In ternationa l Scholars Journa ls African Journal of Pig Farming ISSN 2375-0731 Vol. 2 (11), pp. 001-017, November, 2014. Available online at www.internationalscholarsjournals.org © International Scholars Journals Author(s) retain the copyright of this article. Full Length Research Paper Evolutionary characteristics of swine-origin H1N1 influenza virus that infected humans from sporadic to pandemic Lei Han, Wenying Lu, Yifang Han, Shuhua Li, Jianhua Yin, Jiaxin Xie, Tong Su and Guangwen Cao* Department of Epidemiology, Second Military Medical University, Shanghai 200433, China. Accepted 03 April, 2014 Evolutionary process of swine-origin H1N1 influenza A viruses that infected humans from sporadic to pandemic is of high epidemiological significance but still remains obscure. To understand this process, we performed phylogenetic, bootscan, and adaptive evolution analyses using the sequences of the 8 gene segments from swine-origin H1N1 influenza A viruses that infected humans and the reference viruses. Classic swine H1N1 viruses occasionally infected humans before 1998. Sporadic human infection with the triple-reassortant swine-origin H1N1 viruses was firstly identified in 1998 and has become increasingly frequent since 2005. Except genes encoding the neuraminidase and matrix protein of swine influenza viruses of Eurasian lineage, other 6 genes of A/H1N1/2009 pandemic strain were most closely linked to those of A/Iowa/CEID23/2005(H1N1), a representative swine-origin triple-reassortant virus that infected humans sporadically. Potential positive selections acting on the haemagglutinin gene evolved from classic swine H1N1 viruses to the triple-reassortant H1N1 viruses and on the neuraminidase gene evolved from Eurasian swine viruses to A/H1N1/2009 pandemic viruses might play a role in cross-species transmission and human infection. Surveillance of genetic evolution of influenza A viruses in swine workers might provide useful clues of influenza pandemic. Key words: Swine, H1N1 influenza virus, evolution, sporadic, pandemic. INTRODUCTION Classic swine influenza A viruses cause sporadic human infection via animals to human transmission, especially in swine workers (Myers et al., 2007; Olsen et al., 2002). A swine influenza virus was firstly isolated from autopsy lung tissue of human in 1974 (Smith et al., 1976). Like the infections with avian influenza A viruses, sporadic cases of humans infected with classical swine influenza A viruses in recent decades have rarely resulted in human- to-human spread. Pigs have receptors to swine, avian, and human influenza virus strains and act as a “mixing vessel” in which genetic material of the viruses can be exchanged (Ito et al., 1998). The reassortment of swine, avian, and human influenza viruses in swine possibly *Corresponding author. E-mail: gcao@smmu.edu.cn. Tel: +86- 21-81871060. Fax: +86-21-81871060. results in interspecies transmission of influenza and generation of novel progeny viruses to which humans are immunologically naive and highly susceptible (Scholtissek et al., 1985; Webster et al., 1992). The emergence of pandemic H1N1/2009 influenza demon-strated that pandemic viruses could be generated in pigs. The novel H1N1/2009, triple-reassortant swine-origin influenza A virus, shows a strong ability to transmit from human to human and has caused influenza A pandemic worldwide since its first emergence in Mexico in March 2009 (MMWR Morb Mortal Weekly Report, 2009). This novel virus contains 8 gene segments encoding haema- gglutinin (HA), nucleoprotein (NP), and nonstructural protein (NS) from classic swine influenza A virus of North American lineage, the polymerase basic 2 (PB2) and the polymerase acidic (PA) from avian influenza of North American lineage, the polymerase basic 1 (PB1) from human seasonal influenza A H3N2, and neuraminidase (NA) and matrix protein (MP) from swine influenza A of Eurasian lineage (Babakir-Mina et al., 2009; Dawood et al., 2009; Garten et al., 2009; Lu et al., 2009). The reassortment of swine lineages may have occurred years before emergence in humans. However, the nature and location of the genetically closest swine viruses reveal little about the immediate origin of the epidemic (Smith et al., 2009). Between the 1930s and the 1990s, classic swine H1N1 influenza A underwent little change. However, by the late 1990s, multiple strains and subtypes (H1N1, H3N2, and H1N2) of triple-reassortant swine influenza A (H1) viruses whose genomes included combinations of avian, swine, and human influenza virus gene segments had emerged in pigs (Olsen, 2002). Since 2005, the triple-reassortant swine-origin