 115.648 663.247 Td /F1 9.8 Tf [(Silvia Meschi)] TJ ET 0.271 0.267 0.267 rg BT 172.529 663.247 Td /F1 9.8 Tf [(, )] TJ ET 0.267 0.267 0.267 rg BT 177.950 663.247 Td /F1 9.8 Tf [(Marina Selleri)] TJ ET 0.271 0.267 0.267 rg BT 237.542 663.247 Td /F1 9.8 Tf [(, )] TJ ET 0.267 0.267 0.267 rg BT 242.963 663.247 Td /F1 9.8 Tf [(Paola Zaccaro)] TJ ET 0.271 0.267 0.267 rg BT 305.821 663.247 Td /F1 9.8 Tf [(, )] TJ ET 0.267 0.267 0.267 rg BT 311.242 663.247 Td /F1 9.8 Tf [(Giuseppe Ippolito)] TJ ET 0.271 0.267 0.267 rg BT 387.117 663.247 Td /F1 9.8 Tf [(, )] TJ ET 0.267 0.267 0.267 rg BT 392.538 663.247 Td /F1 9.8 Tf [(Maria Rosaria Capobianchi)] TJ ET 0.271 0.267 0.267 rg BT 510.113 663.247 Td /F1 9.8 Tf [(, )] TJ ET 0.267 0.267 0.267 rg BT 515.534 663.247 Td /F1 9.8 Tf [(Stefano Menzo)] TJ ET 0.271 0.267 0.267 rg BT 26.250 651.342 Td /F1 9.8 Tf [(Valli MB, Meschi S, Selleri M, Zaccaro P, Ippolito G, Capobianchi MR, Menzo S. Evolutionary pattern of pandemic influenza )] TJ ET BT 26.250 639.438 Td /F1 9.8 Tf [(\(H1N1\) 2009 virus in the late phases of the 2009 pandemic.. PLOS Currents Influenza. 2010 Mar 4 . Edition 1. doi: )] TJ ET BT 26.250 627.533 Td /F1 9.8 Tf [(10.1371/currents.RRN1149.)] TJ ET q 15.000 -179.563 577.500 804.715 re W n 0.271 0.267 0.267 rg BT 26.250 598.430 Td /F4 12.0 Tf [(Abstract)] TJ ET BT 26.250 578.476 Td /F1 9.8 Tf [(Influenza A\(H1N1\)v has spread rapidly in all parts of the globe in 2009 as a true pandemic, although fortunately a clinically mild )] TJ ET BT 26.250 566.571 Td /F1 9.8 Tf [(one. The relevant evolutionary steps for the new virus to adapt to human populations occurred very early during the pandemic, )] TJ ET BT 26.250 554.667 Td /F1 9.8 Tf [(before the end of April. Of the several resulting clades or clusters, clade 7 appeared later and proved more successful, )] TJ ET BT 26.250 542.762 Td /F1 9.8 Tf [(substituting all other early clades before the bulk of the worldwide infections occurred.)] TJ ET BT 26.250 506.159 Td /F4 12.0 Tf [(Funding Statement)] TJ ET BT 26.250 486.205 Td /F1 9.8 Tf [(This work has been partly supported by grants from the Italian Ministry of Health \(Ricerca Corrente e Finalizzata\).)] TJ ET BT 26.250 457.103 Td /F4 12.0 Tf [(Introduction)] TJ ET BT 26.250 437.148 Td /F1 9.8 Tf [(2009 influenza A\(H1N1\)v pandemic virus has emerged following a recent reassortment event between swine strains )] TJ ET 0.267 0.267 0.267 rg BT 529.116 437.148 Td /F1 9.8 Tf [([1][2])] TJ ET 0.271 0.267 0.267 rg BT 550.800 437.148 Td /F1 9.8 Tf [(. Its )] TJ ET BT 26.250 425.244 Td /F1 9.8 Tf [(jump in the human population has been tentatively dated back to the beginning of the year)] TJ ET 0.267 0.267 0.267 rg BT 414.856 425.244 Td /F1 9.8 Tf [( [3][4])] TJ ET 0.271 0.267 0.267 rg BT 439.250 425.244 Td /F1 9.8 Tf [(, and very early in its history the )] TJ ET BT 26.250 413.339 Td /F1 9.8 Tf [(new virus could be differentiated in clades or, as later defined, clusters. )] TJ ET 0.267 0.267 0.267 rg BT 335.140 413.339 Td /F1 9.8 Tf [([5])] TJ ET 0.271 0.267 0.267 rg BT 345.982 413.339 Td /F1 9.8 Tf [( The significance of these findings is not clear, both in )] TJ ET BT 26.250 401.434 Td /F1 9.8 Tf [(terms of a possible evolutionary pathway of the pandemic virus and in terms of pathogenicity. The early data showed that clade )] TJ ET BT 26.250 389.529 Td /F1 9.8 Tf [(7 \(as in ref. 4, or cluster 2 in ref. 5\) appeared in New York a few weeks after clades 1 and 2 were isolated in Mexico and )] TJ ET BT 26.250 377.625 Td /F1 9.8 Tf [(California, and originated late in March, but all clades were reported to co-circulate in all continents thereafter. After September, )] TJ ET BT 26.250 365.720 Td /F1 9.8 Tf [(a second more intense peak has involved most temperate countries in the Northern Hemisphere. However, viral sequence )] TJ ET BT 26.250 353.815 Td /F1 9.8 Tf [(information on this second outbreak is relatively scant, and no clear trend in viral evolution has been outlined yet. In Italy most )] TJ ET BT 26.250 341.910 Td /F1 9.8 Tf [(clades were circulating in the first months of the pandemic, when the great majority of the infections were imported by travellers )] TJ ET BT 26.250 330.006 Td /F1 9.8 Tf [(\(mostly from North and South America\) who had become infected abroad. As in most European countries, a second, more )] TJ ET BT 26.250 318.101 Td /F1 9.8 Tf [(intense wave of infections occurred in Italy during the period October-November 2009. Unlike the first epidemic peak of )] TJ ET BT 26.250 306.196 Td /F1 9.8 Tf [(imported infections, this peak was powered by the rapid local spread of the virus \(which had been circulating at low intensity )] TJ ET BT 26.250 294.291 Td /F1 9.8 Tf [(during the whole summer\) in children and adolescents \(and their contacts\) due to the opening of schools, kindergartens and )] TJ ET BT 26.250 282.387 Td /F1 9.8 Tf [(other communities after the summer holidays.)] TJ ET BT 26.250 245.784 Td /F4 12.0 Tf [(Results)] TJ ET BT 26.250 225.830 Td /F1 9.8 Tf [(We examined the nucleotide sequences \(amplified from nasal swabs\) of the hemagglutinin \(HA, bases 440-828 of the coding )] TJ ET BT 26.250 213.925 Td /F1 9.8 Tf [(sequence\) and neuraminidase \(NA\) genes \(variable length\) from respectively 19 and 23 influenza A\(H1N1\)v strains isolated in )] TJ ET BT 26.250 202.020 Td /F1 9.8 Tf [(the city of Rome, in the period May-August 2009. At position 658 \(from the start codon\) of HA the frequency of T was 63% )] TJ ET BT 26.250 190.116 Td /F1 9.8 Tf [(\(12/19\), while the frequency of A \(signature of clade 7/cluster2 virus\) was 37%. These percentages were similar to those )] TJ ET BT 26.250 178.211 Td /F1 9.8 Tf [(deduced from 589 HA sequences isolated globally \(mostly in Mexico and in the United States\) before August )] TJ ET 0.267 0.267 0.267 rg BT 497.175 178.211 Td /F1 9.8 Tf [([5])] TJ ET 0.271 0.267 0.267 rg BT 508.017 178.211 Td /F1 9.8 Tf [(. Viral strains )] TJ ET BT 26.250 166.306 Td /F1 9.8 Tf [(isolated after September can be considered genuinely representative of the local evolution of the pandemic. The complete or )] TJ ET BT 26.250 154.401 Td /F1 9.8 Tf [(partial HA and NA nucleotide sequences of an additional 43 influenza A\(H1N1\)v isolates were obtained. The frequency of the )] TJ ET BT 26.250 142.497 Td /F1 9.8 Tf [(signature HA nucleotide 658 variants in those later isolates was 0% and 100% respectively for T and A, documenting the )] TJ ET BT 26.250 130.592 Td /F1 9.8 Tf [(disappearance of other clades in favour of clade 7. Among these sequences, no mutations considered to be biologically )] TJ ET BT 26.250 118.687 Td /F1 9.8 Tf [(significant were detected, including the D222G/N \(position 239 from the start codon in the H1N1 2009 pandemic virus\) in HA )] TJ ET BT 26.250 106.782 Td /F1 9.8 Tf [(and oseltamivir resistance mutations in NA, despite most patients had been subjected to treatment. Fig.1a and b shows )] TJ ET BT 26.250 94.878 Td /F1 9.8 Tf [(Neighbour-Joining phylogenetic trees \(with bootstrap test\) of HA and NA sequences \(respectively\) isolated at INMI from April to )] TJ ET BT 26.250 82.973 Td /F1 9.8 Tf [(December 2009 \(with indicated the month of collection\) and compared to representative sequences of the initial pandemic from )] TJ ET BT 26.250 71.068 Td /F1 9.8 Tf [(North America in April.)] TJ ET 0.965 0.965 0.965 rg 26.250 -179.563 555.000 240.750 re f 0.267 0.267 0.267 rg 0.267 0.267 0.267 RG 26.250 61.187 m 581.250 61.187 l 581.250 60.437 l 26.250 60.437 l f q 168.750 0 0 225.000 35.250 -173.563 cm /I3 Do Q q 35.250 -179.563 537.000 0.000 re W n Q Q q 168.750 0 0 225.000 35.250 -173.563 cm /I3 Do Q q 0.000 0.000 0.000 rg BT 291.710 19.825 Td /F1 11.0 Tf [(1)] TJ ET BT 25.000 19.825 Td /F1 11.0 Tf [(PLOS Currents Influenza)] TJ ET Q endstream endobj 8 0 obj << /Type /Font /Subtype /Type1 /Name /F1 /BaseFont /Helvetica /Encoding /WinAnsiEncoding >> endobj 9 0 obj << /Type /Font /Subtype /Type1 /Name /F2 /BaseFont /Times-Bold /Encoding /WinAnsiEncoding >> endobj 10 0 obj << /Type /Font /Subtype /Type1 /Name /F3 /BaseFont /Times-Italic /Encoding /WinAnsiEncoding >> endobj 11 0 obj << /Type /Font /Subtype /Type1 /Name /F4 /BaseFont /Helvetica-Bold /Encoding /WinAnsiEncoding >> endobj 12 0 obj << /Type /XObject /Subtype /Image /Width 500 /Height 52 /Filter /FlateDecode /DecodeParms << /Predictor 15 /Colors 1 /Columns 500 /BitsPerComponent 8>> /ColorSpace /DeviceGray /BitsPerComponent 8 /Length 144>> stream x1 0 'ݲ؎"e{dzAdzAdzAdzAdzAdzAdzAdzAdzAdzAdzAdzAtlM0\ endstream endobj 13 0 obj << /Type /XObject /Subtype /Image /Width 500 /Height 52 /SMask 12 0 R /Filter /FlateDecode /DecodeParms << /Predictor 15 /Colors 3 /Columns 500 /BitsPerComponent 8>> /ColorSpace /DeviceRGB /BitsPerComponent 8 /Length 3574>> stream x}ƕK*8+0T ֮ S L*0߇d" sl2FBiFwY0 00 0ŝa0 aqgR܋x||lu2 03UL۶BxI 0:UU0x;֝($z%șHڶ ÐVz ]IS> â(.* TQUuL5 $>BvpH0U:xkkGJT{OɗRNyGOΙyn)q7`(~_Ji2vgk]qVꔷ3N'ȴy4Ikm6!q:FJ)-0n܋v!^}_Z l61va_~Bxw8mv;EJY8}zzQ3]Pȹ,2_jfY4MQ[ '.uO O͋kշ9b6.#ߦUUzhf09+nKɿ4 f7Bu7ĨMfrF0`s8429j1}ZWDW;A4ҫ'TQ{7Cb[$ -"ΨMQgzN/2R7Tco*W:N݋6TUu8`RSi O>r;z^m(0w`:m=]գCE&t |ZYQ%{X[nR+zbEFΧ5pzq`ުం Ih{9G 4wMx' nmvEZ sg7?ZQ}&ҎKGuPu.j8UgNKꓙQC)s#$1䧈x[Z(|Xzc;t<~50Hh_T3Ur>a$]B8Fz[?K>Y/rQ=E4?EaLqzpX!(nNut]}e(/S\fz,<ZOEa]0 sCS_}I Y}n۶r(fg7#)h6ա}$ üvq]Aא$c0ddBDQ)ueypU-z~9Hk<$I©y'Tk`yY d}e{䨙ǐu>&w񚓶('GI\rm[G4 Sk7$B pEkYYC/ 9&Ʋ[V9MQ':X|C֨ʉ jt"ޡ+rttBռGdiM FuTEi(łv]fk"8Ldp' ^הMup2`M?dE!|KJ-\.MzکC=&¾HfR7$ev,La0+$OFuEQ"i,ːN?~E*t4qΚF:t`,{RkR]ޜ(Ԕѣ$IbN}5لS1(Ȱ$I$(eWp8Lٱ3߯ky۶&. 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>> endobj 100 0 obj << /Type /Action >> endobj 101 0 obj << /Type /Annot /Subtype /Link /A 102 0 R /Border [0 0 0] /H /I /Rect [ 497.1750 177.3091 508.0170 187.2297 ] >> endobj 102 0 obj << /Type /Action >> endobj 103 0 obj << /Type /Annot /Subtype /Link /A 104 0 R /Border [0 0 0] /H /I /Rect [ 35.2500 -173.5628 204.0000 51.4372 ] >> endobj 104 0 obj << /Type /Action /S /URI /URI (http://currents.plos.org/influenza/files/2010/03/fig1a.tif) >> endobj 105 0 obj << /Type /Page /Parent 3 0 R /Annots [ 107 0 R 110 0 R 112 0 R 114 0 R 116 0 R 118 0 R ] /Contents 106 0 R >> endobj 106 0 obj << /Length 18168 >> stream q 15.000 53.410 577.500 723.590 re W n 0.965 0.965 0.965 rg 26.250 687.232 555.000 89.768 re f 0.267 0.267 0.267 rg 0.267 0.267 0.267 RG 26.250 687.232 m 581.250 687.232 l 581.250 687.982 l 26.250 687.982 l f q 35.250 698.482 537.000 78.518 re W n 0.271 0.267 0.267 rg BT 35.250 767.476 Td /F4 9.8 Tf [(Fig. 1: 1a. Phylogenetic tree of partial HA sequences from this study \(in blue\), indicating the month of collection, in )] TJ ET BT 35.250 755.571 Td /F4 9.8 Tf [(comparison to sequences from North America collected in April.)] TJ ET BT 35.250 736.201 Td /F1 9.8 Tf [(Clade 7 and the HA G222E subclade are indicated. 