Genetic analysis and antigenic characterization of swine origin influenza viruses isolated from humans in the United States, 1990–2010

Virology 422 (2012) 151–160

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Genetic analysis and antigenic characterization of swine origin influenza viruses isolated from humans in the United States, 1990–2010☆ Bo Shu a, Rebecca Garten a, Shannon Emery a, Amanda Balish a, Lynn Cooper b, Wendy Sessions a, Varough Deyde c, Catherine Smith a, LaShondra Berman a, Alexander Klimov a, Stephen Lindstrom a, Xiyan Xu a,⁎ a b c

Influenza Division, National Center for Immunization and Respiratory Disease, Centers for Disease Control and Prevention, Atlanta, USA MITRE Corporation, 7515 Colshire Drive, McLean, VA 22102-7539, USA Centers for Disease Control and Prevention/Nigeria, Abuja, Nigeria

a r t i c l e

i n f o

Article history: Received 29 August 2011 Returned to author for revision 3 October 2011 Accepted 14 October 2011 Availabe online 10 November 2011 Keywords: Swine origin influenza viruses Triple reassortants

a b s t r a c t Swine influenza viruses (SIV) have been recognized as important pathogens for pigs and occasional human infections with swine origin influenza viruses (SOIV) have been reported. Between1990 and 2010, a total of twenty seven human cases of SOIV infections have been identified in the United States. Six viruses isolated from1990 to 1995 were recognized as classical SOIV (cSOIV) A(H1N1). After 1998, twenty-one SOIV recovered from human cases were characterized as triple reassortant (tr_SOIV) inheriting genes from classical swine, avian and human influenza viruses. Of those twenty-one tr_SOIV, thirteen were of A(H1N1), one of A(H1N2), and seven of A(H3N2) subtype. SOIV characterized were antigenically and genetically closely related to the subtypes of influenza viruses circulating in pigs but distinct from contemporary influenza viruses circulating in humans. The diversity of subtypes and genetic lineages in SOIV cases highlights the importance of continued surveillance at the animal–human interface. © 2011 Published by Elsevier Inc.

Introduction Influenza pandemics occur when a novel influenza virus with surface antigens [hemagglutination (HA) and/or neuraminidase (NA)] to which the majority of the human population has little or no preexisting immunity emerges in humans and is able to be transmitted efficiently from person to person. A novel HA or NA can be introduced into the human population either through the direct transmission of an animal (for example, avian) influenza virus to humans, or through reassortment between human and animal viruses, or between different lineages of swine influenza viruses (Garten et al., 2009). Swine influenza virus (SIV) A(H1N1), or so-called classical SIV (cSIV), was first isolated from pigs in the United States (U.S.) in 1930 (Shope, 1931). From the 1930s through the mid-1990s, the epidemiology of SIV within the continental U.S. remained fairly stable with cSIV being the predominant type of viruses isolated

☆ The findings and conclusions in this report are those of the authors and do not necessarily represent the views of the Centers for Disease Control and Prevention. ⁎ Corresponding author at: Virus Surveillance and Diagnostic Branch, Influenza Division, Centers for Disease Control and Prevention, 1600 Clifton Road, Atlanta, GA 30329, MS-G16, USA. Fax: + 1 404 639 0080. E-mail address: [email protected] (X. Xu). 0042-6822/$ – see front matter © 2011 Published by Elsevier Inc. doi:10.1016/j.virol.2011.10.016

