Microbiol Immunol 2014; 58: 327–341 doi: 10.1111/1348-0421.12152

ORIGINAL ARTICLE

Reassortant swine influenza viruses isolated in Japan contain genes from pandemic A(H1N1) 2009 Katsushi Kanehira, Nobuhiro Takemae, Yuko Uchida, Hirokazu Hikono and Takehiko Saito Influenza and Prion Disease Research Center, National Institute of Animal Health, National Agriculture and Food Research Organization (NARO), 3-1-5 Kannondai, Tsukuba, Ibaraki 305-0856, Japan

ABSTRACT In 2013, three reassortant swine influenza viruses (SIVs)—two H1N2 and one H3N2—were isolated from symptomatic pigs in Japan; each contained genes from the pandemic A(H1N1) 2009 virus and endemic SIVs. Phylogenetic analysis revealed that the two H1N2 viruses, A/swine/Gunma/1/2013 and A/swine/Ibaraki/1/2013, were reassortants that contain genes from the following three distinct lineages: (i) H1 and nucleoprotein (NP) genes derived from a classical swine H1 HA lineage uniquely circulating among Japanese SIVs; (ii) neuraminidase (NA) genes from human-like H1N2 swine viruses; and (iii) other genes from pandemic A(H1N1) 2009 viruses. The H3N2 virus, A/swine/ Miyazaki/2/2013, comprised genes from two sources: (i) hemagglutinin (HA) and NA genes derived from human and human-like H3N2 swine viruses and (ii) other genes from pandemic A(H1N1) 2009 viruses. Phylogenetic analysis also indicated that each of the reassortants may have arisen independently in Japanese pigs. A/swine/Miyazaki/2/2013 were found to have strong antigenic reactivities with antisera generated for some seasonal human-lineage viruses isolated during or before 2003, whereas A/swine/Miyazaki/2/2013 reactivities with antisera against viruses isolated after 2004 were clearly weaker. In addition, antisera against some strains of seasonal human-lineage H1 viruses did not react with either A/swine/Gunma/1/2013 or A/swine/Ibaraki/1/2013. These findings indicate that emergence and spread of these reassortant SIVs is a potential public health risk. Key words

pandemic A(H1N1) 2009, reassortant influenza virus, swine influenza virus.

Swine influenza virus belongs to the family Orthomyxoviridae and causes a highly contagious respiratory disease that is typically characterized by high fever, anorexia, inactivity, abdominal breathing and dyspnea (1). SIV, along with porcine reproductive and respiratory syndrome virus and Mycoplasma hyopneumoniae, is a major pathogen that is detected in 10- to 22week-old pigs with clinical signs of porcine respiratory disease complex. Notably, SIV infections can impair reproductive ability; coinfection with SIV and porcine reproductive and respiratory syndrome virus or M. hyopneumoniae can seriously compromise reproductive ability (2, 3). In addition, because the tracheal epithelium in pigs expresses receptors for avian and human

influenza viruses, domestic pigs may be mixing vessels for the emergence of novel reassortant viruses that could potentially cause human pandemics (4). The broad susceptibility of pigs to influenza viruses is also reflected in the predominant SIV lineages. The classical H1N1 SIVs originated from the Spanish influenza virus that caused the devastating human pandemic that occurred from 1918 to 1919; these lineages predominated among pigs in Europe and North America until the 1990s (5). Since 1979, the classical swine H1N1 lineage has largely been replaced in Europe by an H1N1 virus of avian origin that possessed a genome comprising entirely avian virus genes; this avian H1N1 lineage has since become endemic to Europe (6). Notably, H1N2 SIVs have

Correspondence Takehiko Saito, Influenza and Prion Disease Research Center, National Institute of Animal Health National Agriculture and Food Research Organization (NARO), 3-1-5 Kannondai, Tsukuba, Ibaraki 305-0856, Japan. Tel: þ81 29 838 7757; fax: þ81 29 838 7758; e-mail: [email protected] Received 13 February 2014; revised 3 April 2014; accepted 14 April 2014. List of Abbreviations: HA, hemagglutinin; HI, hemagglutination inhibition; NA, neuraminidase; NP, nucleoprotein; SIV, swine influenza virus.

