crossmark

The Molecular Determinants of Antibody Recognition and Antigenic Drift in the H3 Hemagglutinin of Swine Influenza A Virus Eugenio J. Abente,a Jefferson Santos,b Nicola S. Lewis,c Phillip C. Gauger,d Jered Stratton,a Eugene Skepner,c Daniela S. Rajao,a Daniel R. Perez,b Amy L. Vincenta

Tavis K. Anderson,a

Virus and Prion Research Unit, National Animal Disease Center, Agricultural Research Service, U.S. Department of Agriculture, Ames, Iowa, USAa; Department of Population Health, Poultry Diagnostic and Research Center, College of Veterinary Medicine, University of Georgia, Athens, Georgia, USAb; Department of Zoology, University of Cambridge, Cambridge, United Kingdomc; Department of Veterinary Diagnostic and Production Animal Medicine, College of Veterinary Medicine, Iowa State University, Ames, Iowa, USAd

ABSTRACT

Influenza A virus (IAV) of the H3 subtype is an important respiratory pathogen that affects both humans and swine. Vaccination to induce neutralizing antibodies against the surface glycoprotein hemagglutinin (HA) is the primary method used to control disease. However, due to antigenic drift, vaccine strains must be periodically updated. Six of the 7 positions previously identified in human seasonal H3 (positions 145, 155, 156, 158, 159, 189, and 193) were also indicated in swine H3 antigenic evolution. To experimentally test the effect on virus antigenicity of these 7 positions, substitutions were introduced into the HA of an isogenic swine lineage virus. We tested the antigenic effect of these introduced substitutions by using hemagglutination inhibition (HI) data with monovalent swine antisera and antigenic cartography to evaluate the antigenic phenotype of the mutant viruses. Combinations of substitutions within the antigenic motif caused significant changes in antigenicity. One virus mutant that varied at only two positions relative to the wild type had a >4-fold reduction in HI titers compared to homologous antisera. Potential changes in pathogenesis and transmission of the double mutant were evaluated in pigs. Although the double mutant had virus shedding titers and transmissibility comparable to those of the wild type, it caused a significantly lower percentage of lung lesions. Elucidating the antigenic effects of specific amino acid substitutions at these sites in swine H3 IAV has important implications for understanding IAV evolution within pigs as well as for improved vaccine development and control strategies in swine. IMPORTANCE

A key component of influenza virus evolution is antigenic drift mediated by the accumulation of amino acid substitutions in the hemagglutinin (HA) protein, resulting in escape from prior immunity generated by natural infection or vaccination. Understanding which amino acid positions of the HA contribute to the ability of the virus to avoid prior immunity is important for understanding antigenic evolution and informs vaccine efficacy predictions based on the genetic sequence data from currently circulating strains. Following our previous work characterizing antigenic phenotypes of contemporary wild-type swine H3 influenza viruses, we experimentally validated that substitutions at 6 amino acid positions in the HA protein have major effects on antigenicity. An improved understanding of the antigenic diversity of swine influenza will facilitate a rational approach for selecting more effective vaccine components to control the circulation of influenza in pigs and reduce the potential for zoonotic viruses to emerge.

I

nfluenza A virus (IAV) of the H3 subtype is an important pathogen that infects both humans and swine. The main strategy used to prevent or reduce morbidity of IAV in humans is the implementation of vaccine programs (1). Likewise, swine producers employ commercially available and farm-specific autogenous vaccines to prevent IAV clinical disease in swine (2, 3). Current vaccines rely heavily on the immune response targeted to the head of the hemagglutinin (HA) surface glycoprotein to prevent virus entry, although the neuraminidase (NA), the matrix protein 2 (M2), and the stalk of the HA are also targets of candidate vaccines (1, 4). Despite ongoing efforts to monitor IAV circulation in human and animal populations, vaccines are produced largely in retrospect after surveillance programs detect the emergence of a drift variant. Key components of a successful vaccine strain selection program include a comprehensive understanding of the antigenicity of circulating strains and early detection of antigenically drifted viruses against which the current vaccine would be less efficacious, warranting an update of the vaccine formulation if epidemiologic evidence suggests that circulation and spread of the variant have occurred.

8266

jvi.asm.org

The antigenic regions of a pandemic human H3 virus from 1968 were deduced using monoclonal antibodies against naturally occurring and laboratory produced antigenic variants. These “antigenic sites” have long served as a reference for antigenic positions of relevance to antigenic drift on the globular head of H3 HAs (131 positions, referred to as regions A to E) (5, 6). More recently,

Received 20 May 2016 Accepted 28 June 2016 Accepted manuscript posted online 6 July 2016 Citation Abente EJ, Santos J, Lewis NS, Gauger PC, Stratton J, Skepner E, Anderson TK, Rajao DS, Perez DR, Vincent AL. 2016. The molecular determinants of antibody recognition and antigenic drift in the H3 hemagglutinin of swine influenza A virus. J Virol 90:8266 –8280. doi:10.1128/JVI.01002-16. Editor: A. García-Sastre, Icahn School of Medicine at Mount Sinai Address correspondence to Amy L. Vincent, [email protected]. Supplemental material for this article may be found at http://dx.doi.org/10.1128 /JVI.01002-16. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Journal of Virology