H1N1 influenza viruses have been frequently reported to be able to infect humans sporadically (Gray et al., 2007; Newman et al., 2008; Shinde et al., 2009). In this study, we retrieved the representative DNA sequences of swine-origin H1N1 influenza A viruses that infected humans and reference viruses from GenBank and analyzed evolutionary relationships using phylogenetic analysis, bootscan analysis, and adaptive evolution analysis. The reassortment of swine-origin H1N1 with avian influenza virus of North America lineage and human seasonal influenza virus before 2005 in North America was found to be an important step during evolutionary process of A/H1N1/2009 pandemic virus. EXPERIMENTAL Searching for the sequences of influenza A viruses The genome sequences of the novel A/H1N1/2009 pandemic viruses were downloaded from the NCBI Influenza Virus Resource (http://www.ncbi.nlm.nih.gov/genomes/FLU/FLU.html). We searched the PubMed database up to March 2009, the time point before the outbreak of novel influenza A viruses, using the searching terms “swine”, “influenza”, and “human”, to identify articles describing sporadic cases of human infection thought to be caused by swine influenza virus strains. Reference lists from the articles selected by electronic searching were searched to identify further relevant articles. The 8 gene segments of the corresponding viruses were retrieved from GenBank. The sequences of reference influenza A viruses sampled from human, avian and swine in North America, Europe and Asia during the period 1918 to 2009 were also retrieved from GenBank, respectively. Phylogenetic analysis Sequences of the 8 gene segments of the reference viruses, those of all available swine-origin H1N1 influenza A viruses that infected humans sporadically in North America, and those of selected pandemic A/H1N1/2009 viruses were used for phylogenetic analysis, respectively. Sequence alignments for the gene segments of PB2 (full-length 2,281 bp; partial 1,573 bp), PB1 (2,274 bp), PA (full-length 2,151 bp; partial 1,470 bp), HA (1,701 bp), NP (1,497 bp), NA (1,410 bp), MP (982 bp), and NS (842 bp) and phylogenetic analysis were performed using MEGA 4.0 software package (Tamura et al., 2007). We deleted some of the phylogenetically closely related sequences from reference influenza viruses isolated in the same years and locations and then construct phylogenetic tree. A bootstrap resampling process (1000 replicates) using the neighbor-joining (NJ) method was used to assess the robustness of individual nodes on the phylogeny. Bootscan analysis Full-length genomic sequences containing the PB2, PB1, PA, HA, NP, NA, MP, and NS gene segments in order of a given influenza isolate were generated using MEGA 4.0 software. Bootscan analyses over full-length sequences were carried out by using SimPlot software (version 3.5) (200 bp window size, 20 bp step size, 100 bootstrap replicates, using gap-stripped alignments and neighbor-joining analysis) (Cavinta et al., 2009). Intact sequences of the test viruses, a pandemic A/H1N1/2009 strain (A/Wisconsin/629-D01773/2009) and a representative triple- reassortant swine-origin strain (A/Iowa/CEID23/2005), were compared with those of the previously isolated reference viruses whose gene segments were the closest genetic neighbors of the test viruses. Adaptive evolution analysis Positive selection drives viral evolution during cross-species transmission and human infection. To investigate whether A/Iowa/CEID23/2005 (H1N1) and A/H1N1/2009 viruses were driven by positive selections, the standard McDonald–Kreitman test (http://mkt.uab.es/mkt/MKT.asp) was applied to detect the natural selection (Egea et al., 2008). For the analyses of the NA and MP gene segments, the clades of the novel A/H1N1/2009 viruses were added into “species 1” and their nearest genetic clades (Eurasian swine influenza strains) were added into “species 2”, respectively. For the analysis of the HA, two steps of the selections were performed. Firstly, the clade of the novel A/H1N1/2009 viruses was added into “species 1” and the clade of the triple-reassortant swine H1N1 that sporadically infected humans in or after 2005 was added into “species 2”; Secondly, the clade of the triple-reassortant swine H1N1 that sporadically infected humans in or after 2005 was added into “species 1” and the clade of the classic swine H1N1 that sporadically infected humans before 1998 was added into “species 2”. The Neutrality Index (NI) was used to indicate the extent to which the levels of amino acid polymorphism depart from the expected in the neutral model. A NI value < 1 indicated an excess