© 2014 The Societies and Wiley Publishing Asia Pty Ltd

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co-circulated with avian-origin H1N1 SIVs among European pigs; the HA genes of these H1N2 lineages are genetically and antigenically distinct from those of the avian-origin H1N1 SIVs. Moreover, the N2 variants of the NA genes in the H1N2 SIVs derive from a human epidemic strain (7). In 1997, in a H3N2 lineage that originated solely from a human epidemic strain in the USA, two types of reassortant H3N2 SIVs also appeared. One of these reassortant H3N2 lineages is a double reassortant that contains genes from (i) classical swine and (ii) seasonal human influenza viruses (8), whereas the other is a triple reassortant H3N2 virus with a genetic constellation comprising genes from (i) a seasonal human influenza, (ii) a classical swine H1N1 and (iii) avian influenza viruses (9). Following the emergence of pandemic A(H1N1) 2009 (pdm09) virus, a quadruple reassortant containing six genes from a triple reassortant H1N1 virus and two genes—an NA and an M gene— from an avian-origin SIV found in Europe and Asia (10) spread among human populations; moreover, pdm09 viruses have been isolated from pigs in many areas of the world (11, 12). Reportedly, the pdm09 viruses have independently reassorted with endemic SIVs in many regions globally (13–27). Outbreaks of classical H1N1 swine influenza were first observed in the late 1970s in Japan (28). A H1N2 SIV lineage possessing the NA gene segment from human epidemic H3N2 and all other gene segments from classical H1N1 SIV was generated by reassortment soon after the invasion of classical H1N1 (29). This lineage has become the predominant SIV in Japan; this predominance has continued to the present (30). Human-like H3N2 strains originating from a human epidemic strain have only been isolated sporadically in Japan (31), whereas pdm09 viruses have been repeatedly isolated from pigs in Japan since late 2009 (32). Notably, a reassortant SIV that contained pdm09 genes and an endemic SIV gene was isolated from a healthy pig by active surveillance (33); since then, three reassortants that contained pdm09 genes and endemic SIV genes have been isolated independently from symptomatic pigs. Here, the three isolates of this reassortant SIV were analyzed phylogenetically and serologically to acquire information for livestock hygiene and public health purposes.

MATERIALS AND METHODS Viruses MDCK cells were used in the Gunma Prefectural Livestock Health Laboratory to isolate A/swine/ Gunma/1/2013 (H1N2; Gun13) from the lung of a dead pig; in the Ibaraki Prefectural Livestock Hygiene 328

Center to isolate A/swine/Ibaraki/1/2013 (H1N2; Iba13) from the lung of an autopsied pig with respiratory symptoms; and in the Miyazaki Prefectural Livestock Hygiene Center to isolate A/swine/Miyazaki/2/2013 (H3N2; Miy13) from a nasal swab taken from a piglet with respiratory symptoms. Other SIV strains that were used as the reference strains in HI assays are listed in Table 1; these reference SIVs were propagated in embryonated chicken eggs or in MDCK cells to be used in HI assays. Nucleotide sequencing and genetic analysis Gun13, Iba13, Miy13 and three reassortant viruses used as reference viruses in this study, A/swine/Saitama/01/ 2005 (H1N2; Sai05), A/swine/Ehime/1/2002 (H3N2; Ehi02), and A/swine/Nagano/2000 (H3N2; Nag00), were subjected to viral RNA extraction using an RNeasy Mini Kit (Qiagen, Hilden, Germany). Sequencing of full coding sequences of whole genomic segments from Gun13, Iba13 and Miy13, of whole genomic segments except for HA from Sai05 and Nag00, and of HA and NA segments of Ehi02 were determined by direct sequencing of RT-PCR products. These nucleotide coding sequences and sequences of reference viruses from NCBI GenBank, each comprising all eight genetic segments, were used for phylogenetic analyses to determine the genetic relationships among these three reassortant viruses and reference viruses using two software programs, BioEdit software version 7.0.5.3 (34) and Mega 5 (35). Additionally, the deduced amino acid sequence of the HA proteins were compared among the viruses. Antisera Hyperimmune antisera against formalin-inactivated virus were prepared in chickens. Briefly, allantoic fluid or supernatant from MDCK cell cultures that contained infectious virus particles was inactivated with 0.05% or 0.1% formalin and then concentrated by ultracentrifugation; these samples were then subjected to differential centrifugation through a sucrose density gradient. Each concentrated antigen with a protein content of 50 or 100 mg was inoculated i.m. into one chicken, and each chicken was inoculated at least four times with either incomplete or complete Freund’s adjuvant (Sigma– Aldrich, St. Louis, MO, USA). Experimental procedures and animal care were in accordance with the guidelines of the National Institute of Animal Health’s Animal Care and Use and Biosafety Committees. Post-infection ferret antisera against human seasonal influenza strains and homologous inactivated antigens were kindly provided by the National Institute of Infectious Diseases, Japan. © 2014 The Societies and Wiley Publishing Asia Pty Ltd

Reassortants of A(H1N1) pdm09 in pigs

Table 1. Viruses used in this study

Virus H1 viruses A/swine/Gunma/1/2013 A/swine/Ibaraki/1/2013 A/swine/Iowa/15/1930 A/swine/Kyoto/3/1979 A/swine/Niigata/729/2004 A/swine/Saitama/01/2005 A/swine/Tochigi/1/2008 A/swine/Tochigi/2/2011 A/swine/Binh Duong/02-16/2010 A/California/04/2009 A/swine/Narita/aq21/2011 A/New Caledonia/20/1999 A/Solomon Islands/3/2006 A/Brisbane/59/2007 A/duck/Tsukuba/67/2005 H3 viruses A/swine/Miyazaki/2/2013 A/swine/Nagano/2000 A/swine/Wadayama/5/1969 A/swine/Ehime/1/2002 A/swine/Osaka-C/12-20/2008 A/swine/Binh Duong/03-14/2010 A/swine/Yokohama/aq114/2011 A/Wuhan/359/1995 A/Sydoney/5/1997 A/Panama/2007/1999 A/Wyoming/03/2003 A/New York/55/2004 A/Hiroshima/52/2005 A/Uruguay/716/2007 A/Victoria/361/2011 A/budgerigar/Aichi/1/1977