September 2016 Volume 90 Number 18

Antigenicity of H3 Swine Influenza Viruses

TABLE 1 Antigenic phenotypes and corresponding antigenic motifs identified by Lewis et al. (8) Antigenic motif at indicated position Antigenic reference virus tested previously

145

155

156

158

159

189

193

Antigenic phenotype

A/Swine/Texas/4199-2/1998 A/Swine/Iowa/A01432500/2013 A/Swine/Nebraska/A01271549/2012 A/Swine/Colorado/23619/99 A/Swine/Minnesota/A01125993/2012 A/Swine/Minnesota/A01300213/2012 A/Swine/Minnesota/A01327922/2012 A/Swine/Minnesota/A01432544/2013 A/Swine/Iowa/A01300195/2012 A/Swine/Michigan/A01259000/2012 A/Indiana/08/2011 A/Swine/Illinois/A01241469/2012 A/Swine/NY/A01104005/2011 A/Swine/Indiana/A00968373/2012 A/Swine/Illinois/A01201606/2011 A/Swine/Iowa/A01202613/2011 A/Swine/Iowa/A01202889/2011 A/Swine/Pennsylvania/A01076777/2010 A/Swine/Minnesota/A01280592/2013 A/Swine/Nebraska/A01241171/2012 A/Swine/Illinois/A01241066/2012 A/Swine/Iowa/A01203121/2012 A/Swine/Iowa/A01049750/2011 A/Swine/Texas/A01049914/2011 A/Swine/Illinois/02907/2009 A/Swine/Minnesota/02782/2009 A/Swine/Minnesota/01146/2006 A/Swine/Iowa/01700/2007 A/Swine/Michigan/A01432375/2013 A/Swine/Indiana/A01202866/2011 A/Swine/North_Carolina/A01432566/2013 A/Swine/Wyoming/A01444562/2013 A/Swine/Iowa/A01203196/2012 A/Swine/Michigan/A01203498/2012

K K K K K N N N N N N N N N N N N N N K N N N N N N N N N N K K K K

H H H H Y Y Y Y Y Y Y Y Y Y Y H H Y Y H H H H H N H H H H Y Y Y H H

K H K Q N N N N N N N N N N N N N N H K N N N N N S N N H H K N K N

E N N K N N N N N N N N N N N N N N N N N N N N D N N D G G N N N N

Y Y Y Y N Y Y H Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y H H Y Y Y Y

S R S R K K K K K K K K K K K K K K K R R R R R R R R R E E E K E K

S S N S N N S S S S N N N N S S S N N N N N N N N N N S S S N N S S

Light blue Purple Purple Pink Gold Red Red Red Red Red Red Red Red Red Red Red Red Red Red Dark pink Cyan Cyan Cyan Cyan Cyan Cyan Cyan Cyan Brown Brown Dark green Light green Orange Blue

antigenic cartography, a computational method to quantify binding assay data, such as hemagglutination inhibition (HI) data (7), was used to characterize the antigenic evolution of human, swine, and equine H3 IAV strains (8–10). The distance between viruses in the antigenic map is measured in antigenic units (AU), and 1 AU is equivalent to a 2-fold dilution in the HI assay. An antigenic distance of 2 AU is considered significant, and an 8-fold HI difference (equivalent to 3 AU) is typically sufficient to consider updating the human seasonal vaccine strain (11–13). The evolution of human influenza H3N2 viruses circulating from 1968 to 2003 resulted in 11 discrete antigenic clusters of antigenically similar viruses. The distance between each antigenic cluster was sufficient to require an update in the human influenza virus seasonal vaccine strain, and 67 amino acid positions in the HA protein were associated with the transition from one cluster to the next. It was determined using site-directed mutagenesis that a single amino acid change largely accounted for the phenotypic change that resulted in the emergence of a new antigenic cluster. Although more than 130 positions on the HA were previously characterized as putative antigenic sites, major substitutions leading to the emergence of antigenically distinct seasonal epidemic viruses occurred at just 7 amino acids in the HA (positions 145, 155, 156, 158, 159, 189, and 193) (14). Similarly, antigenic cartography of equine H3

September 2016 Volume 90 Number 18

virus from 1968 to 2007 identified 3 antigenic clusters, and again, a single amino acid substitution was likely responsible for cluster transitions that occurred at 2 of the same 7 positions identified for human H3 (positions 159 and 189) (10). Analyses of the antigenic evolution of swine H3 field isolates from the United States identified two major antigenic clusters (red and cyan) (8, 15) and several distinct outlier groups or strains. By combining antigenic cartography with sequence analysis, it was determined that 6 of the same 7 positions identified for human H3 antigenic cluster transitions (positions 145, 155, 156, 158, 159, and 189) (8) were associated with cluster/outlier transitions in the swine H3 wild-type viruses, and the amino acid pattern observed in individual viruses at these positions are herein referred to as an “antigenic motif.” Twelve antigenic motifs were previously determined based on amino acids located at positions 145, 155, 156, 158, 159, and 189 and designated according to a color-coded naming scheme (Table 1) (8). Reporting of HA sequences from circulating swine influenza viruses in the United States has increased dramatically, in large part due to efforts coordinated and funded by the U.S. Department of Agriculture (USDA) voluntary IAV swine surveillance system and operated through the National Animal Health Laboratory Network and the National Veterinary Service Laboratories.

Journal of Virology

jvi.asm.org

8267

Abente et al.