from pigs (Karasin et al., 2000, 2002; Sheerar et al., 1989; Zhou et al., 2000). In 1997–98, a human influenza A (H3N2) [huA(H3N2)] virus appeared among North American pigs. As a result of several reassortment events, a triple reassortant(tr) _SIV A(H3N2) emerged among pig populations in North America containing genes from huA(H3N2), cSIV and North American avian lineage influenza viruses (Vincent et al., 2006; Zhou et al., 2000). The internal genes included PB2 and PA from North American avian viruses, PB1 from human viruses, and NP, M and NS from cSIV, termed the triple reassortant internal gene (TRIG) cassette (Vincent, et al., 2008). Reassortment in the pig population continued resulting in the appearance of tr_SIV of subtypes A (H1N1), A(H1N2), A(H3N1) and A(H2N3), all of which retained the TRIG cassette (Karasin et al., 2000, 2002; Lorusso et al., 2011; Ma et al., 2007; Olsen et al., 2000; Webby et al., 2004). Although SIV have been recognized as a common and important pathogen by the swine industry (Dacso et al., 1984; Hinshaw et al., 1978), human infections with swine origin influenza viruses (SOIV) including classical SOIV (cSOIV) and tr_SOIV were rarely detected. Sporadic human cases of SOIV infections have been reported around the world with limited human to human transmission (Gaydos et al., 1977; Myers et al., 2007; Newman et al., 2008; Shinde et al., 2009; Wentworth et al., 1994; Xu et al., 2008). In April 2009, a SOIV A(H1N1) containing a unique combination of gene segments from both North American and Eurasian swine lineages

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was identified from human infections in Mexico and the U.S., the virus rapidly spread worldwide causing the first influenza pandemic of the 21st century (CDC, 2009; Garten et al., 2009). The emergence of the 2009 A (H1N1) pandemic influenza [A(H1N1)pdm09] provided strong evidence that SIV have public health importance and pandemic potential (Smith et al., 2009). The systematic surveillance and characterization of SOIV among humans as well as SIV in pigs are critical for the early detection of viruses with pandemic potential. In this report, we present the genetic and antigenic characterization of twenty-seven SOIV isolated from humans in the U.S. between 1990 and 2010.

Results The twenty-seven SOIV examined in this study are listed in Table 1 along with subtype, cluster, and brief case information. In Table S1, GenBank/GISAID EPIFLU sequence accession numbers are provided.

Genetic and antigenic analyses of cSOIV isolated in 1990–95 Six human cases of influenza A(H1N1) cSOIV (A/Oklahoma/11/90, A/Maryland/12/91, A/Nebraska/01/92, A/Indiana/01/94, A/Minnesota/ 18/95, A/Indiana/05/95) isolated between 1990 and 1995 underwent phylogenetic and antigenic analysis. The HA genes of the six isolates were highly homogenous, grouping with the swine H1α cluster of cSIV described previously (Vincent et al., 2009) (Fig. 1). Whole genome phylogenetic analysis demonstrated that all eight gene segments were of cSIV origin and closely related to cSIV circulating among pigs in the U.S. at that time (Figs. 1, 3, S1). Antigenic analysis of the six cSOIV by hemagglutination inhibition (HI) test including reference ferret antisera, demonstrated antigenic homogeneity to each other and close antigenic relatedness to cSIV

Table 1 Swine origin influenza viruses isolated from humans analyzed in this study. Virus strain

A/Oklahoma/11/1990 A/Maryland/12/1991 A/Nebraska/01/1992 A/Indiana/01/1994 A/Minnesota/18/1995 A/Indiana/05/1995 A/Wisconsin/10/1998 A/Wisconsin/87/2005 A/Missouri/04/2006 A/Iowa/01/2006 A/Ohio/01/2007 A/Ohio/02/2007 A/Illinois/09/2007 A/Iowa/05/2007 A/Minnesota/03/2008 A/South Dakota/03/2008 A/Texas/14/2008 A/Iowa/02/2009 A/Minnesota/10/2009 A/Michigan/09/2007 A/Kansas/13/2009 A/Iowa/16/2009 A/Minnesota/09/2010 A/Wisconsin/12/2010 A/Pennsylvania/14/2010 A/Minnesota/11/2010 A/Pennsylvania/40/2010

Information of patient Age/sex

Exposure

Outcome

13 yr/M 37 yr/M 4 yr/F 37 yr/F 37 yr/F 4 yr/F 57 yr/M 17 yr/M 7 yr/M 4 yr/F 36 yr/M 10 yr/F 49 yr/F 2 yr/M 26 yr/F 19 yr/M 14 yr/M 3 yr/M 29 yr/F 1 yr/M 12 yr/M 12 yr/M 1 yr/F 9 mo/M 45 yr/M 31 yr/M 3 yr/F