Abbreviation

Subtype

Genetic lineage of HA gene

Country of origin

HA gene

Gun13 Iba13 Iow30 Kyo79 Nii04 Sai05 Toc08 Toc11 BD16/10 Cal09 Nar11 NC99 SI06 Bri07 Tsu05

H1N2 H1N2 H1N1 H1N1 H1N2 H1N2 H1N2 H1N2 H1N2 H1N1 H1N1 H1N1 H1N1 H1N1 H1N1

Classical swine Classical swine Classical swine Classical swine Classical swine Classical swine Classical swine Classical swine Human-like swine A(H1N1) pdm09 A(H1N1) pdm09 Seasonal human Seasonal human Seasonal human Avian

Japan Japan USA Japan Japan Japan Japan Japan Vietnam USA Japan (Denmark)† New Caledonia Solomon Islands Australia Japan

AB921005 AB921004 AF091308 AB434384 AB600850 AB762401 AB514929 AB741007 AB762408 FJ966082 AB741039 CY033622 EU124177 CY058487 n.a.

Miy13 Nag00 Wad69 Ehi02 Osa08 BD14/10 Yok11

H3N2 H3N2 H3N2 H3N2 H3N2 H3N2 H3N2

Japan Japan Japan Japan Japan Vietnam Japan

AB921006 AB762409 D21183 AB762411 AB762413 AB598503 AB741023

Wuh95 Syd97 Pan99 Wyo03 NY04 Hir05 Uru07 Vic11 Aic77

H3N2 H3N2 H3N2 H3N2 H3N2 H3N2 H3N2 H3N2 H3N8

Human-like swine Human-like swine Human-like swine Human-like swine Human-like swine Human-like swine Human-like swine (triple reassortant SIV: cluster IV) Seasonal human Seasonal human Seasonal human Seasonal human Seasonal human Seasonal human Seasonal human Seasonal human Avian

China Australia Panama USA USA Japan Uruguay Australia Japan

CY112821 CY039079 DQ508865 EU268227 CY033638 EU501660 EU716426 KC306165 n.a.

†

, isolated from swine imported from Denmark. n.a., not available.

Serological analysis Previously described HI assays were used to assess the antigenic reactivities of each H1 or H3 virus (27). Briefly, hyperimmune antisera were treated for 20 hr with receptor-destroying enzyme from Vibrio cholera (Denka Seiken, Tokyo, Japan) to remove non-specific inhibitors of hemagglutination, after which complement system proteins and the receptor-destroying enzyme in these mixtures were heat-inactivated at 56°C for 30 min. Subsequently, antisera were absorbed with packed guinea pig red blood cells for 60 min at room temperature. For each treated antiserum, a dilution series was generated via serial 10-fold dilutions with PBS; each dilution series was used for HI tests. Guinea pig erythrocytes were resuspended in PBS (0.5% v/v) and © 2014 The Societies and Wiley Publishing Asia Pty Ltd

used in each HI assay. A cutoff value of 1:20 was adopted to avoid false positives caused by non-specific reactions.

RESULTS Genomic analysis of SIVs isolated in 2013 in Japan Three type A influenza viruses—Gun13, Iba13 and Miy13 —were subjected to genomic sequencing and phylogenetic analysis; the neighbor-joining method was used to classify ancestries of whole genomic segments of these viruses. The HA gene of Gun13 and Iba13 was found to have originated from a classical swine H1 HA lineage that has been circulating only among Japanese SIVs (Fig. 1a). These HA genes had clustered together and formed a sub329