4 additional HA G222E sequences \(collected after June\) from USA and )] TJ ET BT 35.250 722.465 Td /F1 9.8 Tf [(Sweden, clustering with sequences from Rome, are indicated by green and orange arrows, respectively. The sequences )] TJ ET BT 35.250 708.729 Td /F1 9.8 Tf [(from this study are indicated by the names deposited in GenBank, abbreviated \(Italy/xxx or Rome /xxx\).)] TJ ET Q 0.965 0.965 0.965 rg 26.250 337.814 555.000 341.918 re f 0.267 0.267 0.267 rg 0.267 0.267 0.267 RG 26.250 679.732 m 581.250 679.732 l 581.250 678.982 l 26.250 678.982 l f 26.250 337.814 m 581.250 337.814 l 581.250 338.564 l 26.250 338.564 l f q 168.750 0 0 225.000 35.250 444.982 cm /I4 Do Q q 35.250 349.064 537.000 89.918 re W n 0.271 0.267 0.267 rg BT 35.250 427.993 Td /F4 9.8 Tf [(1b. Phylogenetic tree of complete \(with a few exceptions\) NA sequences from this study \(in blue\), indicating the )] TJ ET BT 35.250 414.256 Td /F4 9.8 Tf [(month of collection, in comparison to sequences from North America collected in April.)] TJ ET BT 440.509 414.256 Td /F1 9.8 Tf [( Clade 7 and the HA G222E )] TJ ET BT 35.250 400.520 Td /F1 9.8 Tf [(subclade are indicated. NA sequences from the same HAG222E isolates as in Fig 1a from USA and Sweden are indicated )] TJ ET BT 35.250 386.784 Td /F1 9.8 Tf [(by green and orange arrows, respectively. Blue arrows indicate early clade 7 sequences from Rome clustering with non )] TJ ET BT 35.250 373.048 Td /F1 9.8 Tf [(clade 7 sequences. The sequences from this study are indicated by the names deposited in GenBank, abbreviated )] TJ ET BT 35.250 359.311 Td /F1 9.8 Tf [(\(Italy/xxx or Rome /xxx\).)] TJ ET Q BT 26.250 320.790 Td /F1 9.8 Tf [(The HA tree confirms the clade shift during the summer. To be noted that a separate subclade of clade 7 consists of 12 )] TJ ET BT 26.250 308.886 Td /F1 9.8 Tf [(sequences with the D222E substitution, which has been found at higher frequency in Italy, in Turkey and in Sweden, and whose )] TJ ET BT 26.250 296.981 Td /F1 9.8 Tf [(biological meaning is still unknown. In contrast, a phylogenetic tree comparing NA sequences \(full-length with a few exceptions\), )] TJ ET BT 26.250 285.076 Td /F1 9.8 Tf [(shows co-clustering of most clade 7 sequences with those from other clades until June, followed by progressive divergence of )] TJ ET BT 26.250 273.171 Td /F1 9.8 Tf [(the late clade 7 Italian sequences from early New York sequences. By the end of June, clade 7 sequences from Rome isolates )] TJ ET BT 26.250 261.267 Td /F1 9.8 Tf [(were clustering in three distinct subclades of clade 7. One of these was apparently the most successful as it was associated )] TJ ET BT 26.250 249.362 Td /F1 9.8 Tf [(with the big autumn wave of infections. NA sequences from viruses bearing the HA G222E cluster together \(including those )] TJ ET BT 26.250 237.457 Td /F1 9.8 Tf [(from USA and Sweden\), indicating that this mutation did not appear by converging evolution of different strains but rather may )] TJ ET BT 26.250 225.552 Td /F1 9.8 Tf [(represent a signature of an authentic subclade within clade 7 sequences.)] TJ ET BT 26.250 206.148 Td /F1 9.8 Tf [(To establish whether the significant clade shift was due to the local epidemic rather than a global phenomenon, and to identify )] TJ ET BT 26.250 194.243 Td /F1 9.8 Tf [(the time course and the local trends of this evolution in different parts of the world, the total set of HA sequences downloadable )] TJ ET BT 26.250 182.338 Td /F1 9.8 Tf [(from GISAID database \(December 2009\) was divided by country \(from countries with a reasonable number of sequences )] TJ ET BT 26.250 170.433 Td /F1 9.8 Tf [(available over a period of at least three months\) or geographical area and by month, and analyzed at the signature nucleotide )] TJ ET BT 26.250 158.529 Td /F1 9.8 Tf [(658 in the HA sequence. There are a few limits to such a database analysis: 1\) the geographical origin of the isolates does not )] TJ ET BT 26.250 146.624 Td /F1 9.8 Tf [(necessarily indicate the actual origin of the infection, rather merely the origin of the infected person, especially for early )] TJ ET BT 26.250 134.719 Td /F1 9.8 Tf [(European and Asian isolates; 2\) the collection times have been submitted with a variable degree of precision and some might )] TJ ET BT 26.250 122.814 Td /F1 9.8 Tf [(be unreliable; 3\) the majority of isolates have been sequenced in the first months of the epidemic, while very few sequences of )] TJ ET BT 26.250 110.910 Td /F1 9.8 Tf [(later isolates have been published. Despite these limits, the pattern of cluster substitution appears quite clearly everywhere. Fig. )] TJ ET BT 26.250 99.005 Td /F1 9.8 Tf [(2 shows the increasing proportion of clade 7 sequences in different countries from April to December. These patterns are )] TJ ET BT 26.250 87.100 Td /F1 9.8 Tf [(completely superimposable if other signature nucleotides )] TJ ET 0.267 0.267 0.267 rg BT 274.436 87.100 Td /F1 9.8 Tf [([4][5])] TJ ET 0.271 0.267 0.267 rg BT 296.120 87.100 Td /F1 9.8 Tf [( of clade 7/cluster2 virus are analyzed \(not shown\): NS position )] TJ ET BT 26.250 75.195 Td /F1 9.8 Tf [(367, MA positions 492 and 600, PB2 position 2163, confirming the stability of the clade and the absence of late reassortment )] TJ ET BT 26.250 63.291 Td /F1 9.8 Tf [(events for these segments.)] TJ ET Q q 15.000 53.410 577.500 723.590 re W n 0.965 0.965 0.965 rg 26.250 687.232 555.000 89.768 re f 0.267 0.267 0.267 rg 0.267 0.267 0.267 RG 26.250 687.232 m 581.250 687.232 l 581.250 687.982 l 26.250 687.982 l f q 35.250 698.482 537.000 78.518 re W n 0.271 0.267 0.267 rg BT 35.250 767.476 Td /F4 9.8 Tf [(Fig. 1: 1a. Phylogenetic tree of partial HA sequences from this study \(in blue\), indicating the month of collection, in )] TJ ET BT 35.250 755.571 Td /F4 9.8 Tf [(comparison to sequences from North America collected in April.)] TJ ET BT 35.250 736.201 Td /F1 9.8 Tf [(Clade 7 and the HA G222E subclade are indicated. 4 additional HA G222E sequences \(collected after June\) from USA and )] TJ ET BT 35.250 722.465 Td /F1 9.8 Tf [(Sweden, clustering with sequences from Rome, are indicated by green and orange arrows, respectively. The sequences )] TJ ET BT 35.250 708.729 Td /F1 9.8 Tf [(from this study are indicated by the names deposited in GenBank, abbreviated \(Italy/xxx or Rome /xxx\).)] TJ ET Q 0.965 0.965 0.965 rg 26.250 337.814 555.000 341.918 re f 0.267 0.267 0.267 rg 0.267 0.267 0.267 RG 26.250 679.732 m 581.250 679.732 l 581.250 678.982 l 26.250 678.982 l f 26.250 337.814 m 581.250 337.814 l 581.250 338.564 l 26.250 338.564 l f q 168.750 0 0 225.000 35.250 444.982 cm /I4 Do Q q 35.250 349.064 537.000 89.918 re W n 0.271 0.267 0.267 rg BT 35.250 427.993 Td /F4 9.8 Tf [(1b. Phylogenetic tree of complete \(with a few exceptions\) NA sequences from this study \(in blue\), indicating the )] TJ ET BT 35.250 414.256 Td /F4 9.8 Tf [(month of collection, in comparison to sequences from North America collected in April.)] TJ ET BT 440.509 414.256 Td /F1 9.8 Tf [( Clade 7 and the HA G222E )] TJ ET BT 35.250 400.520 Td /F1 9.8 Tf [(subclade are indicated. NA sequences from the same HAG222E isolates as in Fig 1a from USA and Sweden are indicated )] TJ ET BT 35.250 386.784 Td /F1 9.8 Tf [(by green and orange arrows, respectively. Blue arrows indicate early clade 7 sequences from Rome clustering with non )] TJ ET BT 35.250 373.048 Td /F1 9.8 Tf [(clade 7 sequences. The sequences from this study are indicated by the names deposited in GenBank, abbreviated )] TJ ET BT 35.250 359.311 Td /F1 9.8 Tf [(\(Italy/xxx or Rome /xxx\).)] TJ ET Q BT 26.250 320.790 Td /F1 9.8 Tf [(The HA tree confirms the clade shift during the summer. To be noted that a separate subclade of clade 7 consists of 12 )] TJ ET BT 26.250 308.886 Td /F1 9.8 Tf [(sequences with the D222E substitution, which has been found at higher frequency in Italy, in Turkey and in Sweden, and whose )] TJ ET BT 26.250 296.981 Td /F1 9.8 Tf [(biological meaning is still unknown. In contrast, a phylogenetic tree comparing NA sequences \(full-length with a few exceptions\), )] TJ ET BT 26.250 285.076 Td /F1 9.8 Tf [(shows co-clustering of most clade 7 sequences with those from other clades until June, followed by progressive divergence of )] TJ ET BT 26.250 273.171 Td /F1 9.8 Tf [(the late clade 7 Italian sequences from early New York sequences. By the end of June, clade 7 sequences from Rome isolates )] TJ ET BT 26.250 261.267 Td /F1 9.8 Tf [(were clustering in three distinct subclades of clade 7. One of these was apparently the most successful as it was associated )] TJ ET BT 26.250 249.362 Td /F1 9.8 Tf [(with the big autumn wave of infections. NA sequences from viruses bearing the HA G222E cluster together \(including those )] TJ ET BT 26.250 237.457 Td /F1 9.8 Tf [(from USA and Sweden\), indicating that this mutation did not appear by converging evolution of different strains but rather may )] TJ ET BT 26.250 225.552 Td /F1 9.8 Tf [(represent a signature of an authentic subclade within clade 7 sequences.)] TJ ET BT 26.250 206.148 Td /F1 9.8 Tf [(To establish whether the significant clade shift was due to the local epidemic rather than a global phenomenon, and to identify )] TJ ET BT 26.250 194.243 Td /F1 9.8 Tf [(the time course and the local trends of this evolution in different parts of the world, the total set of HA sequences downloadable )] TJ ET BT 26.250 182.338 Td /F1 9.8 Tf [(from GISAID database \(December 2009\) was divided by country \(from countries with a reasonable number of sequences )] TJ ET BT 26.250 170.433 Td /F1 9.8 Tf [(available over a period of at least three months\) or geographical area and by month, and analyzed at the signature nucleotide )] TJ ET BT 26.250 158.529 Td /F1 9.8 Tf [(658 in the HA sequence. There are a few limits to such a database analysis: 1\) the geographical origin of the isolates does not )] TJ ET BT 26.250 146.624 Td /F1 9.8 Tf [(necessarily indicate the actual origin of the infection, rather merely the origin of the infected person, especially for early )] TJ ET BT 26.250 134.719 Td /F1 9.8 Tf [(European and Asian isolates; 2\) the collection times have been submitted with a variable degree of precision and some might )] TJ ET BT 26.250 122.814 Td /F1 9.8 Tf [(be unreliable; 3\) the majority of isolates have been sequenced in the first months of the epidemic, while very few sequences of )] TJ ET BT 26.250 110.910 Td /F1 9.8 Tf [(later isolates have been published. Despite these limits, the pattern of cluster substitution appears quite clearly everywhere. Fig. )] TJ ET BT 26.250 99.005 Td /F1 9.8 Tf [(2 shows the increasing proportion of clade 7 sequences in different countries from April to December. These patterns are )] TJ ET BT 26.250 87.100 Td /F1 9.8 Tf [(completely superimposable if other signature nucleotides )] TJ ET 0.267 0.267 0.267 rg BT 274.436 87.100 Td /F1 9.8 Tf [([4][5])] TJ ET 0.271 0.267 0.267 rg BT 296.120 87.100 Td /F1 9.8 Tf [( of clade 7/cluster2 virus are analyzed \(not shown\): NS position )] TJ ET BT 26.250 75.195 Td /F1 9.8 Tf [(367, MA positions 492 and 600, PB2 position 2163, confirming the stability of the clade and the absence of late reassortment )] TJ ET BT 26.250 63.291 Td /F1 9.8 Tf [(events for these segments.)] TJ ET Q q 15.000 53.410 577.500 723.590 re W n 0.965 0.965 0.965 rg 26.250 687.232 555.000 89.768 re f 0.267 0.267 0.267 rg 0.267 0.267 0.267 RG 26.250 687.232 m 581.250 687.232 l 581.250 687.982 l 26.250 687.982 l f q 35.250 698.482 537.000 78.518 re W n 0.271 0.267 0.267 rg BT 35.250 767.476 Td /F4 9.8 Tf [(Fig. 1: 1a. Phylogenetic tree of partial HA sequences from this study \(in blue\), indicating the month of collection, in )] TJ ET BT 35.250 755.571 Td /F4 9.8 Tf [(comparison to sequences from North America collected in April.)] TJ ET BT 35.250 736.201 Td /F1 9.8 Tf [(Clade 7 and the HA G222E subclade are indicated. 