Yes Yes Yes Yes Yes Yes Yes Yes No No Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes No No Yes No Yes Yes

Recovered Fatal Recovered Recovered Recovered Recovered Recovered Recovered Recovered Recovered Recovered Recovered Recovered Recovered Recovered Recovered Recovered Recovered Recovered Recovered Recovered Recovered Recovered Recovered Recovered Recovered Recovered

SOIV subtype

H1 HA cluster

cH1N1 cH1N1 cH1N1 cH1N1 cH1N1 cH1N1 tr_H1N1 tr_H1N1 tr_H1N1 tr_H1N1 tr_H1N1 tr_H1N1 tr_H1N1 tr_H1N1 tr_H1N1 tr_H1N1 tr_H1N1 tr_H1N1 tr_H1N1 tr_H1N2 tr_H3N2 tr_H3N2 tr_H3N2 tr_H3N2 tr_H3N2 tr_H3N2 tr_H3N2

H1 H1 H1 H1 H1 H1 H1 H1 H1 H1 H1 H1 H1 H1 H1 H1 H1 H1 H1 H1

Subtype abbreviation (c = classical; tr = triple reassortant). Mean age: 18.9 yr; male: 59%; female: 41%; pre-exposure to pig: 81%. Recovery rate: 96%.

α α α α α α γ γ β β γ γ γ β β β β γ β δ

circulating in pigs but a lack of antigenic relatedness to human A (H1N1) viruses from the same period (Table S2). Characterization of tr_SOIV A(H1N1), A(H1N2) and A(H3N2) isolated in 1998–2010 Genetic analysis HA and NA genes tr_SOIV A(H1N1). Phylogenetic analyses determined that HA and NA genes of the thirteen tr_SOIV A(H1N1) were genetically related to the tr_SIV which itself evolved from cSOIV A(H1N1) (Fig. 1). The HA genes of tr_SOIV A(H1N1) belong to two distinct clusters, H1γ and H1β, respectively (Vincent et al., 2009). The H1γ cluster contains six tr_SOIV isolates and tr_SIV mostly collected from states located east of the Mississippi River with the exception of A/Iowa/02/2009. The H1β cluster contains seven tr_SOIV and tr_SIV collected primarily from states located west of the Mississippi River (Fig. 1, S2). Numerous amino acid changes allowing differentiation of the two clusters of recent tr_SOIV A(H1N1) were identified as presented in Table S3. Analysis of the HA gene revealed eight potential glycosylation sites that were conserved among the thirteen viruses analyzed. Amino acid residues in the receptor binding site of these tr_SOIV were identical to those of tr_SIV isolated from pigs in North America. Compared with A(H1N1)pdm09, both cSOIV and tr_SOIV as well as SIV analyzed have an alanine at position 227(H3 numbering), while the A(H1N1) pdm09 viruses possess a glutamic acid at this position (Table S3). The phylogeny of the N1 gene of the tr_SOIV showed clustering similar to that seen in HA, with the exception of A/Iowa/02/2009 (H1γ) whose NA clustered with isolates from H1β cluster (Fig. 1 and Table S4). tr_SOIV A(H1N2). A single case of tr_SOIV A(H1N2) infection was detected in the U.S. in 2007. The HA gene of this isolate, A/Michigan/ 09/2007 A(H1N2), was closely related to human seasonal A(H1N1) viruses within cluster H1δ (Fig. 1). Nucleotide divergence between A/Michigan/09/2007 and seasonal A(H1N1) vaccine strain A/Brisbane/ 59/2007 was 5.1%, illustrating the close relatedness of the two HAs. When compared with tr_SOIV H1γ and H1β, the nucleotide divergences were 27.6–29.2%, respectively (Table S5). The NA gene of the tr_SOIV A(H1N2) virus was closely related to recent tr_SOIV A(H3N2) (Fig. 2) with the lowest nucleotide divergence (1.5%) to A/Kansas/ 13/2009, a tr_SOIV A(H3N2) (Fig. 1 and Table S6). tr_SOIV A(H3N2). HA genes of the seven tr_SOIV A(H3N2) clustered with tr_SIV A(H3N2) viruses currently circulating in North America. The tr_SIV A(H3N2) were introduced into the swine population in the late 1990s and have since evolved separately from the human seasonal A(H3N2) viruses (Fig. 2). Four tr_SOIV A(H3N2) viruses isolated in 2010 (A/Pennsylvania/40/2010, A/Minnesota/09/ 2010, A/Minnesota/11/2010 and A/Wisconsin/12/2010) clustered together in the HA phylogenetic tree. A/Iowa/16/2009 grouped with tr_SIV A(H3N2) viruses from Québec, Canada (Fig. 2 and Table S7). A/Kansa/13/2009 was located in a distinct subgroup with other U.S. tr_SIV A(H3N2) viruses sharing six characteristic amino acid changes (Table S7). Compared to the HA gene, increases in nucleotide and amino acid divergence and different phylogenetic clustering patterns were evident among the NA genes of tr_SOIV A(H3N2) (Fig. 2 and Tables S7, S8). The NA genes of six tr_SOIV A(H3N2) were found in three separate branches, with the majority clustering with A/Michigan/09/2007A(H1N2). A/Minnesota/11/2010 and A/Pennsylvania/ 14/2010 were in the same subgroup and both possessed long side branches. The N2 of A/Iowa/16/2009 clusters with other tr_SIV but separate from the recent tr_SOIV isolates (Fig. 2, Table S8). Internal genes of tr_SOIV. All 21 tr_SOIV viruses had internal gene constellations as North American tr_SIV viruses, bearing the TRIG cassette described previously (Vincent et al., 2008). However, phylogenetic groups of the internal genes were not consistent with those of