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A/Athens/INS554/2011(H1N1) A/swine/Narita/aq21/2011(H1N1) A/Florida/06/2012(H1N1) A/swine/Yamagata/11/2010(H1N1) A/Aichi/202/2009(H1N1) A/swine/Osaka/1/2009(H1N1) A/California/04/2009(H1N1) A/swine/Gunma/1/2012(H1N2) A/swine/Guangxi/13/2006(H1N2) A/swine/OH/511445/2007(H1N1) A/Swine/Indiana/9K035/99(H1N2) A/Swine/Ohio/891/01(H1N2) A/Swine/North Carolina/93523/01(H1N2) A/swine/Korea/CAS08/2005(H1N1) A/swine/Shanghai/1/2007(H1N2) A/swine/Ratchaburi/NIAH1481/2000(H1N1) A/swine/Ontario/57561/03(H1N1) A/swine/Ontario/23866/04(H1N1) A/swine/Okinawa/1/2005(H1N1) A/swine/Saraburi/NIAH13021/2005(H1N2) A/swine/Saraburi/NIAH100761-22/2009(H1N1) A/swine/Chonburi/NIAH589/2005(H1N1) A/swine/Niigata/729/2004(H1N2) A/swine/Tochigi/1/2008(H1N2) A/swine/Aomori/1/2005(H1N2) A/swine/Miyazaki/1/2006(H1N2) A/swine/Saitama/21/2004(H1N2) A/swine/Ibaraki/1/2013(H1N2) A/swine/Gunma/1/2013(H1N2) A/swine/Tochigi/2/2011(H1N2) A/swine/Mie/R02/2012(H1N2) A/swine/Saitama/1996(H1N2) A/swine/Saitama/1/2000(H1N2) A/swine/Saitama/01/2005(H1N2) A/swine/Kyoto/3/1979(H1N1) A/swine/Niigata/1/1977(H1N1) A/swine/Iowa/4/1976(H1N1) A/swine/Wisconsin/629/1980(H1N1) A/swine/Kentucky/1/1976(H1N1) A/swine/Hokkaido/2/1981(H1N1) A/swine/Ehime/1/1980(H1N2) A/swine/Iowa/1/1976(H1N1) A/New Jersey/1976(H1N1) A/swine/Illinois/1/1975(H1N1) A/swine/Iowa/1973(H1N1) A/swine/Wisconsin/2/1966(H1N1) A/swine/Wisconsin/1/1957(H1N1) A/swine/1931(H1N1) A/Swine/Iowa/15/30(H1N1) A/swine/Ohio/23/1935(H1N1) A/Illinois/UR06-0093/2007(H1N1) A/California/UR06-0435/2007(H1N1) A/South Australia/57/2005(H1N1) A/New Caledonia/20/1999(H1N1) A/swine/BinhDuong/02-16/2010(H1N2) A/New York/3467/2009(H1N1) A/Brisbane/59/2007(H1N1) A/England/594/2006(H1N1) A/New York/UR06 0326/2007(H1N1) A/Thailand/CU51/2006(H1N1) A/Solomon Islands/3/2006(H1N1) A/Taiwan/2645/2006(H1N1)

Fig. 1. Phylogenetic trees of (a) H1 HA, (b) N2 NA (c) M (d) NP and (e) H3 HA genes from swine and human viruslineages. Swine viruses analyzed in this study are shown in rectangular boxes. The A/swine/Gunma/1/2012 virus is underlined. Bootstrap values greater than 90 are shown.

lineage within the Japanese strains. Notably, these HA genes were distinguishable from HA gene sequences of A/chicken/Gunma/1/2012 (H1N2; Gun12), which had been isolated in the same prefecture in Japan the year before, 2012 (33); the HA gene of Gun12 was found to belong to a pdm09 lineage. NA genes of Gun13 and Iba13 330

were most closely related to the NA genes of human-like H1N2 swine viruses and were, in contrast to the HA genes, distinguishable from each other within the lineage (Fig. 1b). Phylogenetic analyses of the six internal genes of Gun13 and of Iba13 revealed that, except for the NP genes, each internal gene was of pdm09 origin (Fig. 1c, Table 2; © 2014 The Societies and Wiley Publishing Asia Pty Ltd