of fixation of non-neutral replacements due to positive selection, 1 means under neutral selection, and > 1 reflected negative selection that prevented the fixation of harmful mutations (Egea et al., 2008; Ding et al., 2009). RESULTS Genetic characteristics of swine-origin influenza viruses that sporadically infected humans before 2009 A total of 49 sporadic influenza patients caused by swine- origin influenza viruses were reported in North America. Four infections occurred in 1980s and 5 in 1990s. However, 15 infections occurred between December 2005 and February 2009 and 10 of which occurred in or after 2007 (Table 1). Some nucleotide sequences of the 8 gene segments of swine-origin influenza viruses spora- dically infected humans in North America were available in GenBank. Of the 49 cases, 46 were infected with H1N1 Table 1. Cases of human infections with swine-origin H1N1 influenza viruses in North America and their corresponding viral gene segments. Patient Reference Year Residence Subtype Access no. of gene segments in GenBank PB2 PB1 PA HA NP NA MP NS 1 Myers et al. (2007) 1974 Minnesota H1N1 - - - - - - - - 2 Myers et al. (2007) 1975 Wisconsin H1N1 - - - - - - - - 3 Myers et al. (2007) 1975 Virginia H1N1 - - - - - - - - 4 Myers et al. (2007) 1975 Virginia/NY H1N1 - - - - - - - - 5 Myers et al. (2007) 1975 Tennessee H1N1 - - - - - - - - 6 Myers et al. (2007) 1976 Missouri H1N1 - - - - - - - - 7 Myers et al. (2007) 1976 Wisconsin H1N1 - - - - - - - - 8 Myers et al. (2007) 1976 Wisconsin H1N1 - - - - - - - - 9 Myers et al. (2007) 1976 NA H1N1 - - - - - - - - 10-23 Myers et al. (2007) 1976 New Jersey H1N1 - - - - - - - - 24 Shinde et al. (2009) 1976 Wisconsin H1N1 CY026146 CY026145 CY026144 CY026139 CY026142 CY026141 CY026140 CY026143 25 Myers et al. (2007) 1979 Texas H1N1 - - - - - - - - 26 Myers et al. (2007) 1980 Texas H1N1 - - - - - - - - 27 Myers et al. (2007) 1982 Nevada H1N1 - - - - - - - - 28 Shinde et al. (2009) 1988 Ohio H1N1 CY024932 CY024931 CY024930 CY024925 CY024928 CY024927 CY024926 CY024929 29 Myers et al. (2007) 1988 Wisconsin H1N1 - - - - - - - - 30 Myers et al. (2007) 1991 Maryland H1N1 CY039916 CY039915 CY039914 CY039909 CY039912 CY039911 CY039910 CY039913 31 Myers et al. (2007) 1994 Wisconsin H1N1 U53159* U53157* U53161* U53163 U53165* U53167* U53169 U53171 32 Myers et al. (2007) 1994 Wisconsin H1N1 U53158* U53156* U53160* U53162 U53164* U53166 U53168 U53170 33 Myers et al. (2007) 1995 Minnesota H1N1 - - - - - - - - 34 Myers et al. (2007) 1998 Wisconsin H1N1 AF342824* AF342823 AF342822* AF342821* AF342819 AF342820 AF342818 AF342817 35 Gray et al. (2007) 2005 Iowa H1N1 DQ889682 DQ889683 DQ889684 DQ889689 DQ889686 DQ889687 DQ889688 DQ889685 36 Newman et al.(2008) 2005 Wisconsin H1N1 - - - FJ986619 - - - - 37 Olsen et al.(2006) 2005 Canada H3N2 DQ469955 DQ469956 DQ469957 DQ469962 DQ469959 DQ469960 DQ469961 DQ469958 38 Shinde et al. (2009) 2006 Missouri H1N1 - - - - - - - - 39 Shinde et al. (2009) 2006 Iowa H1N1 - - - FJ986618 - - - - 40 Shinde et al. (2009) 2007 Ohio H1N1 - - - FJ986620 - - - - 41 Shinde et al. (2009) 2007 Ohio H1N1 - - - FJ986621 - - - - 42 Shinde et al. (2009) 2007 Michigan H1N2 - - - FJ986622 - - - - 43 Shinde et al. (2009) 2007 Illinois H1N1 - - - - - - - - 44 Shinde et al. (2009) 2007 Iowa H1N1 - - - - - - - - 45 Vincent et al. (2009) 2007 Ohio HIN1 EU604691 EU604692 EU604693 EU604689 EU604694 EU604690 EU604695 EU604696 46 Bastien et al. (20098) 2007 Canada H3N2 EU399758 EU399757 EU399756 EU399751 EU399754 EU399753 EU399752 EU399755 47 Shinde et al. (2009) 2008 Minnesota H1N1 - - - - - - - - 48 Shinde et al. (2009) 2008 Texas H1N1 - - - - - - - - 49 Shinde et al. (2009) 2009 Iowa H1N1 - - - - - - - - “-”, not available in GenBank; “*”, partial sequences. influenza A. Phylogenetic analysis with 30, 27, 27, 35, 28, 28, 30, and 30 full-length sequences of PB2, PB1, PA, HA, NP, NA, MP, and NS genes of the influenza viruses are performed, respectively (Figure 1). All of the 8 gene segments of swine H1N1 viruses that sporadically infected humans in 1970s to 1990s clustered with those of classic swine influenza viruses of North American lineage, except A/Wisconsin/10/98, an unreported isolate from a human case in Wisconsin in 1998. Partial PB2 (1,573 bp) and PA (1,470 bp) segments and other 6 full- length gene segments of A/Wisconsin/10/98 were retrieved from GenBank. Phylogenetic analyses with the partial sequences indicated that the PB2 and PA segments of A/Wisconsin/10/98 were closely related to that of A/Iowa/CEID23/2005(H1N1) (Figure 2). The full- length PB1 gene of A/Wisconsin/10/98 was closely related to that of