4 additional HA G222E sequences \(collected after June\) from USA and )] TJ ET BT 35.250 722.465 Td /F1 9.8 Tf [(Sweden, clustering with sequences from Rome, are indicated by green and orange arrows, respectively. The sequences )] TJ ET BT 35.250 708.729 Td /F1 9.8 Tf [(from this study are indicated by the names deposited in GenBank, abbreviated \(Italy/xxx or Rome /xxx\).)] TJ ET Q 0.965 0.965 0.965 rg 26.250 337.814 555.000 341.918 re f 0.267 0.267 0.267 rg 0.267 0.267 0.267 RG 26.250 679.732 m 581.250 679.732 l 581.250 678.982 l 26.250 678.982 l f 26.250 337.814 m 581.250 337.814 l 581.250 338.564 l 26.250 338.564 l f q 168.750 0 0 225.000 35.250 444.982 cm /I4 Do Q q 35.250 349.064 537.000 89.918 re W n 0.271 0.267 0.267 rg BT 35.250 427.993 Td /F4 9.8 Tf [(1b. Phylogenetic tree of complete \(with a few exceptions\) NA sequences from this study \(in blue\), indicating the )] TJ ET BT 35.250 414.256 Td /F4 9.8 Tf [(month of collection, in comparison to sequences from North America collected in April.)] TJ ET BT 440.509 414.256 Td /F1 9.8 Tf [( Clade 7 and the HA G222E )] TJ ET BT 35.250 400.520 Td /F1 9.8 Tf [(subclade are indicated. NA sequences from the same HAG222E isolates as in Fig 1a from USA and Sweden are indicated )] TJ ET BT 35.250 386.784 Td /F1 9.8 Tf [(by green and orange arrows, respectively. Blue arrows indicate early clade 7 sequences from Rome clustering with non )] TJ ET BT 35.250 373.048 Td /F1 9.8 Tf [(clade 7 sequences. The sequences from this study are indicated by the names deposited in GenBank, abbreviated )] TJ ET BT 35.250 359.311 Td /F1 9.8 Tf [(\(Italy/xxx or Rome /xxx\).)] TJ ET Q BT 26.250 320.790 Td /F1 9.8 Tf [(The HA tree confirms the clade shift during the summer. To be noted that a separate subclade of clade 7 consists of 12 )] TJ ET BT 26.250 308.886 Td /F1 9.8 Tf [(sequences with the D222E substitution, which has been found at higher frequency in Italy, in Turkey and in Sweden, and whose )] TJ ET BT 26.250 296.981 Td /F1 9.8 Tf [(biological meaning is still unknown. In contrast, a phylogenetic tree comparing NA sequences \(full-length with a few exceptions\), )] TJ ET BT 26.250 285.076 Td /F1 9.8 Tf [(shows co-clustering of most clade 7 sequences with those from other clades until June, followed by progressive divergence of )] TJ ET BT 26.250 273.171 Td /F1 9.8 Tf [(the late clade 7 Italian sequences from early New York sequences. By the end of June, clade 7 sequences from Rome isolates )] TJ ET BT 26.250 261.267 Td /F1 9.8 Tf [(were clustering in three distinct subclades of clade 7. One of these was apparently the most successful as it was associated )] TJ ET BT 26.250 249.362 Td /F1 9.8 Tf [(with the big autumn wave of infections. NA sequences from viruses bearing the HA G222E cluster together \(including those )] TJ ET BT 26.250 237.457 Td /F1 9.8 Tf [(from USA and Sweden\), indicating that this mutation did not appear by converging evolution of different strains but rather may )] TJ ET BT 26.250 225.552 Td /F1 9.8 Tf [(represent a signature of an authentic subclade within clade 7 sequences.)] TJ ET BT 26.250 206.148 Td /F1 9.8 Tf [(To establish whether the significant clade shift was due to the local epidemic rather than a global phenomenon, and to identify )] TJ ET BT 26.250 194.243 Td /F1 9.8 Tf [(the time course and the local trends of this evolution in different parts of the world, the total set of HA sequences downloadable )] TJ ET BT 26.250 182.338 Td /F1 9.8 Tf [(from GISAID database \(December 2009\) was divided by country \(from countries with a reasonable number of sequences )] TJ ET BT 26.250 170.433 Td /F1 9.8 Tf [(available over a period of at least three months\) or geographical area and by month, and analyzed at the signature nucleotide )] TJ ET BT 26.250 158.529 Td /F1 9.8 Tf [(658 in the HA sequence. There are a few limits to such a database analysis: 1\) the geographical origin of the isolates does not )] TJ ET BT 26.250 146.624 Td /F1 9.8 Tf [(necessarily indicate the actual origin of the infection, rather merely the origin of the infected person, especially for early )] TJ ET BT 26.250 134.719 Td /F1 9.8 Tf [(European and Asian isolates; 2\) the collection times have been submitted with a variable degree of precision and some might )] TJ ET BT 26.250 122.814 Td /F1 9.8 Tf [(be unreliable; 3\) the majority of isolates have been sequenced in the first months of the epidemic, while very few sequences of )] TJ ET BT 26.250 110.910 Td /F1 9.8 Tf [(later isolates have been published. Despite these limits, the pattern of cluster substitution appears quite clearly everywhere. Fig. )] TJ ET BT 26.250 99.005 Td /F1 9.8 Tf [(2 shows the increasing proportion of clade 7 sequences in different countries from April to December. These patterns are )] TJ ET BT 26.250 87.100 Td /F1 9.8 Tf [(completely superimposable if other signature nucleotides )] TJ ET 0.267 0.267 0.267 rg BT 274.436 87.100 Td /F1 9.8 Tf [([4][5])] TJ ET 0.271 0.267 0.267 rg BT 296.120 87.100 Td /F1 9.8 Tf [( of clade 7/cluster2 virus are analyzed \(not shown\): NS position )] TJ ET BT 26.250 75.195 Td /F1 9.8 Tf [(367, MA positions 492 and 600, PB2 position 2163, confirming the stability of the clade and the absence of late reassortment )] TJ ET BT 26.250 63.291 Td /F1 9.8 Tf [(events for these segments.)] TJ ET Q q 168.750 0 0 225.000 35.250 444.982 cm /I4 Do Q q 0.000 0.000 0.000 rg BT 291.710 19.825 Td /F1 11.0 Tf [(2)] TJ ET BT 25.000 19.825 Td /F1 11.0 Tf [(PLOS Currents Influenza)] TJ ET Q endstream endobj 107 0 obj << /Type /Annot /Subtype /Link /A 108 0 R /Border [0 0 0] /H /I /Rect [ 35.2500 444.9817 204.0000 669.9817 ] >> endobj 108 0 obj << /Type /Action /S /URI /URI (http://currents.plos.org/influenza/files/2010/03/fig1b2.tif) >> endobj 109 0 obj << /Type /XObject /Subtype /Image /Width 225 /Height 300 /ColorSpace /DeviceRGB /Filter /DCTDecode /BitsPerComponent 8 /Length 14895>> stream JFIF;CREATOR: gd-jpeg v1.0 (using IJG JPEG v62), quality = 90 C     C   ," }!1AQa"q2#BR$3br %&'()*456789:CDEFGHIJSTUVWXYZcdefghijstuvwxyz w!1AQaq"2B #3Rbr $4%&'()*56789:CDEFGHIJSTUVWXYZcdefghijstuvwxyz ?Q(( Q? _€b€ Q? 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XƩk9IIgXymB2 Bo;TpylmycWh_! W?_ݸpoCfPXXrܚ@7n@)+!Ea[7 ]aaVa}V`ڌu=@g\r V-㵂8bE(""  (((Z()(/ImoxV/[|n0Km`jcF~fn"9gؖW'G ٕ'zާxVg'7" &"[v6$kc+ыr垓[꛳vDw O:>;"x n!2@ϸ/k[xiD:[Ѷ3Ej? 1q 4#%ҵvK4QGG\eEPGG@QEQEQEQE-Q@ GEbxB%-dL=M.KYn4&HIc*^e=;f?!_A?ޓTαuM+[k {ހ8CWa:x|Z}޽/u؏)o)e-q`t?xmSL׋XZRɊ4 NW |OQT Vz]ҦL>PXͼSyٷ lIN9$tRҴw}jwv[ΑK9$H˷ `t:nyaKЮ|]|6!_$!c; V x@!xN_ A7GLu xAӬ &glIJt 8I{c ^lxԼ_uC$1+c9_Dm xuQ/VTԮC#;p=jtcx$X q=818xØPiG'KҝzԤNm5Mg5} m|%OFdG,#'f_K熩5G'Tq4 VuUKD'%?TMxj)Tq_G&# wMqm+컍ER f1?/ ns3 |}z:\wiPYW7d(c?᷌4K cHmޛ|9dY,+r^ FM9Kދ^[K,Ŷ0ȡ1P"7 GQU$`9]?΋}&+/#DKt1%s"#X=$V_&/ xwRAoe{ncR(3JJqCqkD!OƖQ@Q@Q@Q@ EPRR@ KoI_Tãjֺqa ;Gqd򲰉#ᄫ#zoV4)Eԑ8FQO\1^=߉/XZϬu{L0YcDE,$"7:sz Ky&fڅ>W'@X]aE\x[RRjL7aɏ#9#+@}ޯ[(/2i n q !x![7^ۤK`BztDY-jyo pĬaiݐrI5Kt7y@-d&hB#뜀$#ʿ< Ht9&&D,vmi%$ HϤ:_/Kx%tzV(*Z7PWyJkӴ=GXm쵟x& W Нe#4\ y/[ñ&X8m&her3IV]&¶tѽY5U[Gm/1ؿѦMC^㑢Hb! 2\ôcH\bi ڄ~-"͜ů)jqNќ`U g'#+K\ğ%K+pǫF8`9'T*&@pϗ˓wLn[G@/r=VœGMm5t(=ԒA.̲028ʆ4/[iQ\Z\ )Vaj@H5û?f [ cq;Iy8/@[oI~˸)c9Oֿ K? _&&K@4*IcdZީW+hcg `}^a6K ԯQQfdʨ&N22vi+eZA0DHPt|S4Lj I}\(Y. IeD6H?\ c&9^;wgn|9OGEx nC8ո> uƩhci>_ x23EgP\PC l;} y z^iBiw A]1|3MYf(diwαc$Z%l֊xj)t_a<5O6O? i]Awtˍ|(1*|bɷY`q&qPUUTǝņ6}EvWGHiM+ZuB?(8  A8 p |Y|i +MFrKbaaps0I[9s SËN-od_F "#mm2>rq^ӨǦ7hiX/$8Zm恩I>$eh Leq@d8N|#ԣ{}5fXKhUg!M ÜV_<ؠ4B"줊%\`lxFҲn>'.;fH>|Kh9`Uo0qF: x~<,Dym%Չ.˅ޠLD<OjjQ]1x%+gV)5 @U(J^wKހ +_Xg!xg p `p>YՖvK0, w }(X"{lQ-aʒ0p~K|#knRޤUWwViFCF`#g9oNjx^kJ\ X7 B\W@!Zve/W6?Kγ#DeNaPP9%u_xvMΝo.O]C2Goo¶K_[?m 3JLTTlr#5/'UQV#> endobj 111 0 obj << /Type /Action >> endobj 112 0 obj << /Type /Annot /Subtype /Link /A 113 0 R /Border [0 0 0] /H /I /Rect [ 35.2500 444.9817 204.0000 669.9817 ] >> endobj 113 0 obj << /Type /Action /S /URI /URI (http://currents.plos.org/influenza/files/2010/03/fig1b2.tif) >> endobj 114 0 obj << /Type /Annot /Subtype /Link /A 115 0 R /Border [0 0 0] /H /I /Rect [ 274.4362 86.1983 296.1202 96.1189 ] >> endobj 115 0 obj << /Type /Action >> endobj 116 0 obj << /Type /Annot /Subtype /Link /A 117 0 R /Border [0 0 0] /H /I /Rect [ 35.2500 444.9817 204.0000 669.9817 ] >> endobj 117 0 obj << /Type /Action /S /URI /URI (http://currents.plos.org/influenza/files/2010/03/fig1b2.tif) >> endobj 118 0 obj << /Type /Annot /Subtype /Link /A 119 0 R /Border [0 0 0] /H /I /Rect [ 274.4362 86.1983 296.1202 96.1189 ] >> endobj 119 0 obj << /Type /Action >> endobj 120 0 obj << /Type /Page /Parent 3 0 R /Annots [ 122 0 R 125 0 R 127 0 R 129 0 R 131 0 R 134 0 R 136 0 R 138 0 R 140 0 R 142 0 R 144 0 R 146 0 R 148 0 R 150 0 R 152 0 R ] /Contents 121 0 R >> endobj 121 0 obj << /Length 17840 >> stream 0.271 0.267 0.267 rg q 15.000 59.567 577.500 717.433 re W n 0.965 0.965 0.965 rg 26.250 598.359 555.000 178.641 re f 0.267 0.267 0.267 rg 0.267 0.267 0.267 RG 26.250 777.000 m 581.250 777.000 l 581.250 776.250 l 26.250 776.250 l f 26.250 598.359 m 581.250 598.359 l 581.250 599.109 l 26.250 599.109 l f q 112.500 0 0 112.500 35.250 654.750 cm /I5 Do Q q 35.250 609.609 537.000 39.141 re W n 0.271 0.267 0.267 rg BT 35.250 639.226 Td /F4 9.8 Tf [(Fig. 2: The increasing proportion of clade 7 sequences in different countries from April to December.)] TJ ET BT 35.250 619.856 Td /F1 9.8 Tf [(Proportion \(%\) of clade 7 sequences \(HA\) for each month in different countries or geographical areas.)] TJ ET Q BT 26.250 581.335 Td /F1 9.8 Tf [(The time frame of this phenomenon was different depending on the country. In the Southern hemisphere \(Oceania and South )] TJ ET BT 26.250 569.430 Td /F1 9.8 Tf [(America\) clade 7 was already predominant by the end of April, reflecting perhaps the faster spread of the epidemic in the )] TJ ET BT 26.250 557.526 Td /F1 9.8 Tf [(Southern \(winter\) part of the globe. In Singapore, China