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the HA genes. All internal genes of the tr_SOIV grouped separately from A(H1N1)pdm09 viruses. Polymerase genes of the tr_SOIV were consistent with the conserved TRIG cassette; PB2 and PA genes of all tr_SOIV were of avian lineage and PB1 genes were of human origin, closely related to those of human A(H3N2). Amino acid 627 of the PB2 genes of all tr_SOIV remained as glutamic acid. All but A/Kansas/13/2009 have a full length PB1-F2 open reading frame encoding 90 amino acids

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(aa); the PB1-F2 of A/Kansas/13/2009 virus was truncated, encoding a 57 aa peptide. NP, M and NS genes. Phylogenetic analyses found that the NP, M and NS genes of these tr_SOIV isolates clustered closely with cSOIV (Fig. S1), consistent with the TRIG cassette. The NS1 genes of the 21 tr_SIV lack the deletion at positions 80–84 or a glutamic acid at position 92 previously shown to be potential virulence factors (Long et al., 2008; Seo et al., 2002).

Fig. 1. Phylogenetic analysis of the H1HA, N1NA genes of SOIV in comparison with SIV, human seasonal A(H1N1) and 2009 A (H1N1) pandemic influenza viruses. HA phylogenetic tree with four clusters of related viruses, swH1α, swH1β, swH1γ and swH1δ indicated by the bars on the right; NA phylogenetic tree (according to HA clusters) with three clusters, swH1α, swH1β, swH1γ and swH1δ indicated by the bars on the right. The SOIV analyzed in this study are shown in bold and italic face, 2009 A (H1N1) pandemic influenza virus strain A/California/07/2009 with underline. The bootstrap values are shown next to the branches. Abbreviations are as follows: sw = swine; hu = human; H1pdm09 = 2009 A (H1N1) pandemic influenza virus.

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Fig. 1. (continued).

Known genetic markers conferring antiviral resistance to adamantanes or neuraminidase-inhibitors were not detected in the M2 or NA genes of the 27 SOIV, respectively.