Reassortants of A(H1N1) pdm09 in pigs

A/Ontario/RV1273/2005(H3N2) A/turkey/Ontario/31232/2005(H3N2) A/turkey/Ontario/FAV-10/2011(H3N2) A/swine/Minnesota/65767/2006(H3N2) A/swine/QC/1685-1/2009(H3N2) A/Ontario/1252/2007(H3N2) A/swine/Alberta/14722/2005(H3N2) A/swine/Manitoba/12707/2005(H3N2) A/turkey/Illinois/2004(H3N2) A/swine/North Carolina/2003(H3N2) A/swine/Yokohama/aq114/2011(H3N2) A/Genoa/1/2002(H3N2) A/Dunedin/38/2003(H3N2) A/New York/83/2001(H3N2) A/Waikato/5/2002(H3N2) A/New York/55/01(H3N2) A/swine/Miyazaki/2/2013(H3N2) A/New York/150/2000(H3N2) A/Denmark/35/2000(H3N2) A/swine/Nagano/2000(H3N2) A/Waikato/1/2000(H3N2) A/Hong Kong/CUHK53327/2002(H3N2) A/TW/872/02(H3N2) A/swine/Binh Duong/03-06/2010(H3N2) A/swine/Binh Duong/03-14/2010(H3N2) A/Japan/WRAIR1037P/2009(H3N2) A/TW/220/04(H3N2) A/New York/365/2004(H3N2) A/New York/3/2006(H3N2) A/Wisconsin/67/2005(H3N2) A/Hiroshima/52/2005(H3N2) A/swine/Korea/CAS05/2004(H3N2) A/Sydney/5/1997(H3N2) A/swine/Korea/JNS06/2004(H3N2) A/Swine/Ohio/891/01(H1N2) A/swine/Korea/CY10/2007(H3N2) A/Swine/Indiana/9K035/99(H1N2) A/swine/Guangxi/13/2006(H1N2) A/swine/Shanghai/1/2007(H1N2) A/swine/Korea/CY04/2007(H3N2) A/Swine/Nebraska/209/98(H3N2) A/swine/Korea/CAS09/2006(H3N2) A/Swine/North Carolina/93523/01(H1N2) A/swine/Saraburi/NIAH109713-36/2009(H3N2) A/swine/Ratchaburi/NIAH874/2005(H3N2) A/Memphis/24/95(H3N2) A/WUHAN/359/1995(H3N2) A/swine/Ontario/00130/97(H3N2) A/New York/564/1997(H3N2) A/New York/641/1996(H3N2) A/New York/754/1993(H3N2) A/Hong Kong/14/92(H3N2) A/Memphis/1/90(H3N2) A/Hong Kong/7/87(H3N2) A/swine/Obihiro/10/1985(H3N2) A/swine/Saraburi/NIAH13021/2005(H1N2) A/Albany/1/1970(H3N2) A/Hong Kong/1/1968(H3N2) A/Memphis/1/68(H3N2) A/swine/Tochigi/1/2008(H1N2) A/swine/Aomori/1/2005(H1N2) A/swine/Miyazaki/1/2006(H1N2) A/swine/Ehime/1/1980(H1N2) A/swine/Gunma/1/2013(H1N2) A/swine/Gunma/1/2012(H1N2) A/swine/Tochigi/2/2011(H1N2) A/swine/Mie/R02/2012(H1N2) A/swine/Ibaraki/1/2013(H1N2) A/swine/Saitama/21/2004(H1N2) A/swine/Niigata/729/2004(H1N2) A/swine/Saitama/1996(H1N2) A/swine/Saitama/01/2005(H1N2) A/swine/Saitama/1/2000(H1N2) A/swine/Fujian/43/2007(H3N2) A/mallard/Quebec/11045/2006(H3N2) A/duck/Italy/194659/2006(H3N2)

Fig. 1. (Continued)

phylogenetic trees of PB2, PB1, PA and NS are not shown). The NP gene of Gun13 or Iba13 was found to be most closely related to the corresponding genes from the Japanese H1N2 SIV lineage (Fig. 1d). HA and NA genes of Miy13 have descended from human seasonal influenza viruses and showed the highest homology with corresponding genes from human and © 2014 The Societies and Wiley Publishing Asia Pty Ltd

human-like H3N2 swine viruses, which were isolated in 2000 (Fig. 1b,e, Table 2). All internal viral protein genes of Miy13 were most closely related to corresponding genes from pdm09 viruses (Fig. 1c,d, Table 2; phylogenetic trees of PB2, PB1, PA and NS are not shown). Taken together, it was concluded that the three SIVs (Gun13, Iba13 and Miy13) were each reassortants 331

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A/swine/Iowa/15/1930(H1N1) A/swine/1931(H1N1) A/swine/Ohio/23/1935(H1N1) A/swine/Wisconsin/2/1966(H1N1) A/swine/Wisconsin/1/1957(H1N1) A/swine/Saitama/1/2000(H1N2) A/swine/Saitama/01/2005(H1N2) A/swine/Saitama/1996(H1N2) A/swine/Saitama/21/2004(H1N2) A/swine/Niigata/729/2004(H1N2) A/swine/Miyazaki/1/2006(H1N2) A/swine/Aomori/1/2005(H1N2) A/swine/Okinawa/1/2005(H1N1) A/swine/Kyoto/3/1979(H1N1) A/swine/Hokkaido/2/1981(H1N1) A/swine/Ehime/1/1980(H1N2) A/swine/Niigata/1/1977(H1N1) A/swine/Iowa/4/1976(H1N1) A/swine/Kentucky/1/1976(H1N1) A/swine/Iowa/1/1976(H1N1) A/swine/Illinois/1/1975(H1N1) A/swine/Wisconsin/629/1980(H1N1) A/swine/Ontario/57561/03(H1N1) A/swine/Ontario/23866/04(H1N1) A/Swine/Nebraska/209/98(H3N2) A/swine/Korea/CY10/2007(H3N2) A/swine/Korea/CAS05/2004(H3N2) A/swine/Shanghai/1/2007(H1N2) A/swine/Guangxi/13/2006(H1N2) A/swine/Binh Duong/03-14/2010(H3N2) A/swine/Binh Duong/03-06/2010(H3N2) A/swine/Korea/CY04/2007(H3N2) A/swine/Korea/CAS09/2006(H3N2) A/Swine/Indiana/9K035/99(H1N2) A/swine/Korea/CAS08/2005(H1N1) A/swine/MI/PU243/04(H3N1) A/swine/Minnesota/65767/2006(H3N2) A/swine/Yokohama/aq114/2011(H3N2) A/swine/Alberta/14722/2005(H3N2) A/swine/OH/511445/2007(H1N1) A/turkey/Ontario/31232/2005(H3N2) A/turkey/Illinois/2004(H3N2) A/swine/North Carolina/2003(H3N2) A/Ontario/RV1273/2005(H3N2) A/swine/Manitoba/12707/2005(H3N2) A/Ontario/1252/2007(H3N2) A/swine/QC/1685-1/2009(H3N2) A/northern shoveler/California/HKWF1046C/2007(H3N5) A/swine/Ontario/K01477/01(H3N3) A/mallard/Quebec/11045/2006(H3N2) A/common eider/Netherlands/1/2006(H3N8) A/duck/Italy/194659/2006(H3N2) A/turnstone/Netherlands/1/2007(H3N8) A/swine/Saraburi/NIAH13021/2005(H1N2) A/swine/Ratchaburi/NIAH874/2005(H3N2) A/swine/Chonburi/NIAH589/2005(H1N1) A/swine/Saraburi/NIAH100761-22/2009(H1N1) A/swine/Ratchaburi/NIAH1481/2000(H1N1) A/swine/Saraburi/NIAH109713-36/2009(H3N2) A/swine/Gunma/1/2013(H1N2) A/swine/Ibaraki/1/2013(H1N2) A/turkey/Ontario/FAV-10/2011(H3N2) A/swine/Gunma/1/2012(H1N2) A/swine/Tochigi/2/2011(H1N2) A/Florida/06/2012(H1N1) A/Athens/INS554/2011(H1N1) A/swine/Miyazaki/2/2013(H3N2) A/California/04/2009(H1N1) A/swine/Mie/R02/2012(H1N2) A/Aichi/202/2009(H1N1) A/swine/Narita/aq21/2011(H1N1) A/swine/Osaka/1/2009(H1N1)