A/Iowa/CEID23/2005(H1N1) (Figure 1). Bootscan analysis indicated that A/Iowa/CEID23/2005 (H1N1), a representative swine-origin triple-reassortant influenza that sporadically infected humans in North America, shared 92.3 and 93.0% identities in the PB2 and PA genes with avian influenza virus of North American lineage, 94.6% identity in the PB1 gene with human H3N2 virus, and 92.8 to 96.5% identities in another 5 genes with classic swine influenza H1N1 virus of North American lineage, respectively, as shown in Table 2. The 8 fragments of a strain isolated in 2007, A/swine/OH/511445/2007(H1N1), were phylogenetically linked to those of A/Iowa/CEID23/2005(H1N1) (Figure 1). The PB2 and PA segments, PB1 segment, and other 5 gene segments of swine-origin H1N1 viruses that infected humans sporadically between 2005 and 2007 (or 2009) were closely related to those of avian influenza viruses of North American lineage, human seasonal H3N2 influenza viruses, and classic swine influenza viruses of North American lineage, respectively. Since human infections with the triple-reassortant swine-origin H1N1 viruses had been increasingly frequent several years before the outbreak of the novel H1N1 influenza in 2009, we hypothesize that introduction of the PB2 and PA genes of avian influenza viruses and the PB1 gene from human seasonal H3N2 viruses facilitate the sporadic infection of swine-origin H1N1 influenza A virus in humans since 2005. Evolutionary relationship of swine-origin H1N1 influenza viruses that infected humans from sporadic to pandemic As shown in Figure 1, the A/H1N1/2009 pandemic strains clustered in a unique clade for each of the 8 gene segments, respectively. Within the clades of the nearest genetic neighbors, the PB2, PB1, PA, HA, NP, and NS gene segments of the novel H1N1/2009 strains were most closely linked to those of A/Iowa/CEID23/2005 (H1N1) and those of other triple-reassortant swine-origin viruses that sporadically infected humans since 2005 (Figures 1 and 2). The PB1, NP, and NS were also closely linked to those of a triple-reassortant swine-origin virus A/Wisconsin/10/98 (H1N1) that sporadically infected humans. The HA was also closely linked to the gene of triple-reassortant swine viruses A/Ohio/01/2007 (H1N1), A/Ohio/02/2007 (H1N1), A/Wisconsin/87/2005 (H1N1), and A/Iowa/01/2006 (H1N1) that sporadically infected humans in North America. The NA and MP were closely related to those of influenza A H1N1 viruses circulating in swine populations in Eurasia. Bootscan analysis showed that the A/H1N1/2009 pandemic strain shared great homology with A/Iowa/CEID23/2005 (H1N1) in the PB2 (95.3%), PB1 (95.1%), PA (95.2%), HA (90.3%), NP (95.2%), and NS (94.7%) genes. The NA and MP genes of the A/H1N1/2009 pandemic strain were most closely related to those of A/Swine/Spain/50047/2003 (H1N1), a swine virus of Eurasian lineage, sharing 90.2 and 94.4% identities, respectively, as shown in Figure 3. The genetic components of the swine-origin H1N1 viruses that infected humans from sporadic to pandemic at different phases were shown in Figure 4. Positive selection drives the evolution of swine-origin H1N1 viruses that infected humans from sporadic to pandemic Table 3 shows the results of the McDonald– Kreitman tests. A HI value of 0.817 was observed in the HA segment between the classic swine H1N1 viruses that occasionally infected humans before 1998 and the triple- reassortant H1N1 viruses that sporadically infected humans in or after 2005, implying potential positive selection acting on this gene evolved from the classic swine H1N1 viruses to the triple-reassortant H1N1 viruses. However, positive selection was not observed on this gene between the triple-reassortant H1N1 viruses and A/H1N1/2009 pandemic viruses. Moreover, a HI value of 0.773 was observed acting on the NA gene between A/H1N1/2009 viruses and their nearest genetic neighbors (Euroasian swine H1N1 viruses), implying a positive selection was acting on this gene before or during the reassortment of this gene segment into A/H1N1/2009 pandemic viruses. DISCUSSION The reassortments between gene segments are essential steps for the evolution of influenza viruses. This study indicated that swine-origin H1N1 influenza viruses that infected humans from sporadic to pandemic have experienced two critical steps of the reassortments. The first critical step is the introduction of the PB2 and PA genes of avian influenza viruses and the PB1 gene from human seasonal H3N2 viruses which might facilitate the PB2 100 A/Karasuk/01/2010(H1N1) A/Wisconsin/629-D01773/2009(H1N1) 100 57 A/Beijing/718/2009(H1N1) A/Ontario/RV1273/2005(H3N2) 