Antigenic analysis Subsets of tr_SOIV were antigenically characterized in HI tests using post-infection ferret sera raised against representative swine and/or human influenza viruses. As shown in Table 3, representative A(H1N1) tr_SOIV isolates were well inhibited by antisera raised against A(H1N1) tr_SOIV as well as by the antiserum to A(H1N1) pdm09 vaccine strain A/California/07/2009 but were not inhibited by antiserum raised against A/Brisbane/59/2007, a previous seasonal A(H1N1) vaccine strain. These results indicated that the HA glycoprotein of A(H1N1) tr_SOIV was antigenically distinct from seasonal A(H1N1) viruses. A(H1N1) tr_SOIV were antigenically homogenous; viruses from the H1γ genetic group were antigenically indistinguishable from those in the H1β group. In contrast, A/Michigan/09/2007 (H1N2) virus was inhibited only by its own homologous antiserum and the antiserum raised against A/Brisbane/59/2007, indicating

that A/Michigan/09/2007 virus was more antigenically related to contemporary human seasonal A(H1N1) viruses (Table 2). Six tr_SOIV A(H3N2) tested were poorly inhibited by antisera raised against recent seasonal A(H3N2) viruses (Table 3) including the current vaccine strain A/Perth/16/2009. It is also noticed that two SOIV viruses demonstrated cross reactivity with antisera to A(H3N2) viruses which circulated in the early 1990s. This finding indicates that A(H3N2) tr_SOIV were antigenically distinct from currently circulating human A(H3N2) viruses. With the exception of one tr_SOIV A(H3N2), all were antigenically homogenous; A/Kansa/13/2009 demonstrated an 8-fold or greater reduction in HI titers to antisera raised against other tr_SOIV A(H3N2) viruses. Conversely, most tr_SOIV were weakly inhibited by antiserum raised against A/Kansas/13/2009, demonstrating the antigenic divergence of A(H3N2) tr_SOIV. Discussion Human cases of SOIV infection have been reported previously from Europe, Asia and North America including the US (Claas et al., 1994; Komadina et al., 2007; Myers et al., 2007), those cases were identified by either serological investigation and/or by virus isolation.

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SOIV from Asia possessed an HA that is similar to classical swine H1 viruses circulated in Asia and North America while their remaining genes were found to be more closely related to viruses circulating in European swine (Komadina et al., 2007); SOIV isolated from Europe were avian–human reassortant viruses with their HA and NA genes from a human H3N2 viruses and remaining genes from an avian virus (Claas et al., 1994). Between 1990 and 2010, a total of twenty seven cases of human infections with SOIV were detected by CDC. Six cases identified before 1995 were cSOIV A(H1N1); while the remaining twenty one were tr_SOIV of varying subtype, including thirteen A(H1N1), one A(H1N2) and seven A(H3N2). All but one tr_SOIV (A/Wisconsin/10/1998) were identified between 2005 and 2010. The increased number of documented cases since 2005 may be partially due to improved virologic surveillance, implementation of new testing technologies, and the introduction of requirements for reporting of novel (different from seasonal) virus infections in the U.S. Although tr_SIV A(H3N2) was first detected in pigs in 1998, the first human cases were not identified until 2005 in Canada (Olsen et al., 2006) and 2009 in the U.S. (Cox et al., 2011). All seven human cases of tr_SOIV A(H3N2) were identified by using the CDC

155

real-time RT-PCR detection panels which allow differentiation of North American lineage SIV from human seasonal influenza viruses by detection of swine origin NP gene. All the cases were further confirmed by genetic sequence analysis (CDC, 2008; Shu et al., 2011). In this study, we report two new subtypes of tr_SOIV, A(H1N2) and A(H3N2), identified in the U.S. A/Michigan/09/2007 (H1N2) was isolated from an infant and possessed an H1HA from contemporary human seasonal A(H1N1) viruses with the remaining genes from tr_SIV. Introduction of seasonal A(H3N2) into the swine gene pool in the mid 1990s was followed by an evolutionary path for the H3 HA and N2 NA genes in the swine population separate from their human counterparts. The full genomes of the seven tr_SOIV A(H3N2) characterized in this study were closely related to SIV A(H3N2) circulating in American pigs since 1998. The increased diversity of the SOIV gene pool since 1998 is directly linked to the high level of reassortment of SIV with viruses from diverse species. Once established in the swine population, reassortment continues within that host population. Due to inadequate surveillance in pigs, human cases of SOIV have at times served as sentinels for detection of SIV epidemic among pigs resulting in the identification of circulating variants in swine (Cooper et al., 1999).