While evolutionary studies shed light on genetic diversity (16), it is difficult to interpret the antigenic significance of the amino acid diversity among circulating HAs. Unlike the human vaccine strain selection for IAV that is driven by a concerted global effort of surveillance and epidemiologic tracking, multiple commercial vaccines with independently developed proprietary formulations are available for use in swine. Additionally, producers can purchase custom-made farm-specific autogenous vaccines. The HA sequences of the vaccine and field strains might be compared to predict vaccine efficacy. However, there is often poor correlation between the phylogenetic characterization and the antigenic phenotype of the viruses. Relying entirely on phylogenetic similarity to assess the appropriateness of a particular vaccine strain has questionable value. In this study, we sought to provide a better understanding of the molecular basis for antigenic determinants of swine H3 viruses by characterizing the antigenic phenotype of viruses that contained 1 substitution or a combination of up to 4 substitutions at positions 145, 155, 156, 158, 159, 189, and 193. We introduced site-directed mutations in the H3 HA and derived viruses on an isogenic backbone through the use of reverse genetics. To understand the variability or relative predominance of these antigenic sites in wild-type IAV circulating in pigs, publically available swine H3 sequences were additionally analyzed for patterns of evolution and relative prevalence of different antigenic motifs over time. MATERIALS AND METHODS Viruses. The reverse genetics methods used to generate virus mutants from an isogenic backbone, H3N2 A/turkey/OH/313053/2004 (OH/04), were described previously (17, 18). One substitution or combinations of up to four substitutions per mutant were introduced into the OH/04 HA by PCR-based reverse genetics (19). Changes in putative glycosylation sites were determined using NetNGlyc (http://www.cbs.dtu.dk/services /NetNGlyc/). Recovered OH/04 HA mutants were confirmed by sequencing, and virus stocks from subsequent passages were also sequenced (Table 2). Viruses were propagated in Madin-Darby canine kidney (MDCK) cells. Mutant OH/04 viruses grew efficiently in MDCK cells, with titers that ranged from 106 to 107 50% tissue culture infective doses (TCID50) per ml. Harvested cell culture supernatant was clarified by centrifugation to prepare virus stocks. A previously described panel of monovalent swine sera raised against historical and contemporary swine viruses and representative human vaccine strains (8) was used to titrate hemagglutination inhibition (HI) activity with the reverse genetics-derived wild-type (wt) OH/04, 37 OH/04 mutant viruses, and 2 swine reference viruses as antigens (Table 2). Swine antiserum production. In addition to the previously described swine serum panel (8), swine antiserum against OH/04 was raised as previously described (8). Briefly, two pigs were immunized with 128 to 256 hemagglutinin units (HAU) of UV-inactivated wt OH/04 combined with 20% commercial adjuvant (Emulsigen D; MVP Laboratories, NE, USA) by the intramuscular route and boosted 2 to 3 weeks apart. When HI titers to homologous virus reached at least 160, the pigs were humanely euthanized with pentobarbital sodium (Fatal Plus; Vortech Pharmaceuticals, MI, USA) for blood collection. Sera were collected and stored at ⫺20°C. Virus antigenic characterization. HI assays using postvaccination pig antisera were performed to compare the antigenic properties of OH/04 mutants with those of wt OH/04 and previously characterized swine viruses. HI assays were performed with a subset of a previously described reference antiserum panel (8). Prior to HI testing, sera were treated with receptor-destroying enzyme (Sigma-Aldrich, MO, USA), heat inactivated at 56°C for 30 min, and adsorbed with 50% turkey red blood cells (RBCs) to remove nonspecific inhibitors of hemagglutination. Serial 2-fold dilu-