98 91 100 A/Ontario/1252/2007(H3N2) A/swine/OH/511445/2007(H1N1) 99 A/Iowa/CEID23/2005(H1N1) 79 A/blue winged teal/LA/B228/1986(H1N1) 100 A/duck/Alberta/35/1976(H1N1) A/mallard/MD/02-184/2002(H1N1) 93 98 A/mallard/Minnesota/282/2000(H3N2) A/mallard/Ohio/424/1988(H3N2) 90 A/Alabama/UR06-0455/2007(H1N1) 100 100 A/Albany/20/1978(H1N1) A/AA/Huston/1945(H1N1) A/Auckland/609/2002(H3N2) 80 A/swine/Iowa/1/1976(H1N1) 96 88 A/Wisconsin/301/1976(H1N1) A/swine/Wisconsin/641/1980(H1N1) 100 A/Maryland/12/1991(H1N1) A/swine/Kansas/3024/1987(H1N1) 100 84 A/Ohio/3559/1988(H1N1) 96 A/swine/Bakum/IDT1769/2003(H3N2) 100 A/swine/Chonburi/NIAH9469/2004(H1N1) A/Swine/Cotes dArmour/3633 100 A/mallard/Netherlands/1/2007(H3N2) A/chicken/Israel/1055/2008(H5N1) 100 100 A/cygnus olor/Italy/808/2006(H5N1) 2009 A(H1N1) Triple-assortment swine influenza North America Avian Human Seasonal North America Classic Swine Euroasian Swine Euroasian Avian 0.02 A/Mexico/48N/2009(H1N1) 100 PB1 A/New York/1669/2009(H1N1) 72 A/Taiwan/206/2009(H1N1) 99 99 A/Ontario/RV1273/2005(H3N2) 100 A/Ontario/1252/2007(H3N2) 100 A/swine/OH/511445/2007(H1N1) 97 A/Iowa/CEID23/2005(H1N1) 74 A/Wisconsin/10/98 (H1N1) 68 A/Denmark/35/00(H3N2) 100 A/Alabama/UR06-0482/2007(H3N2) 91 A/Ashburton/280/2004(H3N2) A/mallard/Minnesota/282/2000(H3N2) A/blue-winged teal/Ohio/31/1999(H3N2) 100 39 A/mallard/Maryland/691/2005(H3N2) 54 A/swine/Bakum/5/95(H1N1) 100 A/swine/Jena/5/96(H3N2) A/swine/Hungary/13509/2007(H3N2) 99 A/buzzard/Denmark/6370/06(H5N1) A/Chicken/HongKong/FY150/01-MB(H5N1) 100 100 A/Goose/Guangdong/1/96(H5N1) 100 A/Alabama/UR06-0536/2007(H1N1) A/Albany/20/1978(H1N1) 100 A/swine/Arizona/148/1977(H1N1) 100 A/Wisconsin/301/1976(H1N1) A/swine/Alberta/56626/03(H1N1) 100 A/Maryland/12/1991(H1N1) 92 99 A/Ohio/3559/1988(H1N1) 0.01 2009 A(H1N1) Triple-assortment swine influenza Human Seasonal H3N2 North America Avian Euroasian Swine Euroasian Avian Human Seasonal H1N1 North America Classic Swine PA 100 A/Taiwan/206/2009(H1N1) A/New York/1669/2009(H1N1) 100 95 A/Mexico/48N/2009(H1N1) A/Ontario/RV1273/2005(H3N2) 99 100 A/Ontario/1252/2007(H3N2) 99 A/swine/OH/511445/2007(H1N1) 100 A/Iowa/CEID23/2005(H1N1) 100 A/duck/NY/13152-13/1994(H1N1) A/black duck/Carolina/675-075/2004(H3N2) 100 98 A/mallard/MD/53/2003(H1N1) A/Bar-headed Goose/Qinghai/12/05(H5N1) 100 A/Chicken/Hong Kong/317.5/2001(H5N1) 100 A/Beijing/01/2003(H5N1) 85 100 A/duck/Italy/69238/2007(H1N1) A/swine/Chonburi/NIAH9469/2004(H1N1) A/Swine/Italy/1513-1/98(H1N1) 100 93 A/swine/Spain/42386/2002(H3N2) 100 A/Wisconsin/301/1976(H1N1) A/swine/Wisconsin/464/98(H1N1) A/Maryland/12/1991(H1N1) 96 A/Ohio/3559/1988(H1N1) 98 99 A/swine/Iowa/1/1987(H1N1) 100 A/Auckland/583/2000(H3N2) 74 A/Boston/10/2008(H3N2) A/Albany/1/1970(H3N2) 100 A/Alabama/UR06-0455/2007(H1N1) 69 A/Arizona/14/1978(H1N1) 0.01 2009 A(H1N1) Triple-assortment swine influenza North America Avian Euroasian Avian Euroasian Swine North America Classic Swine Human Seasonal HA 99 A/Italy/05/2009(H1N1) A/New York/1669/2009(H1N1) 97 69 A/Beijing/718/2009(H1N1) A/swine/OH/511445/2007(H1N1) 100 A/Ohio/01/2007(H1N1) 84 A/Ohio/02/2007(H1N1) 100 A/Wisconsin/87/2005(H1N1) 92 A/Iowa/CEID23/2005(H1N1) 100 A/Iowa/01/2006(H1N1) A/Swine/Wisconsin/136/97(H1N1) A/Maryland/12/1991(H1N1) 77 95 A/Ohio/3559/1988(H1N1) 99 A/WI/4755/1994(H1N1) 95 A/WI/4754/1994(H1N1) 100 A/swine/Indiana/1726/1988(H1N1) 60 A/swine/Arizona/148/1977(H1N1) 96 A/Wisconsin/301/1976(H1N1) A/AA/Huston/1945(H1N1) A/USSR/46/1979(H1N1) 100 A/Denmark/14/2001(H1N1) 97 A/Norway/166/2008(H1N1) 64 100 A/Michigan/09/2007(H1N2) 71 A/Alabama/UR06-0536/2007(H1N1) 99 A/blue winged teal/TX/27/2002(H1N1) 99 A/mallard/MD/390/2002(H1N1) A/black duck/Ohio/95/1993(H1N1) 100 A/swine/Beijing/21/2008(H1N1) 99 A/swine/France/WVL13/1995(H1N1) 93 A/duck/Australia/749/80(H1N1) 63 A/duck/Miyagi/66/1977(H1N1) A/swan/Hokkaido/55/1996(H1N1) 99 A/Anas crecca/Spain/1384/2007(H1N1) 94 99 A/goose/Italy/296426/2003(H1N1) 100 A/Ontario/RV1273/2005(H3N2) A/Ontario/1252/2007(H3N2) 2009 A(H1N1) Triple-assortment swine influenza North America Classic Swine Human Seasonal North America Avian Euroasian Swine Euroasian Avian 0.05 NP A/Italy/85/2009(H1N1) 100 A/New York/1682/2009(H1N1) 95 A/Beijing/01/2009(H1N1) 93 100 A/Ontario/RV1273/2005(H3N2) 99 A/Ontario/1252/2007(H3N2) A/swine/OH/511445/2007(H1N1) 82 50 A/Iowa/CEID23/2005(H1N1) 97 A/Wisconsin/10/98 (H1N1) A/Ohio/3559/1988(H1N1) 100 96 A/swine/California/T9001707/1991(H1N1) 39 A/Swine/Wisconsin/168/97(H1N1) 98 A/Maryland/12/1991(H1N1) A/swine/Ontaria/2/1981(H1N1) 100 A/swine/Iowa/1/1976(H1N1) 100 A/Wisconsin/301/1976(H1N1) 100 A/Denmark/11/2001(H1N1) A/Alabama/UR06-0536/2007(H1N1) 