JVI Accepts, published online ahead of print on 1 May 2013 J. Virol. doi:10.1128/JVI.00804-13 Copyright © 2013, American Society for Microbiology. All Rights Reserved.
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Antigenic characterization of H3N2 influenza A viruses from Ohio agricultural fairs Running title: Antigenic profile of swine-origin H3N2 IAVs Zhixin Feng1,2$, Janet Gomez1$, Andrew S. Bowman3, Jianqiang Ye1, Li-Ping Long1, Sarah W. Nelson3, Jialiang Yang1, Brigitte Martin1, Kun Jia1, Jacqueline M. Nolting3, Fred Cunningham4, Carol Cardona5, Jianqiang Zhang6, Kyoung-Jin Yoon6, Richard D. Slemons3, and Xiu-Feng Wan1* 1
Department of Basic Sciences, College of Veterinary Medicine, Mississippi State University, Mississippi State, MS 39762, the United States; 2Institute of Veterinary Medicine, Jiangsu Academy of Agricultural Sciences, Nanjing 210014, People's Republic of China; 3Department of Veterinary Preventive Medicine, College of Veterinary Medicine, The Ohio State University, Columbus, OH 43210, the United States; 4USDA/APHIS/WS, National Wildlife Research Center, 5 Mississippi Field Station, Mississippi State, MS 39762, the United States; College of Veterinary Medicine, University of Minnesota, St. Paul, MN 55108, the United States; 6Department of Veterinary Diagnostic and Production Animal Medicine, College of Veterinary Medicine, Iowa State University, Ames, IA 50011, the United States $
Equal contribution to this work.
*Correspondence:
[email protected] or
[email protected].
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Abstract
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viruses (IAVs) and swine exposure at agricultural fairs has raised concerns about the human
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health risk posed by IAV-infected swine. Understanding the antigenic profiles of IAVs
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circulating in pigs at agricultural fairs is critical to developing effective prevention and control
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strategies. Here 68 H3N2 IAV isolates recovered from pigs at Ohio fairs (2009 to 2011) were
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antigenically characterized. These isolates were compared with other H3 IAVs recovered from
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commercial swine, wild birds, and canines, along with human seasonal and variant H3N2 IAVs.
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Antigenic cartography demonstrated that H3N2 IAV isolates from Ohio fairs could be divided
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into two antigenic groups: 1) the 2009 fair isolates and 2) the 2010 and 2011 fair isolates. These
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same two antigenic clusters have also been observed in commercial swine populations in recent
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years. Human H3N2v isolates from 2010 and 2011 are antigenically clustered with swine-origin
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IAVs from the same time period. The isolates recovered from pigs at fairs did not cross react
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with ferret antisera produced against the human seasonal H3N2 IAVs circulating during the past
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decade, raising the question of the degree of immunity the human population has to swine-origin
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H3N2 IAVs. Our results demonstrate that H3N2 IAVs infecting pigs at fairs and H3N2v isolates
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were antigenically similar to the IAVs circulating in commercial swine, demonstrating that
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exhibition swine can function as a bridge between commercial swine and the human population.
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The demonstrated link between the emergence of H3N2 variant (H3N2v) influenza A
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Introduction
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bi-directional interspecies transmission of influenza A viruses (IAVs). Numerous cases of variant
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influenza A have been linked to swine exposure at agricultural fairs across the United States and
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the first documented transmissions of the 2009 pandemic (pdm09) H1N1 IAV from humans to
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pigs also occurred at agricultural fairs (1-5). Since July 2011, more than 325 confirmed human
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cases of H3N2 variant (H3N2v) influenza A have occurred in 14 states (6). Many of these cases
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reported direct or indirect contact to exhibited swine with limited subsequent human-to-human
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transmission of H3N2v IAVs (7, 8).
The swine-human interface at agricultural fairs has proven to be an important site for the
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Influenza A viruses can cause sporadic, epidemic, and pandemic respiratory disease in
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humans and from mild to severe respiratory diseases in horses, pigs, domestic poultry, and sea
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mammals (9-12). Because the tracheal epithelial cells of swine contain both avian-like alpha 2,3-
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linked sialic acid receptors and human-like alpha 2,6-linked sialic acid receptors (13, 14), pigs
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can be infected by avian- and mammalian-origin IAVs which may facilitate reassortment events
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and result in the subsequent generation of novel IAV capable of infecting humans (13, 14).
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Currently, multiple antigenically and genetically divergent IAVs co-circulate in swine
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populations and antigenic recycling of IAVs can occur in modern swine management systems
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where there is a continuous supply of naive pigs (15). Antigenic characterization of the IAV
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(H1N1)pdm09 demonstrated that the H1 had been circulating among swine for decades prior to
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the onset of the 2009 pandemic (16) confirming the potential for the emergence of pandemic
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IAV from pigs.