and Scandinavian countries the shift occurred a few weeks later, while )] TJ ET BT 26.250 545.621 Td /F1 9.8 Tf [(in the majority of northern hemisphere countries it occurred mostly between June and July. The apparent fall of clade 7 )] TJ ET BT 26.250 533.716 Td /F1 9.8 Tf [(sequences from April to May 2008 in the USA, UK and Canada, might be considered an artefact due to the overrepresentation )] TJ ET BT 26.250 521.811 Td /F1 9.8 Tf [(of sequences from the New York clade 7 outbreak in April, which ignited the boom of scientific and public interest. The )] TJ ET BT 26.250 509.907 Td /F1 9.8 Tf [(somewhat erratic behaviour of South American sequences \(mostly from Brazil, Chile and Argentina\) can be attributed to the fact )] TJ ET BT 26.250 498.002 Td /F1 9.8 Tf [(that the aggregation of data from different countries with such a huge North-South extension and with different timing in the )] TJ ET BT 26.250 486.097 Td /F1 9.8 Tf [(epidemic peaks \(necessary because of the low number of available sequences\) might not be reliably representative of the )] TJ ET BT 26.250 474.192 Td /F1 9.8 Tf [(whole area epidemic. The selection of clade 7 virus in Italian isolates \(data from our lab, n= 66, aggregated to those published )] TJ ET BT 26.250 462.288 Td /F1 9.8 Tf [(by other labs, n= 133 \) appears slightly anticipated compared to other Northern Hemisphere countries such as Mexico, USA, )] TJ ET BT 26.250 450.383 Td /F1 9.8 Tf [(Japan, Spain. In USA and in Japan a few sequences other than clade 7 were collected apparently as late as October/November.)] TJ ET BT 26.250 430.978 Td /F1 9.8 Tf [(While the dynamics of the epidemics suggests a possible selective advantage of clade 7 virus over other early clades, the )] TJ ET BT 26.250 419.073 Td /F1 9.8 Tf [(evolutionary trends within clade 7 need further investigation. One interesting analysis consists in the quantification of the )] TJ ET BT 26.250 407.169 Td /F1 9.8 Tf [(selective pressure acting on the single genetic segments of the virus. For this purpose, 2 groups of clade 7 sequences \(for each )] TJ ET BT 26.250 395.264 Td /F1 9.8 Tf [(genomic segment\) were selected, respectively collected in May \(mostly from the initial New York outbreak\) and in November )] TJ ET BT 26.250 383.359 Td /F1 9.8 Tf [(\(from all parts of the world, after the October peak\). May sequences were randomly selected to match the much smaller number )] TJ ET BT 26.250 371.454 Td /F1 9.8 Tf [(of November sequences. The selective pressure acting on each segment was computed as the ratio between the rate of non )] TJ ET BT 26.250 359.550 Td /F1 9.8 Tf [(synonymous substitutions per non synonymous site and the rate of synonymous substitutions per synonymous sites \(Ka/Ks\) )] TJ ET BT 26.250 347.645 Td /F1 9.8 Tf [(using the Nei and Gojobori substitution model \()] TJ ET 0.267 0.267 0.267 rg BT 228.923 347.645 Td /F1 9.8 Tf [([6])] TJ ET 0.271 0.267 0.267 rg BT 239.765 347.645 Td /F1 9.8 Tf [( with the Jukes-Cantor correction\) implemented in the MEGA 4.0 package )] TJ ET 0.267 0.267 0.267 rg BT 560.560 347.645 Td /F1 9.8 Tf [([7])] TJ ET 0.271 0.267 0.267 rg BT 571.402 347.645 Td /F1 9.8 Tf [(. )] TJ ET BT 26.250 335.740 Td /F1 9.8 Tf [(The Ka/Ks values suggest that a strong purifying selection was active on all segments, in agreement with previous findings for )] TJ ET BT 26.250 323.835 Td /F1 9.8 Tf [(this and other influenza viruses )] TJ ET 0.267 0.267 0.267 rg BT 163.355 323.835 Td /F1 9.8 Tf [([1][8])] TJ ET 0.271 0.267 0.267 rg BT 185.038 323.835 Td /F1 9.8 Tf [(. In particular, purifying selection was extreme \(<0.1\) on NP, MP, PA and PB1, moderate )] TJ ET BT 26.250 311.931 Td /F1 9.8 Tf [(\(>0.2\) on NS and HA. To identify if this evolutionary pattern changed during the course of the pandemic, and to identify the )] TJ ET BT 26.250 300.026 Td /F1 9.8 Tf [(period of the maximum positive selective pressure on the virus, the ratio of average pairwise Ka and Ks was computed )] TJ ET BT 26.250 288.121 Td /F1 9.8 Tf [(separately within the May and the November groups of sequences \(again for each segment\), while the evolutionary step during )] TJ ET BT 26.250 276.216 Td /F1 9.8 Tf [(the period May-November \(that encompassed the greatest number of infections for most countries\) was analyzed as the ratio of )] TJ ET BT 26.250 264.312 Td /F1 9.8 Tf [(average pairwise Ka and Ks between each May and each November sequence \(Fig. 3a\).)] TJ ET 0.965 0.965 0.965 rg 26.250 59.567 555.000 194.864 re f 0.267 0.267 0.267 rg 0.267 0.267 0.267 RG 26.250 254.431 m 581.250 254.431 l 581.250 253.681 l 26.250 253.681 l f q 112.500 0 0 112.500 35.250 132.181 cm /I6 Do Q q 35.250 59.567 537.000 66.614 re W n Q Q q 15.000 59.567 577.500 717.433 re W n 0.965 0.965 0.965 rg 26.250 598.359 555.000 178.641 re f 0.267 0.267 0.267 rg 0.267 0.267 0.267 RG 26.250 777.000 m 581.250 777.000 l 581.250 776.250 l 26.250 776.250 l f 26.250 598.359 m 581.250 598.359 l 581.250 599.109 l 26.250 599.109 l f q 112.500 0 0 112.500 35.250 654.750 cm /I5 Do Q q 35.250 609.609 537.000 39.141 re W n 0.271 0.267 0.267 rg BT 35.250 639.226 Td /F4 9.8 Tf [(Fig. 2: The increasing proportion of clade 7 sequences in different countries from April to December.)] TJ ET BT 35.250 619.856 Td /F1 9.8 Tf [(Proportion \(%\) of clade 7 sequences \(HA\) for each month in different countries or geographical areas.)] TJ ET Q BT 26.250 581.335 Td /F1 9.8 Tf [(The time frame of this phenomenon was different depending on the country. In the Southern hemisphere \(Oceania and South )] TJ ET BT 26.250 569.430 Td /F1 9.8 Tf [(America\) clade 7 was already predominant by the end of April, reflecting perhaps the faster spread of the epidemic in the )] TJ ET BT 26.250 557.526 Td /F1 9.8 Tf [(Southern \(winter\) part of the globe. In Singapore, China and Scandinavian countries the shift occurred a few weeks later, while )] TJ ET BT 26.250 545.621 Td /F1 9.8 Tf [(in the majority of northern hemisphere countries it occurred mostly between June and July. The apparent fall of clade 7 )] TJ ET BT 26.250 533.716 Td /F1 9.8 Tf [(sequences from April to May 2008 in the USA, UK and Canada, might be considered an artefact due to the overrepresentation )] TJ ET BT 26.250 521.811 Td /F1 9.8 Tf [(of sequences from the New York clade 7 outbreak in April, which ignited the boom of scientific and public interest. The )] TJ ET BT 26.250 509.907 Td /F1 9.8 Tf [(somewhat erratic behaviour of South American sequences \(mostly from Brazil, Chile and Argentina\) can be attributed to the fact )] TJ ET BT 26.250 498.002 Td /F1 9.8 Tf [(that the aggregation of data from different countries with such a huge North-South extension and with different timing in the )] TJ ET BT 26.250 486.097 Td /F1 9.8 Tf [(epidemic peaks \(necessary because of the low number of available sequences\) might not be reliably representative of the )] TJ ET BT 26.250 474.192 Td /F1 9.8 Tf [(whole area epidemic. The selection of clade 7 virus in Italian isolates \(data from our lab, n= 66, aggregated to those published )] TJ ET BT 26.250 462.288 Td /F1 9.8 Tf [(by other labs, n= 133 \) appears slightly anticipated compared to other Northern Hemisphere countries such as Mexico, USA, )] TJ ET BT 26.250 450.383 Td /F1 9.8 Tf [(Japan, Spain. In USA and in Japan a few sequences other than clade 7 were collected apparently as late as October/November.)] TJ ET BT 26.250 430.978 Td /F1 9.8 Tf [(While the dynamics of the epidemics suggests a possible selective advantage of clade 7 virus over other early clades, the )] TJ ET BT 26.250 419.073 Td /F1 9.8 Tf [(evolutionary trends within clade 7 need further investigation. One interesting analysis consists in the quantification of the )] TJ ET BT 26.250 407.169 Td /F1 9.8 Tf [(selective pressure acting on the single genetic segments of the virus. For this purpose, 2 groups of clade 7 sequences \(for each )] TJ ET BT 26.250 395.264 Td /F1 9.8 Tf [(genomic segment\) were selected, respectively collected in May \(mostly from the initial New York outbreak\) and in November )] TJ ET BT 26.250 383.359 Td /F1 9.8 Tf [(\(from all parts of the world, after the October peak\). May sequences were randomly selected to match the much smaller number )] TJ ET BT 26.250 371.454 Td /F1 9.8 Tf [(of November sequences. The selective pressure acting on each segment was computed as the ratio between the rate of non )] TJ ET BT 26.250 359.550 Td /F1 9.8 Tf [(synonymous substitutions per non synonymous site and the rate of synonymous substitutions per synonymous sites \(Ka/Ks\) )] TJ ET BT 26.250 347.645 Td /F1 9.8 Tf [(using the Nei and Gojobori substitution model \()] TJ ET 0.267 0.267 0.267 rg BT 228.923 347.645 Td /F1 9.8 Tf [([6])] TJ ET 0.271 0.267 0.267 rg BT 239.765 347.645 Td /F1 9.8 Tf [( with the Jukes-Cantor correction\) implemented in the MEGA 4.0 package )] TJ ET 0.267 0.267 0.267 rg BT 560.560 347.645 Td /F1 9.8 Tf [([7])] TJ ET 0.271 0.267 0.267 rg BT 571.402 347.645 Td /F1 9.8 Tf [(. )] TJ ET BT 26.250 335.740 Td /F1 9.8 Tf [(The Ka/Ks values suggest that a strong purifying selection was active on all segments, in agreement with previous findings for )] TJ ET BT 26.250 323.835 Td /F1 9.8 Tf [(this and other influenza viruses )] TJ ET 0.267 0.267 0.267 rg BT 163.355 323.835 Td /F1 9.8 Tf [([1][8])] TJ ET 0.271 0.267 0.267 rg BT 185.038 323.835 Td /F1 9.8 Tf [(. In particular, purifying selection was extreme \(<0.1\) on NP, MP, PA and PB1, moderate )] TJ ET BT 26.250 311.931 Td /F1 9.8 Tf [(\(>0.2\) on NS and HA. To identify if this evolutionary pattern changed during the course of the pandemic, and to identify the )] TJ ET BT 26.250 300.026 Td /F1 9.8 Tf [(period of the maximum positive selective pressure on the virus, the ratio of average pairwise Ka and Ks was computed )] TJ ET BT 26.250 288.121 Td /F1 9.8 Tf [(separately within the May and the November groups of sequences \(again for each segment\), while the evolutionary step during )] TJ ET BT 26.250 276.216 Td /F1 9.8 Tf [(the period May-November \(that encompassed the greatest number of infections for most countries\) was analyzed as the ratio of )] TJ ET BT 26.250 264.312 Td /F1 9.8 Tf [(average pairwise Ka and Ks between each May and each November sequence \(Fig. 3a\).)] TJ ET 0.965 0.965 0.965 rg 26.250 59.567 555.000 194.864 re f 0.267 0.267 0.267 rg 0.267 0.267 0.267 RG 26.250 254.431 m 581.250 254.431 l 581.250 253.681 l 26.250 253.681 l f q 112.500 0 0 112.500 35.250 132.181 cm /I6 Do Q q 35.250 59.567 537.000 66.614 re W n Q Q q 15.000 59.567 577.500 717.433 re W n 0.965 0.965 0.965 rg 26.250 598.359 555.000 178.641 re f 0.267 0.267 0.267 rg 0.267 0.267 0.267 RG 26.250 777.000 m 581.250 777.000 l 581.250 776.250 l 26.250 776.250 l f 26.250 598.359 m 581.250 598.359 l 581.250 599.109 l 26.250 599.109 l f q 112.500 0 0 112.500 35.250 