100 A/AA/Huston/1945(H1N1) 100 A/Iowa/1943(H1N1) 86 A/blue-winged teal/Ohio/31/1999(H3N2) 100 A/mallard/Maryland/712/2005(H3N2) A/duck/Alberta/35/1976(H1N1) 100 100 A/Bangladesh/207095/2008(H5N1) A/Indonesia/286H/2006(H5N1) A/mallard/Marquenterre/Z237/1983(H1N1) 95 A/swine/Beijing/21/2008(H1N1) 99 A/swine/England/WVL14/1996(H1N1) 99 98 A/swine/Italy/839/1989(H1N1) 2009 A(H1N1) Triple-assortment swine influenza North America Classic Swine Human seasonal North America Avian Euroaisan Avian Euroasian Swine 0.01 NA 0.1 95 A/Italy/05/2009(H1N1) 70 100 A/Beijing/719/2009(H1N1) A/New York/1669/2009(H1N1) 97 A/swine/Germany/SIV05/2007(H1N1) 75 A/swine/Denmark/WVL9/1993(H1N1) 96 95 A/swine/Germany/Vi5698/1995(H1N1) A/swine/Cotes dArmor/1515/1999(H1N1) A/swine/France/WVL8/1992(H1N1) 78 100 A/HongKong/156/97(H5N1) A/Chicken/Hong Kong/220/97 (H5N1) A/Egypt/10217-NAMRU3/2007(H5N1) 62 81 A/Anas crecca/Spain/1402/2007(H1N1) 88 80 A/swan/Hokkaido/55/1996(H1N1) A/chicken/Scotland/59(H5N1) A/Duck/Ohio/194/86 (H1N1) 72 93 A/blue winged teal/TX/27/2002(H1N1) 99 A/Alabama/20/2006(H1N1) A/Kentucky/1/2005(H1N1) 70 A/Iowa/CEID23/2005(H1N1) A/Wisconsin/301/1976(H1N1) 96 A/Wisconsin/10/98(H1N1) 96 A/Maryland/12/1991(H1N1) 98 79 A/Ohio/3559/1988(H1N1) 78 A/swine/OH/511445/2007(H1N1) 41 A/swine/North Carolina/12869/2003(H1N1) 55 A/WI/4754/1994(H1N1) A/Ontario/RV1273/2005(H3N2) 100 A/Ontario/1252/2007(H3N2) 2009 A(H1N1) Euroasian Swine Euroaisan Avian North America Avian Human seasonal North America Classic Swine 81 A/Ontario/RV1273/2005(H3N2) MP 95 A/swine/OH/511445/2007(H1N1) 100 A/Ontario/1252/2007(H3N2) 97 A/Iowa/CEID23/2005(H1N1) 85 A/Wisconsin/10/98 (H1N1) 73 A/Swine/Minnesota/593/99 (H3N2) A/Maryland/12/1991(H1N1) A/Ohio/3559/1988(H1N1) 80 100 A/WI/4755/1994(H1N1) 67 A/WI/4754/1994(H1N1) 72 A/swine/Indiana/1726/1988(H1N1) A/Wisconsin/301/1976(H1N1) 99 A/swine/Arizona/148/1977(H1N1) A/sw/Shizuoka/120/97(H3N2) A/Baylor/11735/82(H1N1) 100 A/Switzerland/5389/95 (H1N1) 99 99 A/Alabama/UR06-0536/2007(H1N1) 70 A/chicken/NY/11602-12/1998(H3N2) 93 A/mallard/Ohio/424/1988(H3N2) A/mallard/MD/168/2002(H1N1) 99 A/duck/Italy/69238/2007(H1N1) 98 100 A/avian/Italy/1485/1997(H5N1) A/chicken/Hong Kong/14/1976(H1N1) A/swine/Italy/v.147/1981(H1N1) 99 A/swine/Brno/1/1992(H1N1) 100 98 A/swine/Schleswig-Holstein/1/1993(H1N1) 68 A/swine/England/WVL10/1993(H1N1) 73 A/Taiwan/T0724/2009(H1N1) A/New York/1669/2009(H1N1) 100 91 A/Italy/05/2009(H1N1) Triple-assortment swine influenza North America Classic Swine Human seasonal North America Avian Euroaisan Avian Euroasian Swine 2009 A(H1N1) 0.01 A/Stockholm/34/2009(H1N1) NS 100 A/Beijing/720/2009(H1N1) 80 A/New York/1669/2009(H1N1) 98 A/Ontario/RV1273/2005(H3N2) 97 100 58 A/Ontario/1252/2007(H3N2) A/swine/OH/511445/2007(H1N1) 78 A/Iowa/CEID23/2005(H1N1) 95 A/Wisconsin/10/98 (H1N1) 97 A/swine/California/T9001707/1991(H1N1) A/Maryland/12/1991(H1N1) 65 A/WI/4754/1994(H1N1) 99 99 A/swine/Indiana/1726/1988(H1N1) 69 A/WI/4755/1994(H1N1) 100 A/swine/Iowa/1/1985(H1N1) 95 A/Ohio/3559/1988(H1N1) A/swine/Niigata/1/1977(H1N1) 100 A/Wisconsin/301/1976(H1N1) 100 A/Denmark/122/2008(H1N1) 51 A/California/UR06-0479/2007(H1N1) 100 A/United Kingdom/157/1982(H1N1) A/Hong Kong/117/77(H1N1) 74 A/black duck/Ohio/95/1993(H1N1) 100 A/mallard/Ohio/424/1988(H3N2) A/mallard/Maryland/802/2007(H5N1) 31 A/mallard/Sweden/S90780/2005(H1N1) 99 75 A/turkey/England/1969(H3N2) A/chicken/Scotland/59(H5N1) 91 A/swine/Cotes d Armor/1482/99(H1N1) A/swine/Italy/1513-1/98(H1N1) 100 A/swine/England/WVL10/1993(H1N1) 59 0.02 2009 A(H1N1) Triple-assortment swine influenza North America Classic Swine Human seasonal North America Avian Euroaisan Avian Euroasian Swine Figure 1. Phylogentic analyses of the 8 full-length gene segments (PB2, PB1, PA, HA, NP, NA, MP, and NS) of the selected pandemic A/H1N1/2009 strains, all available swine-origin influenza viruses that infected humans sporadically, and the selected reference influenza A viruses. ▲ indicates the strains infected humans sporadically. PB2 67 A/Wisconsin/629-D01773/2009(H1N1) 100 A/Beijing/718/2009(H1N1) 100 A/Karasuk/01/2010(H1N1) 89 A/Iowa/CEID23/2005(H1N1) 78 A/Wisconsin/10/98 (H1N1) 94 A/blue winged teal/LA/B228/1986(H1N1) 100 A/duck/Alberta/35/1976(H1N1) A/mallard/MD/02-184/2002(H1N1) 94 98 A/mallard/Minnesota/282/2000(H3N2) A/mallard/Ohio/424/1988(H3N2) 83 A/Alabama/UR06-0455/2007(H1N1) 100 A/Albany/20/1978(H1N1) 100 A/AA/Huston/1945(H1N1) A/Auckland/609/2002(H3N2) 77 A/swine/Iowa/1/1976(H1N1) 89 84 A/Wisconsin/301/1976(H1N1) A/swine/Wisconsin/641/1980(H1N1) 100 A/Maryland/12/1991(H1N1) A/swine/Kansas/3024/1987(H1N1) 100 76 