Fig. 2. Phylogenetic analysis of the HA and NA genes of influenza A(H3N2) viruses. The sequences obtained from this study are shown in bold and italic face. The bootstrap values are shown next to the branches. Abbreviations are as follows: sw = swine; hu = human.

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Fig. 2. (continued).

The majority of SOIV cases described in this report were single isolated infections and patients had a history of direct or indirect exposure to pigs prior to onset of illness, such as visiting animal exhibitions at local agriculture fair, visiting live animal market, butchering pig, etc. Separate clusters of two cases from Ohio in 2007 (H1N1) and Minnesota in 2010 (H3N2) were within families. Both patients from Ohio but only one patient from Minnesota had a history of exposure to pigs. The second case from the Minnesota cluster lacked a confirmed pre-exposure history and thus limited human to human transmission of SIV cannot be excluded. In animal model transmission experiments, Belser and colleagues reported that tr_SOIV A(H1N1) exhibited insufficient transmission among experimental ferrets (Belser et al., 2011). In contrast, A(H1N1)pdm09 viruses transmitted efficiently among ferrets (Maines et al., 2009). Transmission studies of human lineage derived tr_SOIV A(H3N2) are needed for a more complete characterization. In U.S., the majority of pigs were initially produced in the American South-Central and Southeastern regions then follow the main swine transportation routes to the Midwest, a corn-rich commercial center, where they are fattened before slaughter. The Midwest is likely to provide a reservoir allowing multiple, genetically-distinct variants to

cocirculate with the potential to exchange gene segments via reassortment due to the continual importation of SIV from other regions (Nelson et al., 2011; Shields and Mathews, 2010). Of the 21 human cases of tr_SOIV infection, eighteen cases (85%) were from the Midwest and had different gene constellations (Table 1, Fig. S2). This finding further underscores the importance of improving swine– human interface surveillance for the rapid detection of human infections with SIV and identification of new variant SIV. Vincent and colleagues have documented the genetic diversity of North American lineage swine viruses in all gene segments, especially the HA genes in which multiple genetic clusters were identified (Vincent et al., 2009). In our study, seven A(H1N1) tr_SOIV viruses isolated from states located west of the Mississippi River belonged to the previously defined swine H1β genetic cluster, while the remaining six A(H1N1) tr_SOIV viruses isolated east of the Mississippi River belonged to the swine H1γ cluster (Vincent et al., 2009). One isolate, A/Iowa/02/2009, was a reassortant between the tr_SOIV clusters, bearing an HA of H1γ (differing from other Iowa isolates) and an NA of H1β. These observations of geographic distribution patterns for different clades of SIV in the U.S. may be useful for determining the origin of SIV.

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Fig. 3. Genotypes and derivation of swine origin influenza viruses isolated from U.S., 1990–2010. Triple reassortant internal gene (TRIG) cassette was marked as gray.

Although key amino acids in the receptor binding site are conserved among SIV A(H1N1) and A(H1N1)pdm09 viruses, A(H1N1) pdm09 viruses possess a glutamic acid at position 227, similar to contemporary human seasonal A(H1N1) viruses, all other SIV and SOIV A(H1N1) have an alanine at this position. Additional studies on the role of substitutions, such as 227 on host receptor affinity and transmission, are needed. The PB1-F2 protein has been identified as a key virulence factor among influenza A viruses (Gibbs et al., 2003; Horimoto et al., 2010; Munster et al., 2009; Zell et al., 2007), associated with increased pathogenicity of highly pathogenic A(H5N1) and the 1918 pandemic influenza virus (Conenello and Palese, 2007; Conenello et al., 2007). A truncated PB1-F2 ORF has been linked to sustained viral replication and increased proinflammatory responses (Zell et al., 2007). All tr_SOIV except A/Kansas/13/2009 had a typical, full length (90 amino acids) PB1-F2 ORF as generally seen in SIV. In contrast, the A(H1N1)