8268

jvi.asm.org

tions starting at 1:10 were tested for the ability to inhibit the agglutination of 0.5% turkey RBCs with 8 HAU of the swine viruses described above. Antigenic cartography. Quantitative analyses of the antigenic properties of swine and human influenza A H3N2 viruses were performed using antigenic cartography as previously described for human H3, equine H3, and swine influenza A H3 and H1 viruses (8–10, 20–22). To quantify the relative distances of OH/04 mutants, we measured the antigenic distance from wt OH/04 and antigenically representative viruses to the OH/04 mutants. One antigenic unit is equal to a 2-fold loss in HI activity. Receptor binding assay. Turkey RBCs (10%, vol/vol) were pretreated with serially diluted amounts (6,666.7 to 3.3 U/ml) of ␣2-3,6,8 neuraminidase (New England BioLabs, Ipswich, MA; catalog no. P0720) for 1 h at 37°C. The RBCs were then washed twice with phosphate-buffered saline (PBS), and then 1% (vol/vol) RBC solutions were made using PBS. Aliquots (50 ␮l) of each 1% solution were added to 8 agglutinating doses of influenza A virus (as determined on non-neuraminidase-treated RBCs) in a total volume of 100 ␮l. Virus- and neuraminidase-treated red blood cells were allowed to incubate for 1 h at room temperature, and then agglutination was measured. Data are expressed as the maximal concentration of neuraminidase that allowed for full agglutination. Neutralization assays. In vitro serum neutralization (SN) was performed in 96-well flat-bottom plates. Serum was heat inactivated at 56°C for 30 min. Heat-inactivated serum was serially diluted in triplicate, 100 TCID50 units of virus was added, and the mixture was incubated for 1 h at 37°C. The virus-serum mixture was then added to MDCK cells, and virus titers were obtained by following standard procedures that have been described elsewhere (23). Data are reported as log2 of the inverse of the highest dilution that caused neutralization. Pig challenge and transmission study. A total of 35 pigs were obtained from a healthy herd free of IAV, porcine reproductive and respiratory syndrome virus (PRRSV), porcine circovirus type 2 (PCV2), and Mycoplasma hyopneumoniae. Upon arrival, the pigs were confirmed to be seronegative to IAV by performing a nuceoprotein-specific enzymelinked immunosorbent assay (ELISA) (Idexx Laboratories, ME). All pigs were treated with ceftiofur crystalline-free acid (Zoetis Animal Health, Florham Park, NJ) and enrofloxacin (Bayer HealthCare AG, NJ) prior to the start of the study to prevent bacterial infection. Pigs were divided into two groups of 10, and one group of 5 (sham), housed in individual isolation rooms, and cared for in accordance with the of the Institutional Animal Care and Use Committee of the National Animal Disease Center. Ten pigs from each challenge group were inoculated intranasally (1 ml) and intratracheally (2 ml) with 106 TCID50/ml. Inoculation of pigs was performed under anesthesia, using an anesthetic cocktail of ketamine (8 mg/kg), xylazine (4 mg/kg), and tiletamine-zolazepam (Telazol, 6 mg/kg) (Zoetis Animal Health), administered intramuscularly. Five contact pigs were placed in separate decks in the same room with each of the virusinfected groups at 2 days postinfection (dpi) to evaluate the airborne transmissibility of the wt and mutant OH/04 viruses. The pigs were observed daily for clinical signs of respiratory disease. Principal pigs from the challenge group were necropsied at 5 dpi following humane euthanasia with a lethal dose of pentorbarbital sodium (Vortech Pharmaceuticals, Dearborn, MI, USA). Sera were obtained from blood samples collected from contact pigs (17 days postcontact [dpc]) to test for HI responses against homologous and heterologous antigens. Following the pathogenesis and transmission phase of the study, 3 contact pigs from the OH/ 04_N145K⫹R189S group were boosted with whole inactivated virus, and sera from these pigs were collected 7 days later. Viral replication and shedding. Dacron nasal swabs were taken on dpi 1, 3, and 5 from principal pigs and dpc 1 to 5, 7, 9, and 11 from contact pigs to evaluate viral shedding and then placed in 2 ml of minimal essential medium (MEM). Whole lungs were removed from the principal necropsied pigs at 5 dpi, and bronchoalveolar lavage fluid (BALF) was collected for virus titer analysis by performing a lavage with 50 ml of MEM. All nasal swabs and BALF samples were stored at ⫺80°C. Nasal swab and BALF

Journal of Virology

September 2016 Volume 90 Number 18

Antigenicity of H3 Swine Influenza Viruses

TABLE 2 Antigenic distance from all OH/04 mutants to antigenic reference viruses Amino acid at indicated position (antigenic motif)e

Distance from wt OH/04

Antigenic reference virus and OH/04 mutantsd

145

155

156

158

159

189

193

Minnesota/01146/2009 OH/04 OH/04_N158D OH/04_H155Nc OH/04_N158D⫹N193S OH/04_H155N⫹N158Dc

N N N N N N

H H H N H N

N N N N N N

N N D N D D

Y Y Y Y Y Y

R R R R R R

N N N N S N

Michigan/A01432375/2013 OH/04 OH/04_N156H OH/04_N158G OH/04_Y159H OH/04_R189E OH/04_N193S OH/04_N158D⫹Y159H OH/04_H155Y⫹N156H⫹N158G⫹Y159H⫹R189Ec

N N N N N N N N N

Y H H H H H H H Y

H N H N N N N N H

G N N G N N N D G

H Y Y Y H Y Y H H

E R R R R E R R E

S N N N N N S N N

5.3

Iowa/A01203196/2012 OH/04 OH/04_N145K OH/04_N156Kc OH/04_N156K⫹R189E OH/04_N145K⫹R189E OH/04_N145K⫹N156K⫹R189Ec OH/04_N145K⫹N156K⫹R189E⫹N193S

K N K N N K K K

H H H H H H H H

K N N K K N K K

N N N N N N N N

Y Y Y Y Y Y Y Y

E R R R E E E E

S N N N N N N S

2.8

Pennsylvania/A01076777/2010 OH/04 OH/04_H155Y OH/04_R189K OH/04_H155Y⫹N193S OH/04_H155Y⫹R189K OH/04_H155Y⫹N156H⫹R189Kc OH/04_H155Y⫹Y159H⫹R189K⫹N193Sc

N N N N N N N N

Y H Y H Y Y Y Y

N N N N N N H N

N N N N N N N N

Y Y Y Y Y Y Y H

K R R K R K K K

N N N N S N N S

Wyoming/A01444562/2013 OH/04 OH/04_N145K⫹H155Yc OH/04_N145K⫹H155Y⫹R189K

K N K K

Y H Y Y

N N N N

N N N N

Y Y Y Y

K R R K

N N N N

5.1

Minnesota/A011256993/2012 OH/04 OH/04_N145K⫹H155Y⫹Y159N⫹R189Kc

K N K

Y H Y

N N N

N N N

N Y N

K R K

N N N

6.6

Michigan/A01203498/2012 OH/04 OH/04_N145K⫹R189K OH/04_N145K⫹R189K_N193S

K N K K

H H H H

N N N N

N N N N

Y Y Y Y

K R K K

S N N S

4.3

North Carolina/A01432566/2013 OH/04 OH/04_H155Y⫹N156K⫹R189E OH/04_N145K⫹H155Y⫹N156K⫹R189E