654.750 cm /I5 Do Q q 35.250 609.609 537.000 39.141 re W n 0.271 0.267 0.267 rg BT 35.250 639.226 Td /F4 9.8 Tf [(Fig. 2: The increasing proportion of clade 7 sequences in different countries from April to December.)] TJ ET BT 35.250 619.856 Td /F1 9.8 Tf [(Proportion \(%\) of clade 7 sequences \(HA\) for each month in different countries or geographical areas.)] TJ ET Q BT 26.250 581.335 Td /F1 9.8 Tf [(The time frame of this phenomenon was different depending on the country. In the Southern hemisphere \(Oceania and South )] TJ ET BT 26.250 569.430 Td /F1 9.8 Tf [(America\) clade 7 was already predominant by the end of April, reflecting perhaps the faster spread of the epidemic in the )] TJ ET BT 26.250 557.526 Td /F1 9.8 Tf [(Southern \(winter\) part of the globe. In Singapore, China and Scandinavian countries the shift occurred a few weeks later, while )] TJ ET BT 26.250 545.621 Td /F1 9.8 Tf [(in the majority of northern hemisphere countries it occurred mostly between June and July. The apparent fall of clade 7 )] TJ ET BT 26.250 533.716 Td /F1 9.8 Tf [(sequences from April to May 2008 in the USA, UK and Canada, might be considered an artefact due to the overrepresentation )] TJ ET BT 26.250 521.811 Td /F1 9.8 Tf [(of sequences from the New York clade 7 outbreak in April, which ignited the boom of scientific and public interest. The )] TJ ET BT 26.250 509.907 Td /F1 9.8 Tf [(somewhat erratic behaviour of South American sequences \(mostly from Brazil, Chile and Argentina\) can be attributed to the fact )] TJ ET BT 26.250 498.002 Td /F1 9.8 Tf [(that the aggregation of data from different countries with such a huge North-South extension and with different timing in the )] TJ ET BT 26.250 486.097 Td /F1 9.8 Tf [(epidemic peaks \(necessary because of the low number of available sequences\) might not be reliably representative of the )] TJ ET BT 26.250 474.192 Td /F1 9.8 Tf [(whole area epidemic. The selection of clade 7 virus in Italian isolates \(data from our lab, n= 66, aggregated to those published )] TJ ET BT 26.250 462.288 Td /F1 9.8 Tf [(by other labs, n= 133 \) appears slightly anticipated compared to other Northern Hemisphere countries such as Mexico, USA, )] TJ ET BT 26.250 450.383 Td /F1 9.8 Tf [(Japan, Spain. In USA and in Japan a few sequences other than clade 7 were collected apparently as late as October/November.)] TJ ET BT 26.250 430.978 Td /F1 9.8 Tf [(While the dynamics of the epidemics suggests a possible selective advantage of clade 7 virus over other early clades, the )] TJ ET BT 26.250 419.073 Td /F1 9.8 Tf [(evolutionary trends within clade 7 need further investigation. One interesting analysis consists in the quantification of the )] TJ ET BT 26.250 407.169 Td /F1 9.8 Tf [(selective pressure acting on the single genetic segments of the virus. For this purpose, 2 groups of clade 7 sequences \(for each )] TJ ET BT 26.250 395.264 Td /F1 9.8 Tf [(genomic segment\) were selected, respectively collected in May \(mostly from the initial New York outbreak\) and in November )] TJ ET BT 26.250 383.359 Td /F1 9.8 Tf [(\(from all parts of the world, after the October peak\). May sequences were randomly selected to match the much smaller number )] TJ ET BT 26.250 371.454 Td /F1 9.8 Tf [(of November sequences. The selective pressure acting on each segment was computed as the ratio between the rate of non )] TJ ET BT 26.250 359.550 Td /F1 9.8 Tf [(synonymous substitutions per non synonymous site and the rate of synonymous substitutions per synonymous sites \(Ka/Ks\) )] TJ ET BT 26.250 347.645 Td /F1 9.8 Tf [(using the Nei and Gojobori substitution model \()] TJ ET 0.267 0.267 0.267 rg BT 228.923 347.645 Td /F1 9.8 Tf [([6])] TJ ET 0.271 0.267 0.267 rg BT 239.765 347.645 Td /F1 9.8 Tf [( with the Jukes-Cantor correction\) implemented in the MEGA 4.0 package )] TJ ET 0.267 0.267 0.267 rg BT 560.560 347.645 Td /F1 9.8 Tf [([7])] TJ ET 0.271 0.267 0.267 rg BT 571.402 347.645 Td /F1 9.8 Tf [(. )] TJ ET BT 26.250 335.740 Td /F1 9.8 Tf [(The Ka/Ks values suggest that a strong purifying selection was active on all segments, in agreement with previous findings for )] TJ ET BT 26.250 323.835 Td /F1 9.8 Tf [(this and other influenza viruses )] TJ ET 0.267 0.267 0.267 rg BT 163.355 323.835 Td /F1 9.8 Tf [([1][8])] TJ ET 0.271 0.267 0.267 rg BT 185.038 323.835 Td /F1 9.8 Tf [(. In particular, purifying selection was extreme \(<0.1\) on NP, MP, PA and PB1, moderate )] TJ ET BT 26.250 311.931 Td /F1 9.8 Tf [(\(>0.2\) on NS and HA. To identify if this evolutionary pattern changed during the course of the pandemic, and to identify the )] TJ ET BT 26.250 300.026 Td /F1 9.8 Tf [(period of the maximum positive selective pressure on the virus, the ratio of average pairwise Ka and Ks was computed )] TJ ET BT 26.250 288.121 Td /F1 9.8 Tf [(separately within the May and the November groups of sequences \(again for each segment\), while the evolutionary step during )] TJ ET BT 26.250 276.216 Td /F1 9.8 Tf [(the period May-November \(that encompassed the greatest number of infections for most countries\) was analyzed as the ratio of )] TJ ET BT 26.250 264.312 Td /F1 9.8 Tf [(average pairwise Ka and Ks between each May and each November sequence \(Fig. 3a\).)] TJ ET 0.965 0.965 0.965 rg 26.250 59.567 555.000 194.864 re f 0.267 0.267 0.267 rg 0.267 0.267 0.267 RG 26.250 254.431 m 581.250 254.431 l 581.250 253.681 l 26.250 253.681 l f q 112.500 0 0 112.500 35.250 132.181 cm /I6 Do Q q 35.250 59.567 537.000 66.614 re W n Q Q q 112.500 0 0 112.500 35.250 654.750 cm /I5 Do Q q 112.500 0 0 112.500 35.250 132.181 cm /I6 Do Q q 0.000 0.000 0.000 rg BT 291.710 19.825 Td /F1 11.0 Tf [(3)] TJ ET BT 25.000 19.825 Td /F1 11.0 Tf [(PLOS Currents Influenza)] TJ ET Q endstream endobj 122 0 obj << /Type /Annot /Subtype /Link /A 123 0 R /Border [0 0 0] /H /I /Rect [ 35.2500 654.7500 147.7500 767.2500 ] >> endobj 123 0 obj << /Type /Action /S /URI /URI (http://currents.plos.org/influenza/files/2010/03/figure-21.jpg) >> endobj 124 0 obj << /Type /XObject /Subtype /Image /Width 150 /Height 150 /ColorSpace /DeviceRGB /Filter /DCTDecode /BitsPerComponent 8 /Length 6525>> stream JFIF;CREATOR: gd-jpeg v1.0 (using IJG JPEG v62), quality = 90 C     C   " }!1AQa"q2#BR$3br %&'()*456789:CDEFGHIJSTUVWXYZcdefghijstuvwxyz w!1AQaq"2B #3Rbr $4%&'()*56789:CDEFGHIJSTUVWXYZcdefghijstuvwxyz ?RG@%~RR~/GE-v€ ( ((OŠF[Q_|,5WޛhɵkI=G2}`k[Xk>ѫυ?ٖ_2} k[Yk>ѫυ?ٖ_6} k[Yk>ѫυ?ٖ_2} k[Yk>ѫυ?ٖ_2}`k[Yk>ѫυ?ٖ_2} k[Yk>ѫυ?ٖ_2} k[Yk>ѫpUk2}`kj** VF>_jjeƫO ϴjeƨ:(ֿl<;*XXiAm[h* e3;`BdHPEѮt>->[* ]$`&2x`q Yxl[Z֬d+is2Zaњ4]y8T֮+n1T'FM;&zxHRGp[g1?J6㾚HğeTegEUth4*mKqjЮZ 1]B0kϴύW0{_ Zsl0i]̑9,ڣ 8C}}?b\1r{$ߡ|k0<5mR\AK(,α5s@Y.V9&.#2*s\?|Auoy=3UH:XYpH9c#3 I5aOwi6kf..a)4 LzĪZ1olL)iYz7.fhzVF.oӵ6zdň'o&GIaYe Λ8k|Sc<=ē鷈i)b+Č jZ| @Y 3\!+0d qVZy7efTyRJ\Enwӕt׶o k6zd}%xȱn#88_^MJNUqrh;>3ѣ~ŧ?ٵk[8&p˸|jO͎JT0k~}oبn쵻}-m#NCl6F6sQXKk"۴mU `1k/k3٦X[2ayb !~qxqUJ*RQICjZvݾi_5繷=V Tp0rO3fִcl '\ҿ/VjKR`Z8 +RB,E CϙPK:2S߉pAkW[wK=&7;nvR=2{<-SV/--纒Y.z菹_jUokw~~KMk:ggAjwWAcg({:[\؎<(6mGYZ fVdd.>ɃJW7V`6++]GY}7L4 G-f]ͽT*0T1 Cvpxb~ 'T5]X좣}Gp' K*J-KVpe(4}wZ[mcZ]KSI& tFI,*p 9/ÏkZZ鈎)"M/JYbsys g-5ֽ+y-2Z'^G=tn% ]_l(Oܪ漼XY@ RAO[Ahn7Sa+,.d 'WSڊ\j:#X6j֤ #X~Q7㵣EgyռQmkd״.Y&uN!<20q¸j6+kћMvF&;MAvw7eoɚ{jW6f[˩ٞ1<"YUv!TyG~+3j3Bܣ z|ϥRv&RQM^:4A{+rZ$dé}60~3xE{o ^U]ZyfUPDqmʨ'qok˩"!ݞ Hhn_$7+fyѱ$E˒F46t hNXjN+:u{3^Aw&63!UNzpkM`#ӌ~~NXI<+ > bԼIeknoUMl$ke#]wמ HmnQ:85hJ6Q$/r-N{{uR?y=q6K/fñ͝o]#_K 6A[/ OoaMo>.z|]'$Gl%?Ɨm#_H?-ko>.m#_G$Gl%?ƀGk=o>.I Ji?--PAi ;C޶&?+e)5$Zo:x+KiC{}BSw.A򘝘G\$}Xd*^b-C Yd%O@d7^?|4MגrC HkдmK5"EBFO2keNO4RL >*W›}_No-Цb(f!FIQ],6wļzR/7$O0lݕ%[wic-,_?+ʹtϊN6Ma6B+Dj?v1 fx%ʜ(8k}ZiK>]k|94V->+r'l7ˌM#iFA\*1׮i  JI+7P]?PEgҝ +0 !m`T*@+>]G"76Ad< aJ$~.zUG+Wn[/Cާk}mV{nTe l9 EfO\M9->Ϫ63wlG ߨcAmҏ oGfP%MҏĭPz+3PS :5 +;cp<~܁~\Z?o??Ʊ5K?_Z6T]MN :霌~]M!9(o3U!mo!apB9$8ǥt/u$swLVˬ`O>n|^j\=9vHMu d !oϴI>ƺ3=0L"[ڢǼ=Kch3'Ś4pڥhe d} oc&T^v̖er6b{@<_iD m/+|Z;r>q\gwsxnK/ G⻻;ۋ]D,^$ڲ`lA\٭{/ ^SxNG=$䓜뚧xrZ{/_馠?2߷ςN t!g\jpYKfSG?xJxj \趆#gc=d+WpCbo/^;$kK(\v#?.qB足K>%*w {xJC2f}rFmɤ6kwr39Ԣ5 [\qdʗA0})n5mbM(@2Yc ǻY6_S׮浸eUH0*G+F٬'',O q{c(~4~4QGEbhM_R󥾿`ff * qA-F=RkI)ܣ`ȪaԿ$ ?Ə__@t  iXmorǻ1$M7Vuq,RRF}q:KKhNcZڸMFV 3=|.z 'u/ :~&յ oa@O,M#,D8'j-/K|HGej<>]۫36X?aԿ& ?Ə__@Nkufu-N 4)QK:^oxlg6+o[ӌO__Gu/  /x&kFm[5U1c2I'Ox|UjڦawMcn: :Kkh-@<6PwM3ng,vvv[qR-Bqn.%LC?(FO}R0>éMhĊFAN߱j__Gص/  *+7Z-KkhJA5a4Pz?((? 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W ?!W?I j/kvV:Enܑ҅ʕ^Zm?Z+ _{X>'SKwH{mQ6o;sRIIN1i7w _{j:S#MR!u~ E  unz=ާL`4ʨv$5z?4KDk1ZBdx$f$muSsWE*siI3^\(vJ-w \^E/$'+Iz 55ijeHv ?~uΕG䢯'd7 _{[QertUeIf 2̘&lo#,'ʚ57 3%v5$%؏NdWF'h%aITFo _{S‡[|&*nrVHu)Frݣ7 O{Z+w3u5Ǜc %s3Pcq}w g"z (qiXՇ[qjvuKc{%Ug+u=ϵK}Cx`S0eaM#Eoʴ>UVVz ý PKiRᮐ̫Va {vMx~u۴SsGel]J0AU}խa. 29$ze!?!wj̶f$'"E} fiHQ@3viz$ѭtYnr#{ ʾsD"gI%u F$|>oe_Y[Z=&%װ׻~cNaWJG~uhtk-偒FwGPk4:+jVJɸ/㛮3,C)l7ZHVLpʇ̓ÿ?_dGmyд\VX-:~ciIJv^Ybx;›K}{/Nqwqoq]Fez崛[k9Y$ӻ)$W. :j8{dnnmN?+vC.^UσpX(f_\+ˁ6qN=j>tO*|ڵGIDᇉdY.-N^Q:&kx%XZt#aᇉLvr#5?s-PFʲEaLddgK "Ϟ?׹Q>_~,|ESUo5(-&cp?D8Ҽ i_ m S ΢"@bY3Vc{RV4;rI5ȥUquN2I~׉if =))s$m'Ϲ4i<7-i8;.,>;u{ B~R%$y:#zQK%OE֎H6jοbHJ-_p$:2Z4VE;BM_ٝH\/AiդK F%E.WOtӾ<JBgeZ?ixjXV+fF.C`4̨?jxN28 ~??Cm=xP9݄y_?͞~I"?##;ceqrQlA6p=ۋG?/%Ҽ Kk$ 7RH1GӜ_ kL$`4- 1[:\<4aiKV'}^ddq9!.u}PJ.P̄z؞kdko⋸v4{K9l]kqK'Kg&MN2:**&ڵYl?~i5wl+gc(d`nv_5~q_MQ$c'IڸU}YMMc;"1\0=\sKZʂ_$xiJ;s`spU0p׾VN[wԬ%B+NIBQ@"k=f{sy1U?~}xMմ&֤ԡ.v1l2j+˭z%m28:MV53 /OgHUQEr5*)?eQ@#:Fe5*(FY: t (#V_G#:G?Š(gHUQΏ@/O??e3*(FtY: t (#V_G#:G?