pdm09 viruses have a truncated, 11-amino acids ORF (Garten et al., 2009), which appears to be an identifying genetic feature of the swine origin A(H1N1)pdm09 virus. Despite the fact that numerous amino acids differences have been found between the cSOIV and A(H1N1) tr_SOIV, as well as between two recent clusters of A(H1N1) tr_SOIV isolated since 1990, those changes do not appear to be significant antigenically. Conversely, the tr_SOIV A(H3N2) retain antigenic properties more similar to human seasonal viruses from the early 1990s and are thus antigenically distinct from contemporary seasonal A(H3N2) viruses. This observation indicates that the antigenic evolution of SIV may be different from that of human influenza viruses in which host immune pressure often contributes to antigenic variation necessitating revisions of human influenza vaccine components (Boni, 2008; Chen and Deng, 2009). Our results also demonstrate that antiserum raised to the human pandemic strain, A/California/07/2009, the A(H1N1) component of

Table 2 Hemagglutination inhibition reactions of tr_SOIV A (H1N1). Virus strain

H1 β A/Missouri/04/06 A/Minnesota/03/08 A/Texas/14/08 A/South Dakota/03/08 A/sw/Minnesota/02 A/sw/Iowa/06 H1 γ A/Wisconsin/10/98 A/Wisconsin/87/05 A/Ohio/02/07 A/Illinois/09/07 A/Iowa/02/09 A(H1N1)pdm09 A/California/07/09 H1 δ A/Michigan/09/07 A/Brisbane/59/07

Ferret antiserum sw/MN/02

sw/IA/06

WI/10/98

OH/2/07

IL/9/07

CA/07/09

5120 1280 2560 2560 2560 1280

5120 1280 1280 1280 1280 2560

Nta Nta Nta Nta 640 320

5120 5120 5120 5120 5120 5120

1280 640 640 1280 640 640

Nta Nta Nta Nta 2560 320

5 5 5 5 5 5

5 5 5 5 5 5

1280 2560 2560 512 5120

1280 2560 2560 2560 5120

2560 Nta 1280 640 5120

5120 5120 5120 5120 5120

1280 2560 2560 5120 2560

1280 Nta 2560 2560 2560

5 5 5 5 5

5 5 5 5 5

1280

1280

640

2560

1280

2560

5

5

5 5

5 5

5 5

80 5

320 20

2560 640

Bold numbers indicate homologous HI titers. a Not tested.

5 5

5 5

MI/1/07

Bris/59/07

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Table 3 Hemagglutination inhibition reactions of tr_SOIV A(H3N2). Virus strain

tr_-SOIV A/Kansas/13/09 A/Iowa/16/09 A/Minnesota/09/10 A/Wisconsin/12/10 A/Pennsylvania/14/10 A/Minnesota/11/2010 A/sw/Pennsylvania/062170-1/10 Human seasonal A/Beijing/32/92 A/Ann Arbor/09/93 A/Johannesburg/33/94 A/Wuhan/359/95 A/Perth/16/2009a

Ferret antiserum KS/13/09

WI/12/10

PA/14/10

MN/11/10

BJ/32/92

AA/09/93

JOHAN/33/94

WUH/359/95

PE/16/09

2560 320 20 20 80 10 640

320 640 1280 1280 320 1280 2560

640 1280 640 320 1280 640 2560

80 320 640 640 320 640 640

20 160 10 20 80 5 5

10 80 40 5 40 20 10

5 10 5 5 10 5 5

10 5 5 5 5 5 5

5 10 5 5 5 5 5

5 5 5 5 5

5 5 5 5 5

10 5 5 5 5

10 5 5 5 40

80 80 80 80 5

40 320 640 40 5

20 320 320 20 5

5 10 5 320 5

5 5 5 40 1280

Bold numbers indicate homologous HI titers.