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Influenza A virus was first isolated from swine in 1930 (17). This H1N1 strain, likely derived from the 1918 H1N1 pandemic IAV (18), was the only major IAV circulating among US
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swine for more than 60 years and is now known as “classical” swine influenza virus. A triple
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reassortant H3N2 (trH3N2) IAV composed of three genes (HA, NA, and PB1) derived from
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human H3N2 seasonal influenza viruses (huH3N2), two genes (PB2 and PA) from avian-origin
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IAVs, and three genes (NP, MP, and NS) from “classical” swine influenza virus (19, 20)
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emerged in swine populations in the mid-1990s and has since become well-established in North
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America. Phylogenetic analyses of HA genes from H3N2 IAVs isolated from North American
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pigs after 1998 demonstrates that these trH3N2 IAV can be classified into at least four genetic
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groups (clusters I, II, III, and IV) (21) with one lineage (cluster IV) predominating since 2005.
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Neutralization assays with swine antisera demonstrated that these four genetic clusters are also
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antigenically distinct (22). Several reassortment events between trH3N2 IAVs, human-origin
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IAVs, and classical swine influenza virus have occurred since 1998 resulting in multiple gene
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constellations with various HA and NA genes in combination with the Triple Reassortant
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Internal Gene (TRIG) cassette (which includes PB2, PB1, PA, NP, NS, and M segments) (23,
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24). Human cases of infection with swine-origin IAVs reported by the Centers for Disease
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Control and Prevention (CDC) after 1998 were all characterized as containing the TRIG cassette
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and were linked antigenically or genetically, to IAVs concurrently circulating in pigs. Most of
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these cases were isolated infections in which patients reported indirect or direct exposure to pigs
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(25, 26).
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The degree of immunity the human population has to swine-origin H3N2 IAVs is open to
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question (6). Because agricultural fairs bring people into close contact with swine, defining the
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antigenic profiles of the IAVs circulating in exhibited pigs at fairs is critical to developing
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effective strategies to prevent and/or control human infections of swine-origin IAVs.
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Hemagglutination inhibition (HI) tests were used to compare the antigenic profiles of IAV
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isolates recovered from pigs at Ohio agricultural fairs during 2009-2011 with H3 IAV isolates of
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human, swine, canine and avian origins. The antigenic profiles from HI tests were validated by
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microneutralization (MN) assays. The genomic sequences of the HA and NA segments of
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selected swine-origin H3N2 IAV isolates were also analyzed to investigate the genomic changes
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contributing to antigenic profiles.
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Materials and methods
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Viruses and Sera. From 2009 to 2011, a systematically designed surveillance effort was
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performed across 53 agricultural fairs in Ohio, USA (27). From the 1,073 pigs sampled, 155
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were positive for IAV, with 128 H3N2 viruses isolated. Sixty-eight representative (from the 128
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total) H3N2 IAV (TABLE 1) were selected for the present study. The HA, NA, and M genes of
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these isolates were characterized as described previously (27). Additionally, four H3N2v IAV
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isolates (TABLE 1) were kindly provided by CDC, Atlanta, GA. Also included in the study, and
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listed in TABLE 2 and 3, were twelve H3N2 IAV isolates from commercial pigs, two H3N2 IAV
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isolates from wild birds, two H3 IAV isolates from dogs (28, 29), and 12 human seasonal H3N2
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isolates. After being propagated in Madin-Darby canine kidney (MDCK) cells (ATCC,
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Manassas, VA, USA), all IAVs were aliquoted and stored at -70˚C until use.
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Ferret antisera were generated in 6 to 8-week-old ferrets, which had baseline HI titers less
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than 1:10 against A/Brisbane/10/2007(H3N2), A/Brisbane/59/2007(H1N1), and
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A/California/4/2009(H1N1). Each ferret was inoculated intranasally with 106 TCID50 for each
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IAV isolate (TABLE 1). Sera were collected from the ferrets at three weeks post inoculation if
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the HI titer in a ferret was at least 1:160 at 2 weeks post inoculation. Otherwise, a second dose of
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106 TCID50 of the corresponding virus was given and serum was collected five weeks post first
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inoculation. All sera were aliquoted and stored at -70˚C until use.
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Eight swine H3N2 IAV isolates representing six fair events were used to generate ferret
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antisera. The virus designation is summarized in TABLE 4. In addition, ferret antisera were
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produced against 12 human seasonal H3N2 IAV isolates, two H3N2 IAV isolates from wild
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birds, and two canine H3 IAV isolates for comparison. All these isolates are contemporary H3
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IAVs (TABLE 1, 2, and 3).
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Genomic sequencing. Viral RNA was extracted using the RNeasy Mini Kit according to the
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manufacturer’s instructions (QIAGEN Inc., Valencia, CA, USA). Reverse transcription and PCR
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were performed using influenza-specific primers (30). PCR products were separated using
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agorose gel electrophoresis and purified using the QIAquik Gel Extraction Kit (QIAGEN Inc,
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Valencia, CA, USA). Sequencing was performed by the Life Sciences Core Laboratories Center
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at Cornell University using the Applied Biosystems Automated 3730 DNA Analyzer, with Big
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Dye Terminator chemistry and AmpliTaq-FS DNA Polymerase. The HA genes of nine
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agricultural fair isolates are fully sequenced, and the HA genes of 12 commercial swine IAV
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isolates were partially sequenced and at least covered the entire HA1. The GenBank accession
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numbers of these sequences are xxxxxx-xxxxxx (pending).