A/Ohio/3559/1988(H1N1) 96 A/swine/Bakum/IDT1769/2003(H3N2) 100 A/swine/Chonburi/NIAH9469/2004(H1N1) A/Swine/Cotes dArmour/3633 100 A/mallard/Netherlands/1/2007(H3N2) A/chicken/Israel/1055/2008(H5N1) 100 100 A/cygnus olor/Italy/808/2006(H5N1) 0.02 2009 A(H1N1) North America Avian Human Seasonal North America Classic Swine Euroasian Swine Euroasian Avian PA 77 A/New York/1669/2009(H1N1) 100 A/Mexico/48N/2009(H1N1) 100 A/Taiwan/206/2009(H1N1) A/Iowa/CEID23/2005(H1N1) 94 98 A/Wisconsin/10/98 (H1N1) 78 A/black duck/Carolina/675-075/2004(H3N2) A/mallard/MD/53/2003(H1N1) 100 100 A/duck/NY/13152-13/1994(H1N1) 99 A/mallard/Ohio/48/1986(H3N2) A/HongKong/156/97(H5N1) 100 A/Hong Kong/378.1/2001(H5N1) 64 A/Bar-headed Goose/Qinghai/12/05(H5N1) 100 A/Chicken/Hong Kong/317.5/2001(H5N1) 100 A/Beijing/01/2003(H5N1) 79 100 A/duck/Italy/69238/2007(H1N1) A/swine/Belgium/WVL1/1979(H1N1) A/Swine/Italy/1513-1/98(H1N1) 100 A/swine/Chonburi/NIAH9469/2004(H1N1) 93 50 A/swine/Spain/42386/2002(H3N2) 97 A/Ohio/3559/1988(H1N1) 96 A/swine/Iowa/1/1987(H1N1) 92 A/Maryland/12/1991(H1N1) 100 A/swine/Wisconsin/464/98(H1N1) A/Wisconsin/301/1976(H1N1) A/Alaska/1935(H1N1) 83 A/Alabama/UR06-0455/2007(H1N1) 100 A/Arizona/14/1978(H1N1) 68 A/Albany/1/1970(H3N2) 64 A/Auckland/583/2000(H3N2) 100 A/Boston/10/2008(H3N2) 0.01 2009 A(H1N1) North America Avian Euroasian Avian Euroasian Swine North America Classic Swine Human Seasonal Figure 2. Phylogentic analyses of the partial PB2 (1,573 bp) and PA (1,470 bp) sequences of the selected pandemic A/H1N1/2009 strains, swine-origin H1N1 influenza viruses that infected humans sporadically, and the selected reference influenza A viruses. ▲ indicates the strains infected humans sporadically. Table 2. Sequence identities of the 8 gene segments of a representative swine-origin triple reassortant virus that infected humans in 2005 with those of selected reference strains using Bootscan analysis. Reference influenza virus strains Identities (%) with A/Iowa/CEID23/2005 (H1N1) Full PB2 PB1 PA HA NP NA MP NS A/Swine/Spain/50047/2003 (H1N1) 80.6 81.0 83.4 84.3 64.5 80.9 77.2 85.5 78.9 A/Swine/Wisconsin/1915/1988 (H1N1) 86. 8 81.1 75.5 78.9 92.9 94.2 93.1 96.2 94.9 A/Ohio/3559/1988 (H1N1)* 86.9 81.4 75.6 78.5 92.8 96.1 92.8 96.5 93.6 A/mallard/Ohio/48/1986 (H3N2) 83.9 92.3 86.8 92.7 29.9 80.8 29.3 89.7 62.7 A/Turkey/Ontario/84/1983 (H5N1) 83.4 90.0 86.6 93.0 41.4 80.5 78.0 89.4 83.3 A/Yucatan/ME6057/2003 (H3N2)* 73.5 81.4 94.6 79.5 30.0 79.9 2.9 85.9 79.5 * Infected humans sporadically. Figure 3. Bootscan analysis for full-length of A/H1N1/2009 pandemic strain with those of 3 representative reference strains. sporadic infection of swine-origin H1N1 influenza A virus in humans since 2005. The other step is the reassortment with the NA and MP gene segments of swine H1N1 viruses of Eurasian lineage which might enable the triple- reassortant swine-origin H1N1 viruses to cause the pandemic in 2009. Stochastic mutation in nucleotides and directional immune selection may play an important role in deter- mining the direction of the evolution of influenza viruses. The reassortments between gene segments are essential steps for the evolution of influenza viruses. This study shows that swine-origin H1N1 influenza viruses that infected humans from sporadic to pandemic have experienced two critical steps of the reassortments. The gene segments of the swine H1N1 viruses that occasionally infected persons who ever exposed to pigs in North America before 1998 were phylogenetically linked to those of classic swine H1N1 influenza viruses. Phylogenetic analyses with the partial PB2 and PA gene segments and the full-length PB1 gene segment of A/Wisconsin/10/98 indicate that the triple-reassortant H1N1 virus started to infect humans in late 1990s. The swine-origin H1N1 viruses that sporadically infected humans between 2005 and 2009 were of the triple- reassortant viruses (Figure 4). The triple-reassortant virus may be considered as “backbone frame” of the A/H1N1/2009 pandemic strain or “intermediate” in evolutionary process of the A/H1N1/2009 pandemic Swine H1N1 virus that sporadically Triple -reassortant H1N1 virus infected humans in North America that sporadically infected before 1998 humans in No rth America in 1998 and between 2005 and 2009 PB2 PB2 PB1 PB1 PA PA HA HA NP NP NA NA M M PNS PNS Avian, North American lineage Human seasonal H3N2 Classic swine, North American lineage Eurasian swine lineage The novel H1N1 virus caused the pandemic in 2009 PB2 PB1 PA HA NP NA M PNS Figure 4. The genetic components of the swine-origin H1N1 viruses that infected humans from sporadic to pandemic at different