the current influenza vaccine, well inhibited the cSIOV and A(H1N1) tr_SOIV tested, indicating that currently licensed trivalent influenza vaccine may provide adequate protection against cSIV or A(H1N1) tr_SIV infections in humans. However, A(H3N2) tr_SOIV were antigenically distinct from the current A(H3N2) vaccine component (Table 3) and thus vaccination with current trivalent influenza vaccines may not provide protection against A(H3N2) tr_SIV infection in humans. In August 2011, four additional cases of tr_SOIV A(H3N2) infections were reported by the CDC (CDC, 2011). Sequence analysis revealed that those 2011 tr_SOIV A(H3N2) obtained seven gene segments, including both HA and NA genes, from a contemporary tr_SIV A(H3N2) and are similar to that of tr_SOIV A(H3N2) described in this study; while M gene of these 2011 tr_SOIV A(H3N2) viruses was acquired from a A(H1N1)pdm09 virus (CDC, 2011). (Details will be discussed in a separate publication which is currently being drafted) The facts that the first influenza pandemic of the 21st century was caused by a swine origin virus and human infections with various subtypes of SIV continue to be documented in the U.S. and other countries, clearly demonstrate the importance of further strengthening influenza surveillance in both human and pig populations.

Terminator v3.1 Cycle Sequencing Kit with reaction products resolved on an Applied Biosystems Sequencer 3730 DNA Analyzer.

Sequence analysis Sequences analyzed were either obtained from this study or from the NCBI Influenza Resource (http://www.ncbi.nlm.nih.gov/genomes/ FLU/). Nucleotide sequences were aligned using the CLUSTALW program. Sequences alignment results were further analyzed using the BioEdit program (http://www.mbio.ncsu.edu/BioEdit/). Phylogenetic analyses were carried out using Molecular Evolutionary Genetics Analysis software (MEGA, version 5.0) (Tamura et al., 2011). The evolutionary history was inferred using the NeighborJoining method (Saitou and Nei, 1987). The evolutionary distances were computed using the Tamura–Nei method (Tamura and Nei, 1993). The bootstrap value (1000 replicates) of HA and NA genes is shown next to the branches (Felsenstein, 1985) (Figs. 1 and 2). Sequences obtained from this study have been submitted to GenBank and the accession numbers are listed in Table 1 and S1.

Materials and methods

Nucleotide divergence

Clinical specimens and virus isolates

Multiple sequence alignment and evolutionary distance analyses were performed using programs in Accelrys GCG, Version 11.1.2UNIX (Genetic Computer Group, Accelrys Inc., San Diego, CA). The extent of nucleotide heterogeneity was examined by unbiased estimates of nucleotide divergence calculated according to Nei and Li using the program ARLEQUIN (Excoffier and Lischer, 2010). Divergence times were calculated using BEAST (Version 1.4.8; http:// beast.bio.ed.ac.uk/) (Drummond and Rambaut, 2007). Substitution rates for each ORF or region of the genome used in the determination of population dynamics were calculated according to the method of Moratorio et al. (2007). The GTR substitution model with four gamma categories and invariant sites was used for each calculation. Codons were grouped into three partitions and the substitution model was unlinked across codon positions. UPGMA was used to construct a starting tree. Each analysis was run so that the effective sample size was greater than 200, unless otherwise noted.

Original clinical specimens or virus isolates were submitted by the U.S. State or local public health laboratories. Viruses used in this study were propagated in either Madin–Darby canine kidney (MDCK) cells or in the allantoic/amniotic cavities of embryonated chicken eggs. RNA extraction Viral RNA was isolated from 100 μl of original clinical specimen or viral culture medium using the MagNA Pure Compact RNA isolation kit or MagNA Pure Compact Nucleic Acid Isolation Kit I on a MagNA Pure Compact instrument (Roche Applied Science), according to manufacturer's instructions. PCR and DNA sequencing Invitrogen SuperScript™III One-Step RT-PCR System with Platinum® Taq High Fidelity kits were used for PCR amplifications. PCR primers for amplifications of all eight genes are available up to request. PCR products were purified by ExoSAP-IT® for PCR Product Clean-Up kit (USB Corporation, USA). Sequencing reactions were performed using an Applied Biosystems BigDye®

Antigenic analysis The antigenic characteristics of virus isolates were determined by HI tests using post-infection antisera. The HI test was performed as described previously (Kendal and Cate, 1983).

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