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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. 
 

 
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