Š(gHUQE endstream endobj 134 0 obj << /Type /Annot /Subtype /Link /A 135 0 R /Border [0 0 0] /H /I /Rect [ 35.2500 654.7500 147.7500 767.2500 ] >> endobj 135 0 obj << /Type /Action /S /URI /URI (http://currents.plos.org/influenza/files/2010/03/figure-21.jpg) >> endobj 136 0 obj << /Type /Annot /Subtype /Link /A 137 0 R /Border [0 0 0] /H /I /Rect [ 228.9232 346.7431 239.7653 356.6637 ] >> endobj 137 0 obj << /Type /Action >> endobj 138 0 obj << /Type /Annot /Subtype /Link /A 139 0 R /Border [0 0 0] /H /I /Rect [ 560.5598 346.7431 571.4017 356.6637 ] >> endobj 139 0 obj << /Type /Action >> endobj 140 0 obj << /Type /Annot /Subtype /Link /A 141 0 R /Border [0 0 0] /H /I /Rect [ 163.3545 322.9336 185.0385 332.8542 ] >> endobj 141 0 obj << /Type /Action >> endobj 142 0 obj << /Type /Annot /Subtype /Link /A 143 0 R /Border [0 0 0] /H /I /Rect [ 35.2500 132.1807 147.7500 244.6807 ] >> endobj 143 0 obj << /Type /Action /S /URI /URI (http://currents.plos.org/influenza/files/2010/03/figure-31.jpg) >> endobj 144 0 obj << /Type /Annot /Subtype /Link /A 145 0 R /Border [0 0 0] /H /I /Rect [ 35.2500 654.7500 147.7500 767.2500 ] >> endobj 145 0 obj << /Type /Action /S /URI /URI (http://currents.plos.org/influenza/files/2010/03/figure-21.jpg) >> endobj 146 0 obj << /Type /Annot /Subtype /Link /A 147 0 R /Border [0 0 0] /H /I /Rect [ 228.9232 346.7431 239.7653 356.6637 ] >> endobj 147 0 obj << /Type /Action >> endobj 148 0 obj << /Type /Annot /Subtype /Link /A 149 0 R /Border [0 0 0] /H /I /Rect [ 560.5598 346.7431 571.4017 356.6637 ] >> endobj 149 0 obj << /Type /Action >> endobj 150 0 obj << /Type /Annot /Subtype /Link /A 151 0 R /Border [0 0 0] /H /I /Rect [ 163.3545 322.9336 185.0385 332.8542 ] >> endobj 151 0 obj << /Type /Action >> endobj 152 0 obj << /Type /Annot /Subtype /Link /A 153 0 R /Border [0 0 0] /H /I /Rect [ 35.2500 132.1807 147.7500 244.6807 ] >> endobj 153 0 obj << /Type /Action /S /URI /URI (http://currents.plos.org/influenza/files/2010/03/figure-31.jpg) >> endobj 154 0 obj << /Type /Page /Parent 3 0 R /Annots [ 156 0 R 158 0 R 160 0 R 162 0 R 164 0 R 166 0 R 168 0 R 170 0 R 172 0 R ] /Contents 155 0 R >> endobj 155 0 obj << /Length 22057 >> stream q 15.000 23.148 577.500 753.852 re W n 0.965 0.965 0.965 rg 26.250 699.136 555.000 77.864 re f 0.267 0.267 0.267 rg 0.267 0.267 0.267 RG 26.250 699.136 m 581.250 699.136 l 581.250 699.886 l 26.250 699.886 l f q 35.250 710.386 537.000 66.614 re W n 0.271 0.267 0.267 rg BT 35.250 767.476 Td /F4 9.8 Tf [(Fig. 3: Average Ka/Ks values and nucleotide distances within and between the same set of sequences.)] TJ ET BT 35.250 748.106 Td /F1 9.8 Tf [(A\) Average Ka/Ks values within May sequences, between May and November sequences and within November sequences, )] TJ ET BT 35.250 734.370 Td /F1 9.8 Tf [(for each genomic segment. B\) Nucleotide distances within and between the same set of sequences. For each segment, the )] TJ ET BT 35.250 720.634 Td /F1 9.8 Tf [(number of sequences analyzed in the May group, which matches those in November, is indicated.)] TJ ET Q BT 26.250 682.113 Td /F1 9.8 Tf [(Two different patterns can be observed: for PB1, PA, and NA segments Ka/Ks remained very low and constant for the whole )] TJ ET BT 26.250 670.208 Td /F1 9.8 Tf [(period; by contrast, for NS, HA , PB2, NP and MP, Ka/Ks, although highly variable among segments, showed a common )] TJ ET BT 26.250 658.303 Td /F1 9.8 Tf [(decreasing pattern. The dramatic reduction from May to November suggests that the strongest positive selective pressure )] TJ ET BT 26.250 646.398 Td /F1 9.8 Tf [(acted before May on these segments. By contrast, the genetic distance values \(Fig. 3b\) for the same sets of sequences, )] TJ ET BT 26.250 634.494 Td /F1 9.8 Tf [(indicate a progressive increase over time. Taken together, these findings suggest that the October-November peak of )] TJ ET BT 26.250 622.589 Td /F1 9.8 Tf [(infections, which occurred in the Northern Hemisphere, did not impose any significant positive selective pressure on clade 7 )] TJ ET BT 26.250 610.684 Td /F1 9.8 Tf [(virus, but only a random genetic drift in strict purifying selection conditions.)] TJ ET BT 26.250 574.082 Td /F4 12.0 Tf [(Conclusions)] TJ ET BT 26.250 554.127 Td /F1 9.8 Tf [(Our study demonstrates that pandemic \(H1N1\) 2009 virus has evolved worldwide, shifting from an initial mixed clade pattern to )] TJ ET BT 26.250 542.223 Td /F1 9.8 Tf [(the predominance of one clade \(clade 7\) during the course of the pandemic. The virus constituting this clade was therefore )] TJ ET BT 26.250 530.318 Td /F1 9.8 Tf [(responsible for most of the pandemic burden worldwide. After its origin, which remains obscure, clade 7 virus has been )] TJ ET BT 26.250 518.413 Td /F1 9.8 Tf [(subjected to strong purifying selection, with the exception of the earliest phases of its evolution, behaving later as a well-fit virus, )] TJ ET BT 26.250 506.508 Td /F1 9.8 Tf [(similar to viruses circulating in swine or seasonal influenza in humans. Interestingly, the highest Ka/Ks values were associated )] TJ ET BT 26.250 494.604 Td /F1 9.8 Tf [(to HA and NS, key proteins for virus-host interactions, suggesting adaptation to the new host species. As yet, no pathogenetic )] TJ ET BT 26.250 482.699 Td /F1 9.8 Tf [(correlate of this evolution has emerged, since no clear trend in the clinical aspects could be observed between the early and the )] TJ ET BT 26.250 470.794 Td /F1 9.8 Tf [(late peaks of the epidemic)] TJ ET 0.267 0.267 0.267 rg BT 139.516 470.794 Td /F1 9.8 Tf [( [9])] TJ ET 0.271 0.267 0.267 rg BT 153.068 470.794 Td /F1 9.8 Tf [(. Neither was a clear clinical impact demonstrated for HA variants which occurred on clade 7: )] TJ ET BT 26.250 458.889 Td /F1 9.8 Tf [(D222/G/N or E, \(WHO report, 28)] TJ ET BT 167.128 462.778 Td /F1 8.7 Tf [(th)] TJ ET BT 174.356 458.889 Td /F1 9.8 Tf [( December 2009\). The hypothesis that clade 7 virus enjoyed a marked advantage, in terms of )] TJ ET BT 26.250 446.985 Td /F1 9.8 Tf [(transmissibility, over other early clades is intriguing, but has yet to be demonstrated.)] TJ ET BT 26.250 410.382 Td /F4 12.0 Tf [(Materials and Methods)] TJ ET BT 26.250 390.428 Td /F4 9.8 Tf [(Patients and samples)] TJ ET BT 26.250 371.023 Td /F1 9.8 Tf [(Patients with febrile respiratory illness from the southern half of Rome were referred to the National Institute for Infectious )] TJ ET BT 26.250 359.118 Td /F1 9.8 Tf [(Diseases \(INMI\) L.Spallanzani for diagnosis and treatment. All patients underwent nasal and faringeal swab sampling, and )] TJ ET BT 26.250 347.214 Td /F1 9.8 Tf [(both swabs were stirred in the same tube containing RPMI 1640 tissue culture medium with antibiotics. Approximately half of )] TJ ET BT 26.250 335.309 Td /F1 9.8 Tf [(the sequences in this study were obtained from patients with severe respiratory syndromes, the other half were from randomly )] TJ ET BT 26.250 323.404 Td /F1 9.8 Tf [(chosen patients with mild symptoms.)] TJ ET BT 26.250 303.999 Td /F4 9.8 Tf [(RNA extraction, amplification and sequencing)] TJ ET BT 26.250 284.595 Td /F1 9.8 Tf [(Nucleic acids were extracted from the swab fluid by an automated procedure \(Biorobot MDx, Quiagen, Hilden, Germany\) and )] TJ ET BT 26.250 272.690 Td /F1 9.8 Tf [(amplified by in house methods using One-Step qRT-PCR system \(Invitrogen, Carlsbad CA, USA\) to yield partial or full-length )] TJ ET BT 26.250 260.785 Td /F1 9.8 Tf [(sequences of HA and NA. Sequencing was performed on an automated ABI Prism 3130 instrument \(Applied Biosystems, Foster )] TJ ET BT 26.250 248.880 Td /F1 9.8 Tf [(City CA, USA\) by use of Big Dye3.1 cycle sequencing kits provided by the same manufacturer. All sequences have been )] TJ ET BT 26.250 236.976 Td /F1 9.8 Tf [(deposited in Gen Bank with the following accession numbers: from CY052070 to CY052092 and from CY055309 to CY055414.)] TJ ET BT 26.250 217.571 Td /F4 9.8 Tf [(Phylogenetic and selective pressure analysis)] TJ ET BT 26.250 198.166 Td /F1 9.8 Tf [(The sequences were aligned by the Clustal algorithm. Phylogenesis was performed using the MEGA 4 package )] TJ ET 0.267 0.267 0.267 rg BT 510.172 198.166 Td /F1 9.8 Tf [([7])] TJ ET 0.271 0.267 0.267 rg BT 521.014 198.166 Td /F1 9.8 Tf [(. The )] TJ ET BT 26.250 186.261 Td /F1 9.8 Tf [(Neighbor-Joining phylogenetic trees \(300 Bootstrap replicas\) were generated using the Tamura 3 parameters distance option. )] TJ ET BT 26.250 174.357 Td /F1 9.8 Tf [(The selective pressure acting on each segment was computed using the Nei and Gojobori substitution model \()] TJ ET 0.267 0.267 0.267 rg BT 501.504 174.357 Td /F1 9.8 Tf [([6] )] TJ ET 0.271 0.267 0.267 rg BT 515.057 174.357 Td /F1 9.8 Tf [(with the Jukes-)] TJ ET BT 26.250 162.452 Td /F1 9.8 Tf [(Cantor correction\): Ka/Ks values were calculated as the ratio between the average rate of non synonymous substitutions per )] TJ ET BT 26.250 150.547 Td /F1 9.8 Tf [(non synonymous site of all pairwise comparisons \(average Ka\), and the average rate of synonymous substitutions per )] TJ ET BT 26.250 138.642 Td /F1 9.8 Tf [(synonymous site of all pairwise comparisons \(average Ks\). The genetic distances between sequences were calculated by the )] TJ ET BT 26.250 126.738 Td /F1 9.8 Tf [(Tamura 3 parameter method.)] TJ ET BT 26.250 90.135 Td /F4 12.0 Tf [(Competing interests)] TJ ET BT 26.250 70.181 Td /F1 9.8 Tf [(The authors have declared that no competing interests exist.)] TJ ET Q q 15.000 23.148 577.500 753.852 re W n 0.965 0.965 0.965 rg 26.250 699.136 555.000 77.864 re f 0.267 0.267 0.267 rg 0.267 0.267 0.267 RG 26.250 699.136 m 581.250 699.136 l 581.250 699.886 l 26.250 699.886 l f q 35.250 710.386 537.000 66.614 re W n 0.271 0.267 0.267 rg BT 35.250 767.476 Td /F4 9.8 Tf [(Fig. 3: Average Ka/Ks values and nucleotide distances within and between the same set of sequences.)] TJ ET BT 35.250 748.106 Td /F1 9.8 Tf [(A\) Average Ka/Ks values within May sequences, between May and November sequences and within November sequences, )] TJ ET BT 35.250 734.370 Td /F1 9.8 Tf [(for each genomic segment. B\) Nucleotide distances within and between the same set of sequences. For each segment, the )] TJ ET BT 35.250 720.634 Td /F1 9.8 Tf [(number of sequences analyzed in the May group, which matches those in November, is indicated.)] TJ ET Q BT 26.250 682.113 Td /F1 9.8 Tf [(Two different patterns can be observed: for PB1, PA, and NA segments Ka/Ks remained very low and constant for the whole )] TJ ET BT 26.250 670.208 Td /F1 9.8 Tf [(period; by contrast, for NS, HA , PB2, NP and MP, Ka/Ks, although highly variable among segments, showed a common )] TJ ET BT 26.250 658.303 Td /F1 9.8 Tf [(decreasing pattern. The dramatic reduction from May to November suggests that the strongest positive selective pressure )] TJ ET BT 26.250 646.398 Td /F1 9.8 Tf [(acted before May on these segments. By contrast, the genetic distance values \(Fig. 3b\) for the same sets of sequences, )] TJ ET BT 26.250 634.494 Td /F1 9.8 Tf [(indicate a progressive increase over time. Taken together, these findings suggest that the October-November peak of )] TJ ET BT 26.250 622.589 Td /F1 9.8 Tf [(infections, which occurred in the Northern Hemisphere, did not impose any significant positive selective pressure on clade 7 )] TJ ET BT 26.250 610.684 Td /F1 9.8 Tf [(virus, but only a random genetic drift in strict purifying selection conditions.)] TJ ET BT 26.250 574.082 Td /F4 12.0 Tf [(Conclusions)] TJ ET BT 26.250 554.127 Td /F1 9.8 Tf [(Our study demonstrates that pandemic \(H1N1\) 2009 virus has evolved worldwide, shifting from an initial mixed clade pattern to )] TJ ET BT 26.250 542.223 Td /F1 9.8 Tf [(the predominance of one clade \(clade 7\) during the course of the pandemic. The virus constituting this clade was therefore )] TJ ET BT 26.250 530.318 Td /F1 9.8 Tf [(responsible for most of the pandemic burden worldwide. After its origin, which remains obscure, clade 7 virus has been )] TJ ET BT 26.250 518.413 Td /F1 9.8 Tf [(subjected to strong purifying selection, with the exception of the earliest phases of its evolution, behaving later as a well-fit virus, )] TJ ET BT 26.250 506.508 Td /F1 9.8 Tf [(similar to viruses circulating in swine or seasonal influenza in humans. Interestingly, the highest Ka/Ks values were associated )] TJ ET BT 26.250 494.604 Td /F1 9.8 Tf [(to HA and NS, key proteins for virus-host