K N N K

Y H Y Y

K N K K

N N N N

Y Y Y Y

E R E E

N N N N

5.8

Iowa/A01432500/2013/ Nebraska/A01271549/2012 OH/04 OH/04_R189S

K K N N

H H H H

H K N N

N N N N

Y Y Y Y

R S R S

S N N N

5.8/6.8f

Distance from reference virus

Cyana Cyanb

0.2 1.4 NTg 0.9 NT

0.8 0.4 1.4 0.7 1.3 0.8 NT

2.0 NT 1.6 3.0 NT 3.8

0.2 1.3 NT 0.9 NT

Browna Cyanb

5.3 5.9 5.3 6.4 5.3 6.0 6.1 NT

Orangea Cyanb

2.8 1.8 NT 2.2 1.9 NT 3.0

Reda Cyanb

2.9 1.2 1.6 1.1 2.4 NT NT

NT 3.8

NT

1.3 2.4

1.8 6.9

1.3

Antigenic phenotype

2.9 2.3 2.0 2.4 1.9 NT NT

Light greena Cyanb

5.1 NT 1.8

Golda Cyanb

6.6 NT

Bluea Cyanb

4.3 3.6 2.8

Dark greena Cyanb

5.8 4.3 1.1

Purplea Cyanb 5.8/6.8 6.8/7.7 (Continued on following page)

September 2016 Volume 90 Number 18

Journal of Virology

jvi.asm.org

8269

Abente et al.

TABLE 2 (Continued) Amino acid at indicated position (antigenic motif)e Antigenic reference virus and OH/04 mutantsd

145

155

156

158

159

189

193

Distance from wt OH/04

Distance from reference virus

OH/04_N145K⫹N156H OH/04_N145K⫹R189S OH/04_N156K⫹R189S OH/04_N145K⫹N156K⫹R189Sc OH/04_N145K⫹N156H⫹N193Sc

K K N K K

H H H H H

H N K K H

N N N N N

Y Y Y Y Y

R S S S R

N N N N S

2.0 3.3 2.0 NT NT

4.5/5.5 4.6/5.6 6.6/7.6 NT NT

Nebraska/A01241171/2012 OH/04 OH/04_N145K⫹N156Kc

K N K

H H H

K N K

N N N

Y Y Y

R R R

N N N

7.5 NT

7.5 NT

Antigenic phenotype

Dark pinka Cyanb

a

Determined previously by Lewis et al. (8). b Determined in this study. c One or more substitutions reverted after passaging of the virus. d Antigenic reference viruses are indicated in bold. e Substitutions introduced into OH/04 are indicated in bold. f Values separated by a slash correspond to distances from Iowa/A01432500 and Nebraska/A01271549/2012, respectively. g NT, not tested.

samples were processed for virus isolation and virus titers by following standard procedures that have been described elsewhere (23). Pathological examination of lungs. Lungs were removed from euthanized principal pigs to determine the percentage of surface area with purple-red consolidation typical of the pathology caused by IAV. For each lung lobe, an estimate of the percentage of surface area affected with pneumonia was performed visually, and the total percentage for the entire lung was estimated based on weighted proportions of each lobe to total lung volume (24, 25). Tissue samples from the trachea and right middle or affected lung lobe were fixed in 10% buffered formalin for histopathologic examination. Microscopic lesions were evaluated by a veterinary pathologist that was blind to the identity of the treatment groups. Scores were adapted from a previously described scoring system (26), and a composite score was computed with the sum of the six individual scores (0 to 21). Statistical analysis. The percentage of macroscopic lesions, the microscopic lesion scores, and the log10-transformed BALF and NS virus titers of principal pigs were analyzed using ordinary one-way analysis of variance, with a P value of ⱕ0.05 considered significant, followed by Tukey’s multiple-comparison test (GraphPad Prism 6; GraphPad Software, La Jolla, CA). Nasal swab virus titers from contact pigs were analyzed using two-way analysis of variance, with a P value of ⱕ0.05 considered significant, followed by Tukey’s multiple-comparison test. Phylogenetic analysis. A total of 1,341 H3 HA swine IAV (IAV-S) nucleotide sequences available in the Influenza Research Database (IRD) on 9 July 2015 were downloaded for phylogenetic analysis. In addition to the 1,341 sequences originally downloaded, 3 turkey and 12 human H3 IAV isolates were included to use as references for clade designation. A nucleotide alignment was generated using default settings in MUSCLE (27) with subsequent manual correction in Geneious v.6.1.6 (28). A maximum-likelihood tree was inferred using RAxML v.8.1.24 (29) on the Cipres Science Gateway (30) with a general time-reversible (GTR) model of nucleotide substitution with gamma-distributed rate variation among sites. The starting tree was generated using parsimony methods with the best-scoring tree and statistical support obtained using the rapid bootstrap algorithm, with the number of replicates determined automatically using an extended majority-rule consensus tree criterion (31). Each H3 HA IAV-S sequence was assigned to a phylogenetic clade as described by Kitikoon et al. (32). Amino acids at positions 145, 155, 156, 158, 159, and 189 were recorded to analyze antigenic motif diversity. Of the 1,341 sequences, 20 were removed on the basis of missing sequence data for at least one position within the antigenic motif, for not belonging to designated genetic clades, or for being identical sequences. The remaining 1,321 sequences were used to obtain counts of HA genes assigned to

8270

jvi.asm.org

antigenic clusters (e.g., red, light green) in each year and phylogenetic clade.