Fig. 1. (Continued)

between pdm09 viruses and viruses that persist in the Japanese pig population. Antigenic analysis and comparison of deduced amino acid sequences of the Japanese SIVs The HI cross-reactivities of Gun13 and Iba13 viruses across a panel of 13 distinct antisera generated against 332

pdm09 viruses, seasonal human-lineage H1 viruses or avian H1 virus were examined (Table 3). Gun13 and Iba13 reacted similarly with each antiserum examined. Gun13 virus reacted with Sai05 and Toc08 antisera at HA titers of 1280 and 160, respectively; these titers were each fourfold less dilute than the reactive HA titers (5120 and 640) of the native antigens (Sai05 HA and Toc08 HA, respectively). Similarly, Iba13 virus reacted with Toc08 and BD16/10 antisera at HA titers (320 and 160, © 2014 The Societies and Wiley Publishing Asia Pty Ltd

Reassortants of A(H1N1) pdm09 in pigs

A/swine/Yokohama/aq114/2011(H3N2) A/Ontario/1252/2007(H3N2) A/turkey/Ontario/31232/2005(H3N2) A/Ontario/RV1273/2005(H3N2) A/swine/Manitoba/12707/2005(H3N2) A/swine/Alberta/14722/2005(H3N2) A/swine/Minnesota/65767/2006(H3N2) A/swine/QC/1685-1/2009(H3N2) A/swine/MI/PU243/04(H3N1) A/swine/North Carolina/2003(H3N2) A/swine/Korea/CAS08/2005(H1N1) A/swine/OH/511445/2007(H1N1) A/Swine/Ohio/891/01(H1N2) A/swine/Korea/CAS09/2006(H3N2) A/swine/Korea/CY04/2007(H3N2) A/swine/Binh Duong/03-06/2010(H3N2) A/swine/Binh Duong/03-14/2010(H3N2) A/swine/Guangxi/13/2006(H1N2) A/swine/Shanghai/1/2007(H1N2) A/Swine/North Carolina/93523/01(H1N2) A/Swine/Nebraska/209/98(H3N2) A/swine/Korea/CY10/2007(H3N2) A/Swine/Indiana/9K035/99(H1N2) A/swine/Korea/JNS06/2004(H3N2) A/swine/Korea/CAS05/2004(H3N2) A/California/04/2009(H1N1) A/turkey/Ontario/FAV-10/2011(H3N2) A/swine/Miyazaki/2/2013(H3N2) A/swine/Gunma/1/2012(H1N2) A/swine/Tochigi/2/2011(H1N2) A/swine/Mie/R02/2012(H1N2) A/Florida/06/2012(H1N1) A/Athens/INS554/2011(H1N1) A/swine/Narita/aq21/2011(H1N1) A/Aichi/202/2009(H1N1) A/swine/Yamagata/11/2010(H1N1) A/swine/Ontario/23866/04(H1N1) A/swine/Ontario/57561/03(H1N1) A/swine/Saraburi/NIAH109713-36/2009(H3N2) A/swine/Ratchaburi/NIAH874/2005(H3N2) A/swine/Wisconsin/629/1980(H1N1) A/swine/Niigata/1/1977(H1N1) A/swine/Iowa/4/1976(H1N1) A/swine/Iowa/1/1976(H1N1) A/swine/Kentucky/1/1976(H1N1) A/swine/Ehime/1/1980(H1N2) A/swine/Hokkaido/2/1981(H1N1) A/swine/Kyoto/3/1979(H1N1) A/swine/Illinois/1/1975(H1N1) A/swine/Wisconsin/1/1957(H1N1) A/swine/Wisconsin/2/1966(H1N1) A/swine/Okinawa/1/2005(H1N1) A/swine/Saitama/21/2004(H1N2) A/swine/Tochigi/1/2008(H1N2) A/swine/Aomori/1/2005(H1N2) A/swine/Miyazaki/1/2006(H1N2) A/swine/Ibaraki/1/2013(H1N2) A/swine/Gunma/1/2013(H1N2) A/swine/Niigata/729/2004(H1N2) A/swine/Saitama/1996(H1N2) A/swine/Saitama/01/2005(H1N2) A/swine/Saitama/1/2000(H1N2) A/swine/Ohio/23/1935(H1N1) A/swine/Iowa/15/1930(H1N1) A/swine/1931(H1N1)