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Hemagglutination inhibition assay. Before conducting HI tests, ferret antisera were treated
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with receptor-destroying enzyme (RDE) (Denka Seiken Co., Tokyo, Japan) at 37˚C for 18 hours,
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and then heat inactivated at 55˚C for 30 minutes. The sera were then diluted with phosphate-
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buffered saline (for a final dilution of 1:10), and tested by HI assay with 0.5% turkey red blood
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cells according to the WHO manual on animal influenza diagnosis and surveillance
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(http://www.who.int/vaccine_research/diseases/influenza/WHO_manual_on_animal-
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diagnosis_and_surveillance_2002_5.pdf).
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Microneutralization assay. The sera were tested by performing a microneutralization (MN)
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assay in MDCK cells. Neutralizing titers were expressed as the reciprocal of the serum dilution
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that inhibited 50% of viral growth of 100 tissue culture infectious dose (TCID50) of virus. The
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MN titers were determined by HA assay using 0.5% turkey red blood cells (31).
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Antigenic cartography and antigenic cluster identification. Antigenic cartography was
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generated based on HI data using AntigenMap (32, 33) by setting 1:10 as the low reactor. Data
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normalization was performed as previously reported (34-36). Influenza A viruses were classified
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into antigenic clusters using k-mean clustering (37). Student’s t-test was performed to assess the
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significance of antigenic distances.
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Molecular characterization and phylogenic analysis. The multiple sequence alignments were
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conducted by using the MUSCLE software package (38). The phylogenetic analyses were
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performed using maximum likelihood by GARLI version (39), and bootstrap resampling
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analyses were conducted using PAUP* 4.0 Beta (40) with a neighborhood joining method, as
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previously described (41).
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Results
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Genetic characterization of H3N2 influenza A viruses. Phylogenetic analysis of HA genes
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showed the HA genes of four human H3N2v isolates and all H3N2 IAVs isolates from both
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agricultural fairs and commercial swine farms belonged to cluster IV (FIG. 1A) (22). A high
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degree of genetic diversity was observed among the viruses. The amino acid sequence identity
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between the HA amino acid sequence of A/swine/Ohio/10SW130/2010(H3N2) and that of
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A/swine/Ohio/11SW111/2011(H3N2) was 99.3%; the HA protein sequence identity between
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A/swine/Ohio/09SW64/2009(H3N2) and A/swine/Ohio/10SW130/2010(H3N2) and that
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between A/swine/Ohio/09SW64/2009(H3N2) and A/swine/Ohio/11SW111/2011(H3N2) was
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95.8% and 95.1%, respectively and the human H3N2v isolate A/Iowa/07/2011(H3N2) was
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99.5% identical to A/swine/Ohio/11SW111/2011(H3N2) (TABLE 5).
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The phylogenetic trees of influenza NA genes showed that the NA genes of these
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agricultural fair isolates were phylogenetically close to the NA gene of
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A/swine/Ontario/33853/2005 and to those NA genes of other IAVs in the same cluster, as similar
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to the HA genes (FIG. 1B).
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Antigenic diversity of H3N2 IAVs from pigs at agricultural fairs. The antisera generated
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against selected IAV isolates from pigs at Ohio fairs during 2010 and 2011 cross-reacted with
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the isolates from 2010 and 2011 fairs, but were less reactive to IAV recovered from pigs at fairs
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during 2009. Similarly, the antisera generated against IAV isolates from the 2009 fair season
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reacted poorly with 2010 and 2011 swine isolates (TABLE 1). The IAV isolates from pigs at
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agricultural fairs could be divided into two antigenic groups: 1) the 2009 fair isolates (antigenic
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cluster H3N2-alpha), and 2) the 2010 and 2011 fair isolates (antigenic cluster H3N2-beta).
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The k-mean clustering also grouped 84 isolates (68 exhibition swine isolates, 4 human
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H3N2v isolates, and 12 commercial swine isolates) into one of the two identified antigenic
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clusters (35 in cluster alpha and 49 in cluster beta) as summarized in TABLE 1. The average
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distance between the viruses within cluster alpha and cluster beta was 1.45 and 1.07 units,
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respectively. The minimum distance between antigenic clusters was 2.32 units (e.g.,
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A/Pennsylvania/14/2010 and A/swine/Iowa/11333/2006) and the maximum distance between
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clusters was 6.53 units (e.g., A/swine/North Carolina/23592/2009 and
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A/swine/Ohio/11SW214/2011). The average distance between two clusters from their centers
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was 4.18 units. Each unit corresponds to a 2-fold change in HI assay. Statistically, the antigenic
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distances between clusters were significantly different from those within each antigenic cluster
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(p