RESULTS

Isogenic backbone virus and mutants. A/turkey/OH/313053/ 2004(H3N2) (OH/04) is a prototypic swine origin clade IV virus that grows well in cell culture, infects and transmits efficiently in pigs, and is amenable to genetic manipulations using a reverse genetics system (17, 18, 33). One substitution or combinations of up to four substitutions per mutant were introduced into the HA based on previous findings showing that six positions (positions 145, 155, 156, 158, 159, and 189) played a role in major antigenic changes in wild-type swine H3 viruses circulating in the United States (8). Substitutions were also introduced at position 193 because it was shown to be important for human H3 antigenic evolution (14). Thirty-seven mutant viruses generated using a reverse genetics system were selected for this study (Table 2). Substitutions did not affect any putative glycosylation sites found in wt OH/04 nor did they produce novel putative glycosylation sites (data not shown). None of the substitutions were lethal for virus recovery, although reversions at a substitution site were observed in 12 viruses in the first passage after virus recovery (Table 2). Mutant viruses with reversions were omitted from the antigenic analyses. Five mutants encoded antigenic motifs that matched wild-type antigenic cluster/outlier viruses previously identified for swine (Table 3) (8). The remaining 20 mutants were used to explore the impact of amino acid diversity at the motif sites. Analysis of antigenic properties using swine antisera. HI assays were performed using a previously described swine antiserum panel (8), with the addition of sera raised against wild-type (wt) OH/04. The HI data were used to generate an antigenic map. The distances between viruses in the map are measured in antigenic units (AU), and 1 AU is equivalent to a 2-fold dilution in the HI assay. The antigenic position of the wt OH/04 in the context of IAV-S antigenic cartography was not included in the previous report, but it was predicted to fall within the cyan antigenic cluster based on its antigenic motif (Table 3). HI data showed that the OH/04 antigen was indeed located near the previously described

Journal of Virology

September 2016 Volume 90 Number 18

Antigenicity of H3 Swine Influenza Viruses

TABLE 3 Antigenic distance from a subset of OH/04 mutants shown in Fig. 1 relative to reference field isolates Amino acid at indicated position (antigenic motif)e

Distance from wt OH/04b

Distance from reference virusb

Antigenic reference virus or OH/04 mutantd

145

155

156

158

159

189

193

A/Swine/Minnesota/01146/2009 (MN/06) A/Turkey/OH/313053/2004 (OH/04)

N N

H H

N N

N N

Y Y

R R

N N

0.2

A/Swine/Pennsylvania/A01076777/2010 (PA/10) OH/04_H155Y⫹R189K

N N

Y Y

N N

N N

Y Y

K K

N N

2.9 2.4

1.9

A/Swine/Iowa/A01203196/2012 (IA/12) OH/04_N145K⫹N156K⫹R189E⫹N193S

K K

H H

K K

N N

Y Y

E E

S S

2.8 3.8

3.0

A/Swine/Wyoming/A01444562/2013 (WY/13) OH/04_N145K⫹H155Y⫹R189K

K K

Y Y

N N

N N

Y Y

K K

N N

5.1 3.8

1.8

A/Swine/North Carolina/A01432566/2013 (NC/13) OH/04_N145K⫹H155Y⫹N156K⫹R189E

K K

Y Y

K K

N N

Y Y

E E

N N

5.8 6.9

1.1

A/Swine/Michigan/A01203498/2012 (MI/12) OH/04_N145K⫹R189K_N193S

K K

H H

N N

N N

Y Y

K K

S S

4.3 2.4

2.8

A/Swine/Nebraska/A01271549/2013 (NE/12) OH/04_N145K⫹R189Sc

K K

H H

K N

N N

Y Y

S S

N N

6.8 3.3

5.6

Antigenic phenotype Cyana

0.2 Reda

Orangea

Light Greena

Dark Greena

Bluea

Purplea

a

Determined previously by Lewis et al. (8). One unit of antigenic distance is equal to a 2-fold difference in the HI assay. c OH/04 mutant selected to perform fitness analysis in vivo. d Antigenic reference viruses are indicated in bold. e Mutated amino acids are indicated in bold. b