Fig. 1. (Continued)

respectively) that were twofold and fourfold less dilute than the reactive titers of the respective native antigens, respectively. Cross-reactivity between Gun13 or Iba13 virus and antisera against Iow30, Kyo79, Tsu05 or each of three seasonal human-lineage viruses, NC99, SI06, and Bri07, was minimal or undetectable. © 2014 The Societies and Wiley Publishing Asia Pty Ltd

Deduced amino acid residues in the antigenic sites of HA proteins of Gun13, Iba13 and reference viruses tested in the HI assays were compared to identify amino acid residues that confer differential antigenic specificities (Table 4). The HA nucleotide and putative amino acid sequences were 96.9% and 95.9% identical, 333

K. Kanehira et al.

A/Victoria/361/2011(H3N2) A/Japan/WRAIR1037P/2009(H3N2) A/Uruguay/716/2007(H3N2) A/New York/3/2006(H3N2) A/Hiroshima/52/2005(H3N2) A/Wisconsin/67/2005(H3N2) A/swine/Osaka_C/12-20/2008(H3N2) A/New York/365/2004(H3N2) A/New York/55/2004(H3N2) A/swine/Binh Duong/03-14/2010(H3N2) A/swine/Binh Duong/03-06/2010(H3N2) A/TW/220/04(H3N2) A/Dunedin/38/2003(H3N2) A/Wyoming/03/2003(H3N2) A/Hong Kong/CUHK53327/2002(H3N2) A/TW/872/02(H3N2) A/swine/Ehime/1/2002(H3N2) A/Waikato/1/2000(H3N2) A/swine/Korea/CAS05/2004(H3N2) A/Memphis/59/99(H3N2) A/Panama/2007/1999(H3N2) A/Waikato/5/2002(H3N2) A/Genoa/1/2002(H3N2) A/New York/83/2001(H3N2) A/New York/55/01(H3N2) A/swine/Miyazaki/2/2013(H3N2) A/swine/Nagano/2000(H3N2) A/New York/150/2000(H3N2) A/Denmark/35/2000(H3N2) A/swine/Fujian/43/2007(H3N2) A/Sydney/5/1997(H3N2) A/New York/564/1997(H3N2) A/swine/Korea/CY10/2007(H3N2) A/swine/North Carolina/2003(H3N2) A/swine/Korea/JNS06/2004(H3N2) A/turkey/Ontario/FAV-10/2011(H3N2) A/swine/MI/PU243/04(H3N1) A/turkey/Illinois/2004(H3N2) A/Ontario/1252/2007(H3N2) A/swine/QC/1685-1/2009(H3N2) A/swine/Yokohama/aq114/2011(H3N2) A/swine/Minnesota/65767/2006(H3N2) A/turkey/Ontario/31232/2005(H3N2) A/Ontario/RV1273/2005(H3N2) A/swine/Alberta/14722/2005(H3N2) A/swine/Manitoba/12707/2005(H3N2) A/swine/Ontario/00130/97(H3N2) A/WUHAN/359/1995(H3N2) A/swine/Saraburi/NIAH109713-36/2009(H3N2) A/swine/Ratchaburi/NIAH874/2005(H3N2) A/Memphis/24/95(H3N2) A/swine/Korea/CY04/2007(H3N2) A/New York/641/1996(H3N2) A/swine/Korea/CAS09/2006(H3N2) A/Swine/Nebraska/209/98(H3N2) A/New York/754/1993(H3N2) A/Hong Kong/14/92(H3N2) A/Memphis/1/90(H3N2) A/swine/Obihiro/10/1985(H3N2) A/Hong Kong/7/87(H3N2) A/Albany/1/1970(H3N2) A/Memphis/1/68(H3N2) A/swine/Wadayama/51968(H3N2) A/Hong Kong/1/1968(H3N2) A/northern shoveler/California/HKWF1046C/2007(H3N5) A/swine/Ontario/K01477/01(H3N3) A/mallard/Quebec/11045/2006(H3N2) A/common eider/Netherlands/1/2006(H3N8) A/duck/Italy/194659/2006(H3N2) A/turnstone/Netherlands/1/2007(H3N8)