phases. Table 3. McDonald-Kreitman test for the natural selection of 3 gene segments of novel influenza A/H1N1/2009 viruses. Gene Neutral Non-neutral Neutrality Proportion of adaptive p Polymorphism Divergence Polymorphism Divergence Index (NI) substitutions HA* 206 13.30 89 7.02 0.817 0.182 0.677 HA** 126 32.79 71 17.15 1.077 -0.077 0.822 NA 797 25.37 97 16.16 0.773 0.226 0.450 MP 50 14.60 15 4.01 1.091 -0.091 0.890 *The clade of the triple-reassortant swine H1N1 that sporadically infected humans in or after 2005 was added into “species 1” and the clade of the classic swine H1N1 that sporadically infected humans before 1998 was added into “species 2”; ** The clade of the novel A/H1N1/2009 pandemic strains was added into “species 1” and the clade of the triple-reassortant swine H1N1 that sporadically infected humans in or after 2005 was added into “species 2”. strain. As shown in Figure 3, the H1N1/2009 pandemic strain shared great homology with a representative triple- reassortant strain, A/Iowa/CEID23/2005 (H1N1), in the PB2, PB1, PA, HA, NP, and NS gene segments. The reassortment with the NA and MP gene segments of swine H1N1 viruses of Eurasian lineage might enable the triple-reassortant swine-origin H1N1 viruses to cause the pandemic. Since the HA protein is critical for binding to cellular receptors and fusion of the viral and endosomal membranes (Neumann et al., 2009), we investigated the natural selection acting on this gene of the swine-origin viruses that infected humans from sporadic to pandemic. A positive selection might exist during the evolution of the HA gene from the classic swine H1N1 viruses that sporadically infected humans before 1998 to the triple- reassortant H1N1 viruses that sporadically infected humans from 2005 to 2009, possibly driving the adaptation of the classic swine H1N1 viruses to human at this stage. A positive selection might also act on the NA gene segment from the swine H1N1 virus of Eurasian lineage to the A/H1N1/2009 pandemic strain. The NA protein facilitates virus release from infected cells by removing sialic acids from cellular and viral HA and NA proteins (Neumann et al., 2009). Alterations of the NA are needed for the emergence of pandemic influenza viruses (Uhlendorff et al., 2009). The positive selection of the NA gene might drive the adaptation of this gene segment to human. Thus, in addition to the reassortment between gene segments of influenza A viruses of different lineages, nucleotide variations within a given gene segment and subsequent selection might play a role in the evolutionary process of swine-origin H1N1 viruses infected humans from sporadic to pandemic. However, there are population bottlenecks that might limit evidential power of the McDonald-Kreitman tests. This study has a number of limitations. Firstly, the viral sequences of the swine-origin viruses used in this study were mostly from the United States because sporadic infections with classic swine H1N1 before 1998 and with triple-reassortant swine H1N1 influenza A viruses since 2005 in persons with exposure to pigs were well- documented in this country, not in other countries. Secondly, the patients with slight symptom and syndrome might not be reported, possibly resulting in underestimation of human infections with swine-origin H1N1 viruses before the outbreak. Thirdly, not all sequences from the reported cases were available in GenBank, which might have resulted in bias of the analyses based on the incomplete data. In conclusion, this study clearly characterized the evolutionary process of swine-origin H1N1 influenza viruses that infected humans from sporadic to pandemic. In addition to the present knowledge, this study indicates that a representative triple-reassortant strain with intact sequence data of the 8 gene segments, A/Iowa/ CEID23/2005 (H1N1), may be a suitable “intermediate” in the evolutionary process of A/H1N1/2009 pandemic viruses. Monitoring the evolutionary steps of animal-origin influenza A viruses that occasionally infect humans from swine workers is of epidemiological significance for the prediction of novel influenza pandemic. ACKNOWLEDGEMENTS This work is supported by grant 06G65 (to Guangwen Cao) from the General Logistics of the People’s Liberation Army, People’s Republic of China. The fund had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. REFERENCES Babakir-Mina M, Dimonte S, Perno CF, Ciotti M (2009). Origin of the 2009 Mexico influenza virus: a comparative phylogenetic analysis of the principal external antigens and matrix protein. Arch. Virol., 154: 1349-1352. 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