cyan antigens on the antigenic map and was 0.2 AU from the representative cyan antigen (A/Swine/Minnesota/01146/2006) and 2.9 AU from the representative red antigen (A/Swine/Pennsylvania/A01076777/2010) (Fig. 1A). The OH/04 mutants had antigenic phenotypes that differed significantly from that of wt OH/04 (Table 3; Fig. 1A to F). Antigenic changes ranged from 2.4 AU to 6.9 AU. With the sole exception of OH/04_N145K, individual substitutions did not cause significant antigenic changes (⬎2 AU) relative to wt OH/04 (Table 2). One OH/04 double mutant, OH/ 04_N145K⫹R189S, encoding a motif similar to that of the purple outlier group, caused an antigenic change of 3.3 AU relative to OH/04 (Table 3; Fig. 1G). To rule out whether virus-receptor affinity affected the HI titers of the mutant viruses, we tested their ability to agglutinate red blood cells (RBCs) that had been treated with increasing levels of neuraminidase. Mutant viruses and wt OH/04 bound to RBCs treated with similar neuraminidase concentrations (Table 4), indicating that the results observed in the HI assay were not confounded by changes in receptor affinity. Furthermore, serum neutralization (SN) was performed with a subset of sera and viruses, and a positive correlation was observed between the HI and SN results (Table 5). The Spearman’s rank correlation score we observed (r ⫽ 0.72 to 0.81) is comparable to what others have reported (r ⫽ 0.78 to 0.88) when comparing HI titers and neutralization titers (34, 35). This supports the tenet that not only are the introduced amino acid substitutions causing antigenic change and such antigenic change is characterized by the gold standard HI assay but that they are also clinically relevant, as they were associated with loss of cross-HI and cross-neutralizing titers.

September 2016 Volume 90 Number 18

Pathogenesis and transmission of OH/04_N145KⴙR189S. Since mutations at positions 145 and 189 were repeatedly indicated as influential and due to the large antigenic distance resulting from the double mutation, OH/04_N145K⫹R189S was selected to test for phenotypic changes in vivo, as measured by pathogenesis and transmission in comparison with wt OH/04. Pigs infected with OH/04_N145K⫹R189S had typical macroscopic lesions in the lungs, although less than those observed in pigs infected with wt OH/04 (Fig. 2A). Virus was detected in the lungs of both challenge groups, and group mean virus titers were comparable between the two groups (Fig. 2B). Likewise, similar levels of virus were observed in nasal secretions beginning at 1 dpi through 5 dpi and peaking at 3 dpi (Fig. 2C to E). Virus was detected in the nasal swabs from all five contact pigs of both groups beginning at 5 dpc: however, the contact pigs of the OH/ 04_N145K⫹R189S challenge group virus demonstrated a decreased shedding period, with significantly less virus by 9 dpc compared to that for the wt OH/04 group. Consistent with the HI data generated with monovalent reference antisera used in the antigenic map, contact pigs that seroconverted from natural exposure to wt OH/04 had a 14-fold reduction in cross-reactivity to OH/04_N145K⫹R189S antigen compared to that to wt OH/04 when assayed by HI (Table 6). At 17 dpc, contact pigs exposed to OH/04_N145K⫹R189S had low levels of HI titers to homologous antigen, with a geometric mean of 35, perhaps correlated with reduced replication as indicated by the low nasal titers. To increase HI titers for the cross-HI assays, 3 pigs were boosted with an adjuvanted whole inactivated double mutant virus. Sera collected at 7 days postboost reached mean homologous HI titers of 253 to OH/04_N145K⫹R189S and demonstrated a 5-fold reduction in cross-reactivity to the wt OH/04

Journal of Virology

jvi.asm.org

8271

Abente et al.

FIG 1 Three-dimensional (3D) antigenic maps of OH/04 mutants and reference viruses. (A) Relative positions of wild-type OH/04 and reference viruses; (B) OH/04_H155Y⫹R189K and a representative red antigenic virus, A/Swine/Pennsylvania/A01076777/2010 (PA/10); (C) OH/04_N145K⫹N156K⫹ R189E⫹N193S and the orange outlier virus, A/Swine/Iowa/A01203196/2012 (IA/12); (D) OH/04_N145K⫹H155Y⫹R189K and the light green outlier virus, A/Swine/Wyoming/A01444562/2013 (WY/13); (E) OH/04_N145K⫹H155Y⫹N156K⫹R189E and the dark green outlier virus, A/Swine/North Carolina/ A01432566/2013 (NC/13); (F) OH/04_N145K⫹R189K_N193S and the blue outlier virus, A/Swine/Michigan/A01203498/2012 (MI/12); (G) OH/ 04_N145K⫹R189S, the double mutant used to test for pathogenesis and transmission, and A/Swine/Nebraska/A01271549/2013 (NE/12). Wild-type OH/04 and OH/04 mutants are shown as larger spheres, and the corresponding antigenic motifs are labeled. Reference viruses corresponding to antigenic clusters and outliers are color coded in accordance with the color scheme used previously by Lewis et al. (8). The scale bar in each map represents 2 antigenic units (1 antigenic unit corresponds to a 2-fold dilution of antiserum in the HI assay).

8272

jvi.asm.org

Journal of Virology

September 2016 Volume 90 Number 18

Antigenicity of H3 Swine Influenza Viruses

TABLE 4 Relative receptor binding avidities of OH/04 and a subset of OH/04 mutantsa Virus

Relative receptor binding avidity

OH/04 OH/04_H155Y OH/04_R189E OH/04_N145K⫹H155Y⫹R189K OH/04_N145K⫹R189K OH/04_N145K⫹R189S OH/04_H155Y⫹R189K OH/04_N145K⫹H155Y⫹N156K⫹R189E OH/04_N145K⫹N156K⫹R189E⫹N193S

⫹⫹ ⫹⫹ ⫹⫹ ⫹⫹ ⫹⫹ ⫹⫹ ⫹⫹ ⫹⫹ ⫹⫹

a All viruses tested were able to agglutinate RBCs treated with 833 to 1,666 U/ml of neuraminidase (⫹⫹). Data represent the results of 3 independent experiments performed in triplicate. A/Victoria/361/11(H3N2) was included as a reference virus with low receptor binding avidity. A/Victoria/361/11(H3N2) was able to agglutinate RBCs treated with 104.2 U/ml of neuraminidase.