interactions, suggesting adaptation to the new host species. As yet, no pathogenetic )] TJ ET BT 26.250 482.699 Td /F1 9.8 Tf [(correlate of this evolution has emerged, since no clear trend in the clinical aspects could be observed between the early and the )] TJ ET BT 26.250 470.794 Td /F1 9.8 Tf [(late peaks of the epidemic)] TJ ET 0.267 0.267 0.267 rg BT 139.516 470.794 Td /F1 9.8 Tf [( [9])] TJ ET 0.271 0.267 0.267 rg BT 153.068 470.794 Td /F1 9.8 Tf [(. Neither was a clear clinical impact demonstrated for HA variants which occurred on clade 7: )] TJ ET BT 26.250 458.889 Td /F1 9.8 Tf [(D222/G/N or E, \(WHO report, 28)] TJ ET BT 167.128 462.778 Td /F1 8.7 Tf [(th)] TJ ET BT 174.356 458.889 Td /F1 9.8 Tf [( December 2009\). The hypothesis that clade 7 virus enjoyed a marked advantage, in terms of )] TJ ET BT 26.250 446.985 Td /F1 9.8 Tf [(transmissibility, over other early clades is intriguing, but has yet to be demonstrated.)] TJ ET BT 26.250 410.382 Td /F4 12.0 Tf [(Materials and Methods)] TJ ET BT 26.250 390.428 Td /F4 9.8 Tf [(Patients and samples)] TJ ET BT 26.250 371.023 Td /F1 9.8 Tf [(Patients with febrile respiratory illness from the southern half of Rome were referred to the National Institute for Infectious )] TJ ET BT 26.250 359.118 Td /F1 9.8 Tf [(Diseases \(INMI\) L.Spallanzani for diagnosis and treatment. All patients underwent nasal and faringeal swab sampling, and )] TJ ET BT 26.250 347.214 Td /F1 9.8 Tf [(both swabs were stirred in the same tube containing RPMI 1640 tissue culture medium with antibiotics. Approximately half of )] TJ ET BT 26.250 335.309 Td /F1 9.8 Tf [(the sequences in this study were obtained from patients with severe respiratory syndromes, the other half were from randomly )] TJ ET BT 26.250 323.404 Td /F1 9.8 Tf [(chosen patients with mild symptoms.)] TJ ET BT 26.250 303.999 Td /F4 9.8 Tf [(RNA extraction, amplification and sequencing)] TJ ET BT 26.250 284.595 Td /F1 9.8 Tf [(Nucleic acids were extracted from the swab fluid by an automated procedure \(Biorobot MDx, Quiagen, Hilden, Germany\) and )] TJ ET BT 26.250 272.690 Td /F1 9.8 Tf [(amplified by in house methods using One-Step qRT-PCR system \(Invitrogen, Carlsbad CA, USA\) to yield partial or full-length )] TJ ET BT 26.250 260.785 Td /F1 9.8 Tf [(sequences of HA and NA. Sequencing was performed on an automated ABI Prism 3130 instrument \(Applied Biosystems, Foster )] TJ ET BT 26.250 248.880 Td /F1 9.8 Tf [(City CA, USA\) by use of Big Dye3.1 cycle sequencing kits provided by the same manufacturer. All sequences have been )] TJ ET BT 26.250 236.976 Td /F1 9.8 Tf [(deposited in Gen Bank with the following accession numbers: from CY052070 to CY052092 and from CY055309 to CY055414.)] TJ ET BT 26.250 217.571 Td /F4 9.8 Tf [(Phylogenetic and selective pressure analysis)] TJ ET BT 26.250 198.166 Td /F1 9.8 Tf [(The sequences were aligned by the Clustal algorithm. Phylogenesis was performed using the MEGA 4 package )] TJ ET 0.267 0.267 0.267 rg BT 510.172 198.166 Td /F1 9.8 Tf [([7])] TJ ET 0.271 0.267 0.267 rg BT 521.014 198.166 Td /F1 9.8 Tf [(. The )] TJ ET BT 26.250 186.261 Td /F1 9.8 Tf [(Neighbor-Joining phylogenetic trees \(300 Bootstrap replicas\) were generated using the Tamura 3 parameters distance option. )] TJ ET BT 26.250 174.357 Td /F1 9.8 Tf [(The selective pressure acting on each segment was computed using the Nei and Gojobori substitution model \()] TJ ET 0.267 0.267 0.267 rg BT 501.504 174.357 Td /F1 9.8 Tf [([6] )] TJ ET 0.271 0.267 0.267 rg BT 515.057 174.357 Td /F1 9.8 Tf [(with the Jukes-)] TJ ET BT 26.250 162.452 Td /F1 9.8 Tf [(Cantor correction\): Ka/Ks values were calculated as the ratio between the average rate of non synonymous substitutions per )] TJ ET BT 26.250 150.547 Td /F1 9.8 Tf [(non synonymous site of all pairwise comparisons \(average Ka\), and the average rate of synonymous substitutions per )] TJ ET BT 26.250 138.642 Td /F1 9.8 Tf [(synonymous site of all pairwise comparisons \(average Ks\). The genetic distances between sequences were calculated by the )] TJ ET BT 26.250 126.738 Td /F1 9.8 Tf [(Tamura 3 parameter method.)] TJ ET BT 26.250 90.135 Td /F4 12.0 Tf [(Competing interests)] TJ ET BT 26.250 70.181 Td /F1 9.8 Tf [(The authors have declared that no competing interests exist.)] TJ ET Q q 15.000 23.148 577.500 753.852 re W n 0.965 0.965 0.965 rg 26.250 699.136 555.000 77.864 re f 0.267 0.267 0.267 rg 0.267 0.267 0.267 RG 26.250 699.136 m 581.250 699.136 l 581.250 699.886 l 26.250 699.886 l f q 35.250 710.386 537.000 66.614 re W n 0.271 0.267 0.267 rg BT 35.250 767.476 Td /F4 9.8 Tf [(Fig. 3: Average Ka/Ks values and nucleotide distances within and between the same set of sequences.)] TJ ET BT 35.250 748.106 Td /F1 9.8 Tf [(A\) Average Ka/Ks values within May sequences, between May and November sequences and within November sequences, )] TJ ET BT 35.250 734.370 Td /F1 9.8 Tf [(for each genomic segment. B\) Nucleotide distances within and between the same set of sequences. For each segment, the )] TJ ET BT 35.250 720.634 Td /F1 9.8 Tf [(number of sequences analyzed in the May group, which matches those in November, is indicated.)] TJ ET Q BT 26.250 682.113 Td /F1 9.8 Tf [(Two different patterns can be observed: for PB1, PA, and NA segments Ka/Ks remained very low and constant for the whole )] TJ ET BT 26.250 670.208 Td /F1 9.8 Tf [(period; by contrast, for NS, HA , PB2, NP and MP, Ka/Ks, although highly variable among segments, showed a common )] TJ ET BT 26.250 658.303 Td /F1 9.8 Tf [(decreasing pattern. The dramatic reduction from May to November suggests that the strongest positive selective pressure )] TJ ET BT 26.250 646.398 Td /F1 9.8 Tf [(acted before May on these segments. By contrast, the genetic distance values \(Fig. 3b\) for the same sets of sequences, )] TJ ET BT 26.250 634.494 Td /F1 9.8 Tf [(indicate a progressive increase over time. Taken together, these findings suggest that the October-November peak of )] TJ ET BT 26.250 622.589 Td /F1 9.8 Tf [(infections, which occurred in the Northern Hemisphere, did not impose any significant positive selective pressure on clade 7 )] TJ ET BT 26.250 610.684 Td /F1 9.8 Tf [(virus, but only a random genetic drift in strict purifying selection conditions.)] TJ ET BT 26.250 574.082 Td /F4 12.0 Tf [(Conclusions)] TJ ET BT 26.250 554.127 Td /F1 9.8 Tf [(Our study demonstrates that pandemic \(H1N1\) 2009 virus has evolved worldwide, shifting from an initial mixed clade pattern to )] TJ ET BT 26.250 542.223 Td /F1 9.8 Tf [(the predominance of one clade \(clade 7\) during the course of the pandemic. The virus constituting this clade was therefore )] TJ ET BT 26.250 530.318 Td /F1 9.8 Tf [(responsible for most of the pandemic burden worldwide. After its origin, which remains obscure, clade 7 virus has been )] TJ ET BT 26.250 518.413 Td /F1 9.8 Tf [(subjected to strong purifying selection, with the exception of the earliest phases of its evolution, behaving later as a well-fit virus, )] TJ ET BT 26.250 506.508 Td /F1 9.8 Tf [(similar to viruses circulating in swine or seasonal influenza in humans. Interestingly, the highest Ka/Ks values were associated )] TJ ET BT 26.250 494.604 Td /F1 9.8 Tf [(to HA and NS, key proteins for virus-host interactions, suggesting adaptation to the new host species. As yet, no pathogenetic )] TJ ET BT 26.250 482.699 Td /F1 9.8 Tf [(correlate of this evolution has emerged, since no clear trend in the clinical aspects could be observed between the early and the )] TJ ET BT 26.250 470.794 Td /F1 9.8 Tf [(late peaks of the epidemic)] TJ ET 0.267 0.267 0.267 rg BT 139.516 470.794 Td /F1 9.8 Tf [( [9])] TJ ET 0.271 0.267 0.267 rg BT 153.068 470.794 Td /F1 9.8 Tf [(. Neither was a clear clinical impact demonstrated for HA variants which occurred on clade 7: )] TJ ET BT 26.250 458.889 Td /F1 9.8 Tf [(D222/G/N or E, \(WHO report, 28)] TJ ET BT 167.128 462.778 Td /F1 8.7 Tf [(th)] TJ ET BT 174.356 458.889 Td /F1 9.8 Tf [( December 2009\). The hypothesis that clade 7 virus enjoyed a marked advantage, in terms of )] TJ ET BT 26.250 446.985 Td /F1 9.8 Tf [(transmissibility, over other early clades is intriguing, but has yet to be demonstrated.)] TJ ET BT 26.250 410.382 Td /F4 12.0 Tf [(Materials and Methods)] TJ ET BT 26.250 390.428 Td /F4 9.8 Tf [(Patients and samples)] TJ ET BT 26.250 371.023 Td /F1 9.8 Tf [(Patients with febrile respiratory illness from the southern half of Rome were referred to the National Institute for Infectious )] TJ ET BT 26.250 359.118 Td /F1 9.8 Tf [(Diseases \(INMI\) L.Spallanzani for diagnosis and treatment. All patients underwent nasal and faringeal swab sampling, and )] TJ ET BT 26.250 347.214 Td /F1 9.8 Tf [(both swabs were stirred in the same tube containing RPMI 1640 tissue culture medium with antibiotics. Approximately half of )] TJ ET BT 26.250 335.309 Td /F1 9.8 Tf [(the sequences in this study were obtained from patients with severe respiratory syndromes, the other half were from randomly )] TJ ET BT 26.250 323.404 Td /F1 9.8 Tf [(chosen patients with mild symptoms.)] TJ ET BT 26.250 303.999 Td /F4 9.8 Tf [(RNA extraction, amplification and sequencing)] TJ ET BT 26.250 284.595 Td /F1 9.8 Tf [(Nucleic acids were extracted from the swab fluid by an automated procedure \(Biorobot MDx, Quiagen, Hilden, Germany\) and )] TJ ET BT 26.250 272.690 Td /F1 9.8 Tf [(amplified by in house methods using One-Step qRT-PCR system \(Invitrogen, Carlsbad CA, USA\) to yield partial or full-length )] TJ ET BT 26.250 260.785 Td /F1 9.8 Tf [(sequences of HA and NA. Sequencing was performed on an automated ABI Prism 3130 instrument \(Applied Biosystems, Foster )] TJ ET BT 26.250 248.880 Td /F1 9.8 Tf [(City CA, USA\) by use of Big Dye3.1 cycle sequencing kits provided by the same manufacturer. All sequences have been )] TJ ET BT 26.250 236.976 Td /F1 9.8 Tf [(deposited in Gen Bank with the following accession numbers: from CY052070 to CY052092 and from CY055309 to CY055414.)] TJ ET BT 26.250 217.571 Td /F4 9.8 Tf [(Phylogenetic and selective pressure analysis)] TJ ET BT 26.250 198.166 Td /F1 9.8 Tf [(The sequences were aligned by the Clustal algorithm. Phylogenesis was performed using the MEGA 4 package )] TJ ET 0.267 0.267 0.267 rg BT 510.172 198.166 Td /F1 9.8 Tf [([7])] TJ ET 0.271 0.267 0.267 rg BT 521.014 198.166 Td /F1 9.8 Tf [(. The )] TJ ET BT 26.250 186.261 Td /F1 9.8 Tf [(Neighbor-Joining phylogenetic trees \(300 Bootstrap replicas\) were generated using the Tamura 3 parameters distance option. )] TJ ET BT 26.250 174.357 Td /F1 9.8 Tf [(The selective pressure acting on each segment was computed using the Nei and Gojobori substitution model \()] TJ ET 0.267 0.267 0.267 rg BT 501.504 174.357 Td /F1 9.8 Tf [([6] )] TJ ET 0.271 0.267 0.267 rg BT 515.057 174.357 Td /F1 9.8 Tf [(with the Jukes-)] TJ ET BT 26.250 162.452 Td /F1 9.8 Tf [(Cantor correction\): Ka/Ks values were calculated as the ratio between the average rate of non synonymous substitutions per )] TJ ET BT 26.250 150.547 Td /F1 9.8 Tf [(non synonymous site of all pairwise comparisons \(average Ka\), and the average rate of synonymous substitutions per )] TJ ET BT 26.250 138.642 Td /F1 9.8 Tf [(synonymous site of all pairwise comparisons \(average Ks\). The genetic distances between sequences were calculated by the )] TJ ET BT 26.250 126.738 Td /F1 9.8 Tf [(Tamura 3 parameter method.)] TJ ET BT 26.250 90.135 Td /F4 12.0 Tf [(Competing interests)] TJ ET BT 26.250 70.181 Td /F1 9.8 Tf [(The authors have declared that no competing interests exist.)] TJ ET Q q 0.000 0.000 0.000 rg BT 291.710 19.825 Td /F1 11.0 Tf [(4)] TJ ET BT 25.000 19.825 Td /F1 11.0 Tf [(PLOS Currents Influenza)] TJ ET Q endstream endobj 156 0 obj << /Type /Annot /Subtype /Link /A 157 0 R /Border [0 0 0] /H /I /Rect [ 139.5157 469.8923 153.0683 479.8129 ] >> endobj 157 0 obj << /Type /Action >> endobj 158 0 obj << /Type /Annot /Subtype /Link /A 159 0 R /Border [0 0 0] /H /I /Rect [ 510.1717 197.2643 521.0137 207.1849 ] >> endobj 159 0 obj << /Type /Action >> endobj 160 0 obj << /Type /Annot /Subtype /Link /A 161 0 R /Border [0 0 0] /H /I /Rect [ 501.5040 173.4548 515.0565 183.3754 ] >> endobj 161 0 obj << /Type /Action >> endobj 162 0 obj << /Type /Annot /Subtype /Link /A 163 0 R /Border [0 0 0] /H /I /Rect [ 139.5157 469.8923 153.0683 479.8129 ] >> endobj 163 0 obj << /Type /Action >> endobj 164 0 obj << /Type /Annot /Subtype /Link /A 165 0 R /Border [0 0 0] /H /I /Rect [ 510.1717 197.2643 521.0137 207.1849 ] >> endobj 165 0 obj << /Type /Action >> endobj 166 0 obj << /Type /Annot /Subtype /Link /A 167 0 R /Border [0 0 0] /H /I /Rect [ 501.5040 173.4548 515.0565 183.3754 ] >> endobj 167 0 obj << /Type /Action >> endobj 168 0 obj << /Type /Annot /Subtype /Link /A 169 0 R /Border [0 0 0] /H /I /Rect [ 139.5157 469.8923 153.0683 479.8129 ] >> endobj 169 0 obj << /Type /Action >> endobj 170 0 obj << /Type /Annot /Subtype /Link /A 171 0 R /Border [0 0 0] /H /I /Rect [ 510.1717 197.2643 521.0137 207.1849 ] >> endobj 171 0 obj << /Type /Action >> endobj 172 0 obj << /Type /Annot /Subtype /Link /A 173 0 R /Border [0 0 0] /H /I /Rect [ 501.5040 173.4548 515.0565 183.3754 ] >> endobj 173 0 obj << /Type /Action >> endobj 174 0 obj << /Type /Page /Parent 3 0 R /Contents 175 0 R >> endobj 175 0 obj << /Length 8641 >> stream 0.271 0.267 0.267 rg q 15.000 405.910 577.500 371.090 re W n 0.271 0.267 0.267 rg BT 26.250 750.278 Td /F4 12.0 Tf [(Acknowledgements)] TJ ET BT 26.250 730.324 Td /F1 9.8 Tf [(We gratefully acknowledge the editing of the manuscript by Carla Nisii.)] TJ ET BT 26.250 701.222 Td /F4 12.0 Tf [(References)] TJ ET BT 26.250 673.767 Td /F1 9.8 Tf [(1.)] TJ ET BT 38.132 673.767 Td /F1 9.8 Tf [(Smith GJ et al. Origins and evolutionary genomics of the 2009 swine-origin H1N1 influenza A epidemic. Nature. )] TJ ET BT 26.250 661.863 Td /F1 9.8 Tf [(2009;459\(7250\):1122-5.)] TJ ET BT 26.250 642.458 Td /F1 9.8 Tf [(2.)] TJ ET BT 38.132 642.458 Td /F1 9.8 Tf [(Novel Swine-origin Influenza A \(H1N1\) Investigation team, Dawood FS et al. Emergence of a novel swine-origin influenza A )] TJ ET BT 26.250 630.553 Td /F1 9.8 Tf [(\(H1N1\) virus in humans. N Engl J Med. 2009;360\(25\):2605-15.)] TJ ET BT 26.250 611.148 Td /F1 9.8 Tf [(3.)] TJ ET BT 38.132 611.148 Td /F1 9.8 Tf [(Ranbaut A and Holmes E. The early molecular epidemiology of the swine-origin A/H1N1 human influenza pandemic. PLoS )] TJ ET BT 26.250 599.244 Td /F1 9.8 Tf [(Curr Influenza. 2009 August 18:RRN1003)] TJ ET BT 26.250 579.839 Td /F1 9.8 Tf [(4.)] TJ ET BT 38.132 579.839 Td /F1 9.8 Tf [(Nelson M et al. The early diversification of influenza A/H1N1pdm. PLoS Curr Influenza. 2009 Nov 3:RRN1126)] TJ ET BT 26.250 560.434 Td /F1 9.8 Tf [(5.)] TJ ET BT 38.132 560.434 Td /F1 9.8 Tf [(Fereindouni SR, Beer M, Vahlenkamp T, Starick E. Differentiation of two distinct clusters among currently circulating )] TJ ET BT 26.250 548.529 Td /F1 9.8 Tf [(influenza A\(H1N1\) viruses, March-September 2009. Euro Surveill. 2009;14\(46\) pii: 19409.)] TJ ET BT 26.250 529.125 Td /F1 9.8 Tf [(6.)] TJ ET BT 38.132 529.125 Td /F1 9.8 Tf [(Nei M and Gojobori T. Simple methods for estimating the numbers of synonymous and nonsynonymous nucleotide )] TJ ET BT 26.250 517.220 Td /F1 9.8 Tf [(substitutions. Mol Biol Evol. 1986;3\(5\):418-26.)] TJ ET BT 26.250 497.815 Td /F1 9.8 Tf [(7.)] TJ ET BT 38.132 497.815 Td /F1 9.8 Tf [(Tamura K, Dudley J, Nei M and Kumar S MEGA4: Molecular Evolutionary Genetics Analysis \(MEGA\) software version 4.0. )] TJ ET BT 26.250 485.910 Td /F1 9.8 Tf [(Mol Biol Evol. 2007; 24:1596-1599.)] TJ ET BT 26.250 466.506 Td /F1 9.8 Tf [(8.)] TJ ET BT 38.132 466.506 Td /F1 9.8 Tf [(Sinha NK, Roy A, Das B, Das S, Basak S. Evolutionary complexities of swine flu H1N1 gene sequences of 2009. Bioch. )] TJ ET BT 26.250 454.601 Td /F1 9.8 Tf [(Bioph. Res Comm. 2009; 390: 349-351.)] TJ ET BT 26.250 435.196 Td /F1 9.8 Tf [(9.)] TJ ET BT 38.132 435.196 Td /F1 9.8 Tf [(Donaldson LJ et al. Mortality from pandemic A/H1N1 influenza in England public health surveillance study. BMJ. )] TJ ET BT 26.250 423.291 Td /F1 9.8 Tf [(2009;339:b5213.)] TJ ET Q q 15.000 405.910 577.500 371.090 re W n 0.271 0.267 0.267 rg BT 26.250 750.278 Td /F4 12.0 Tf [(Acknowledgements)] TJ ET BT 26.250 730.324 Td /F1 9.8 Tf [(We gratefully acknowledge the editing of the manuscript by Carla Nisii.)] TJ ET BT 26.250 701.222 Td /F4 12.0 Tf [(References)] TJ ET BT 26.250 673.767 Td /F1 9.8 Tf [(1.)] TJ ET BT 38.132 673.767 Td /F1 9.8 Tf [(Smith GJ et al. Origins and evolutionary genomics of the 2009 swine-origin H1N1 influenza A epidemic. Nature. )] TJ ET BT 26.250 661.863 Td /F1 9.8 Tf [(2009;459\(7250\):1122-5.)] TJ ET BT 26.250 642.458 Td /F1 9.8 Tf [(2.)] TJ ET BT 38.132 642.458 Td /F1 9.8 Tf [(Novel Swine-origin Influenza A \(H1N1\) Investigation team, Dawood FS et al. Emergence of a novel swine-origin influenza A )] TJ ET BT 26.250 630.553 Td /F1 9.8 Tf [(\(H1N1\) virus in humans. N Engl J Med. 2009;360\(25\):2605-15.)] TJ ET BT 26.250 611.148 Td /F1 9.8 Tf [(3.)] TJ ET BT 38.132 611.148 Td /F1 9.8 Tf [(Ranbaut A and Holmes E. The early molecular epidemiology of the swine-origin A/H1N1 human influenza pandemic. PLoS )] TJ ET BT 26.250 599.244 Td /F1 9.8 Tf [(Curr Influenza. 2009 August 18:RRN1003)] TJ ET BT 26.250 579.839 Td /F1 9.8 Tf [(4.)] TJ ET BT 38.132 579.839 Td /F1 9.8 Tf [(Nelson M et al. The early diversification of influenza A/H1N1pdm. PLoS Curr Influenza. 2009 Nov 3:RRN1126)] TJ ET BT 26.250 560.434 Td /F1 9.8 Tf [(5.)] TJ ET BT 38.132 560.434 Td /F1 9.8 Tf [(Fereindouni SR, Beer M, Vahlenkamp T, Starick E. Differentiation of two distinct clusters among currently circulating )] TJ ET BT 26.250 548.529 Td /F1 9.8 Tf [(influenza A\(H1N1\) viruses, March-September 2009. Euro Surveill. 2009;14\(46\) pii: 19409.)] TJ ET BT 26.250 529.125 Td /F1 9.8 Tf [(6.)] TJ ET BT 38.132 529.125 Td /F1 9.8 Tf [(Nei M and Gojobori T. Simple methods for estimating the numbers of synonymous and nonsynonymous nucleotide )] TJ ET BT 26.250 517.220 Td /F1 9.8 Tf [(substitutions. Mol Biol Evol. 1986;3\(5\):418-26.)] TJ ET BT 26.250 497.815 Td /F1 9.8 Tf [(7.)] TJ ET BT 38.132 497.815 Td /F1 9.8 Tf [(Tamura K, Dudley J, Nei M and Kumar S MEGA4: Molecular Evolutionary Genetics Analysis \(MEGA\) software version 4.0. )] TJ ET BT 26.250 485.910 Td /F1 9.8 Tf [(Mol Biol Evol. 2007; 24:1596-1599.)] TJ ET BT 26.250 466.506 Td /F1 9.8 Tf [(8.)] TJ ET BT 38.132 466.506 Td /F1 9.8 Tf [(Sinha NK, Roy A, Das B, Das S, Basak S. Evolutionary complexities of swine flu H1N1 gene sequences of 2009. Bioch. )] TJ ET BT 26.250 454.601 Td /F1 9.8 Tf [(Bioph. Res Comm. 2009; 390: 349-351.)] TJ ET BT 26.250 435.196 Td /F1 9.8 Tf [(9.)] TJ ET BT 38.132 435.196 Td /F1 9.8 Tf [(Donaldson LJ et al. Mortality from pandemic A/H1N1 influenza in England public health surveillance study. BMJ. )] TJ ET BT 26.250 423.291 Td /F1 9.8 Tf [(2009;339:b5213.)] TJ ET Q q 15.000 405.910 577.500 371.090 re W n 0.271 0.267 0.267 rg BT 26.250 750.278 Td /F4 12.0 Tf [(Acknowledgements)] TJ ET BT 26.250 730.324 Td /F1 9.8 Tf [(We gratefully acknowledge the editing of the manuscript by Carla Nisii.)] TJ ET BT 26.250 701.222 Td /F4 12.0 Tf [(References)] TJ ET BT 26.250 673.767 Td /F1 9.8 Tf [(1.)] TJ ET BT 38.132 673.767 Td /F1 9.8 Tf [(Smith GJ et al. Origins and evolutionary genomics of the 2009 swine-origin H1N1 influenza A epidemic. Nature. )] TJ ET BT 26.250 661.863 Td /F1 9.8 Tf [(2009;459\(7250\):1122-5.)] TJ ET BT 26.250 642.458 Td /F1 9.8 Tf [(2.)] TJ ET BT 38.132 642.458 Td /F1 9.8 Tf [(Novel Swine-origin Influenza A \(H1N1\) Investigation team, Dawood FS et al. Emergence of a novel swine-origin influenza A )] TJ ET BT 26.250 630.553 Td /F1 9.8 Tf [(\(H1N1\) virus in humans. N Engl J Med. 2009;360\(25\):2605-15.)] TJ ET BT 26.250 611.148 Td /F1 9.8 Tf [(3.)] TJ ET BT 38.132 611.148 Td /F1 9.8 Tf [(Ranbaut A and Holmes E. The early molecular epidemiology of the swine-origin A/H1N1 human influenza pandemic. PLoS )] TJ ET BT 26.250 599.244 Td /F1 9.8 Tf [(Curr Influenza. 2009 August 18:RRN1003)] TJ ET BT 26.250 579.839 Td /F1 9.8 Tf [(4.)] TJ ET BT 38.132 579.839 Td /F1 9.8 Tf [(Nelson M et al. The early diversification of influenza A/H1N1pdm. PLoS Curr Influenza. 2009 Nov 3:RRN1126)] TJ ET BT 26.250 560.434 Td /F1 9.8 Tf [(5.)] TJ ET BT 38.132 560.434 Td /F1 9.8 Tf [(Fereindouni SR, Beer M, Vahlenkamp T, Starick E. Differentiation of two distinct clusters among currently circulating )] TJ ET BT 26.250 548.529 Td /F1 9.8 Tf [(influenza A\(H1N1\) viruses, March-September 2009. Euro Surveill. 2009;14\(46\) pii: 19409.)] TJ ET BT 26.250 529.125 Td /F1 9.8 Tf [(6.)] TJ ET BT 38.132 529.125 Td /F1 9.8 Tf [(Nei M and Gojobori T. Simple methods for estimating the numbers of synonymous and nonsynonymous nucleotide )] TJ ET BT 26.250 517.220 Td /F1 9.8 Tf [(substitutions. Mol Biol Evol. 1986;3\(5\):418-26.)] TJ ET BT 26.250 497.815 Td /F1 9.8 Tf [(7.)] TJ ET BT 38.132 497.815 Td /F1 9.8 Tf [(Tamura K, Dudley J, Nei M and Kumar S MEGA4: Molecular Evolutionary Genetics Analysis \(MEGA\) software version 4.0. )] TJ ET BT 26.250 485.910 Td /F1 9.8 Tf [(Mol Biol Evol. 2007; 24:1596-1599.)] TJ ET BT 26.250 466.506 Td /F1 9.8 Tf [(8.)] TJ ET BT 38.132 466.506 Td /F1 9.8 Tf [(Sinha NK, Roy A, Das B, Das S, Basak S. Evolutionary complexities of swine flu H1N1 gene sequences of 2009. Bioch. )] TJ ET BT 26.250 454.601 Td /F1 9.8 Tf [(Bioph. Res Comm. 2009; 390: 349-351.)] TJ ET BT 26.250 435.196 Td /F1 9.8 Tf [(9.)] TJ ET BT 38.132 435.196 Td /F1 9.8 Tf [(Donaldson LJ et al. Mortality from pandemic A/H1N1 influenza in England public health surveillance study. 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