Fig. 1. (Continued)

respectively, between Gun13 and Iba13. Eleven amino acid substitutions were observed between their antigenic sites in the HA1 domain. Amino acid residues substituted from Gun13 to Iba13 were as follows: P158Q in the Sa site; R152K, G155S, L188I, Q189R and P194T in the Sb site; R141S, F165V and K234E in the Ca site; and S74R and G115E in the Cb site. Gun13 reacted most strongly with anti-Sai05 among the antisera examined; Gun13 HA1 and Sai05 HA1 differed at 17 334

amino acid residues within the antigenic domain. AntiToc08 antiserum also reacted with Gun13 virus at a titer fourfold less dilute than the reactive titer of Toc08 virus, and 20 substitutions between antigenic sites of Gun13 HA1 and Toc08 HA1 were identified. Iba13 HA1 and Toc08 HA1 differed at 19 amino acid residues within the antigenic region. Although only a fourfold reduction from homologous titer was observed with the antiserum against BD16/10, 28 amino acid substitutions in the © 2014 The Societies and Wiley Publishing Asia Pty Ltd

Reassortants of A(H1N1) pdm09 in pigs

Table 2. Origins of viral gene segments Segment

A/swine/Ibaraki/1/2013 A/swine/Gunma/1/2013 A/swine/Gunma/1/2012† A/swine/Miyazaki/2/2013 A/swine/Nagano/2000

Sub type

1 PB2

2 PB1

3 PA

4 HA

5 NP

6 NA

7 MP

8 NS

H1N2 H1N2 H1N2 H3N2 H3N2

pdm09 pdm09 pdm09 pdm09 hlS H3N2

pdm09 pdm09 pdm09 pdm09 hlS H3N2

pdm09 pdm09 pdm09 pdm09 hlS H3N2

cSIV cSIV pdm09 hlS H3N2 hlS H3N2

cSIV cSIV pdm09 pdm09 hlS H3N2

hlS H1 hlS H1 hlS H1 hlS H3N2 hlS H3N2

pdm09 pdm09 pdm09 pdm09 hlS H3N2

pdm09 pdm09 pdm09 pdm09 hlS H3N2

† , this virus was isolated at Gunma Prefectural Institute of Public Health and Environmental Sciences. pdm09, pandemic A(H1N1) 2009; cSIV, classical swine influenza virus; hlS, human-like swine influenza virus.

antigenic region were identified between Iba13 and BD16/10. Antigenic analysis of Miy13 was also performed with a panel of 13 antisera generated against four individual human-like H3 swine viruses, eight individual seasonal human-like H3 viruses or an avian H3 virus (Table 5). Miy13 virus reacted with anti-Wyo03 serum at the same titer as did Wyo03 virus. Miy13 reacted with anti-Wuh95 serum at a twofold higher titer than did Wuh95 virus. Notably, Nag00 virus is one of the SIVs that has an HA gene that is most homologous with the Miy13 HA gene. When tested with anti-BD14/10, Wuh95, Wyo03 or NY04 antisera, the reactive Nag00 HA titer was always

twofold lower than the reactive Miy13 titer; moreover, the reactive Nag00 titer was twofold higher than the reactive Miy13 titer with anti-Osa08, anti-Hir05 or antiUru07; eightfold higher with anti-Pan99; and 16-fold higher with anti-Syd97. Variations in deduced amino acid residues in the HA1 domains of 15 H3-subtype HA proteins are listed in Table 6. The antigenic sites in the HA1 domain of the HA proteins from Miy13 and Nag00 differed at 11 amino acid residues-four residues in site A, four in B, two in C and one in E. Moreover, Miy13 HA had lost a potential glycosylation site (aa133) in site A, whereas the corresponding amino acid of Nag00 HA had retained

Table 3. Hemagglutination inhibition (HI) titers with swine and human H1 viruses HI titers of sera from chicken† and ferret‡ infected with Classical SIV Kyo79†

Seasonal human-lineage virus

Avian virus

Nii04†

Sai05†

Toc08†

Toc11†

Cal09†

Nar11†

BD16/10†

NC99‡

SI06‡

Bri07‡

Tsu05†

Gun13 20 40 40 Iba13 20 40 160 Classical SIV Iow30 5120 640 80 Kyo79 5120 5120 80 Nii04 160 320 1280 Sai05 320 640 320 Toc08 2560 1280 320 Toc11 1280 640 160 A(H1N1) pdm09 virus Cal09 2560 1280 80 Nar11 5120 1280 320 Seasonal human-lineage virus BD16/10 20 20 20 NC99 40 80 40 SI06 40 40 20 Bri07 80 40 20 Avian virus Tsu05 2560 80 80

1280 320

160 320

320 160

80 40

320 160

80 160