antigen, which was more consistent with the converse wild-type antiserum reactivity to the mutant antigen (Table 6). Frequency of antigenic motifs in contemporary viruses. From the 1,341 sequences downloaded from the IRD, 1,180 swine H3 HA sequences are from viruses collected from 2009 to 2015 in the United States; these contemporary isolates were used to determine the relative proportions of the different antigenic motifs (Fig. 3A and B). Approximately 50% had an antigenic motif associated with the red antigenic cluster (593), 11% had an antigenic motif associated with the cyan antigenic cluster (127), 12% had an antigenic motif associated with the light green antigenic outlier group (139), and the remaining 27% (321) were comprised of viruses with 92 unique antigenic motifs, referred to as “other,” that did not match the predicted red, cyan, or light green antigenic motifs. Temporally, the antigenic motif corresponding to the cyan antigenic cluster was predominant in 2009 (43/56; 77%) but was detected less frequently in subsequent years (Fig. 3A). The antigenic motif associated with the red antigenic cluster was detected as early as 2009 (4/56; 7%) and became numerically dominant

from 2010 through 2015, ranging from 35% to 68% of reported viruses. Of note, strains expressing an antigenic motif corresponding to the light green antigenic outlier rose to 33% in 2013, which is nearly as predominant as the red antigenic strains at 40%, but circulated at lower frequencies of 13% in 2014 and 12% in 2015. Among the more recent 2015 viruses available in public databases, an increasing number (i.e., 321) contained unique putative antigenic motifs that did not match our defined set (Fig. 3A). Only 50 (16%) of “other” viruses have an antigenic motif that matches a previously described antigenic outlier, and the remaining 271 viruses are referred to as “unknown” in Fig. 3B. There were 60 different possible antigenic motifs among the 271 virus strains classified as “unknown,” and while they varied in frequency, the most common antigenic motif represented only 14% of the “other” subtotal (38) (Table 7). These “other” motifs indicate an unexpectedly large amount of potential antigenic diversity in circulating swine H3N2 viruses and suggest a steady expansion of antigenic diversity since 2011. A total of 1,341 publicly available swine H3 HA sequences from the United States as of 9 July 2015 were obtained and used to perform a phylogenetic analysis to examine if specific antigenic motifs correlated with genetic clades. Twenty of the 1,341 HA sequences were removed for antigenic motif analysis because of missing sequence data for at least one position within the antigenic motif or for being identical sequences, and therefore 1,321 HA sequences were used to assign putative antigenic clusters. The antigenic motif patterns of swine H3 did not strongly correlate with previously determined phylogenetic clade IV or clades IV-A to -F (Fig. 3C). While no particular antigenic motif was restricted to a single clade, some patterns were observed. Clade IV contained mostly viruses with a cyan antigenic motif (141/244; 58%), followed by “other” (88/244; 36%), and a small proportion of red antigenic motif viruses (15/244; 6%). Clade IV-A had mostly red antigenic motif viruses (477/691; 69%), followed by light green (131/691; 19%), “other” (82/691; 12%), and cyan antigenic motif viruses (1/691; ⬍1%). Clade IV-B had comparable proportions of red antigenic motif viruses (80/170; 47%) and “other” (86/170;

TABLE 5 Relative log2 change of SN and HI reciprocal of geometric mean titers compared to homologous titersa Relative log2 change of GMT of indicated serum sample OH/04b (r ⫽ 0.75)d Test antigen OH/04 MN/06 NY/11 WY/13 OH/04_H155Y OH/04_R189E OH/04_N145K⫹ H155Y⫹R189K OH/04_N145K⫹ R189K OH/04_N145K⫹ R189S OH/04_H155Y⫹ R189K OH/04_N145K⫹ H155Y⫹N156K⫹ R189E OH/04_N145K⫹ N156K⫹R189E⫹ N193S

SN ⫺2.3 ⫺3.0 ⫺4.0 1.0 ⫺1.4 ⫺3.3 ⫺3.0 ⫺2.3 ⫺3.0 ⫺4.0 ⫺3.3

NY/11 (r ⫽ 0.81)

WY/13b (r ⫽ 0.72)

SN

HI

SN

HI

SN

HI

⫺1.7 ⫺3.0

⫺2 ⫺2

⫺0.7 ⫺0.7 ⫺1.4 ⫺1.7 ⫺1.4 ⫺4.7 ⫺1.0 ⫺4.7 ⫺4.7

⫺2 ⫺1 ⫺2 ⫺1 ⫺2 ⫺4 ⫺1 ⫺5 ⫺4

⫺5.7 ⫺5.7 ⫺5.0

⫺6 ⫺5 ⫺4

⫺4.7 ⫺5.0 ⫺3.7 ⫺4.0 ⫺3.3 ⫺5.0 ⫺4.0 ⫺5.0

⫺5 ⫺7 ⫺2 ⫺3 ⫺3 ⫺5 ⫺3 ⫺2

⫺2.7 ⫺2.3 ⫺1.7 0.3 ⫺2.0 ⫺2.3 ⫺0.7 ⫺0.3

⫺2 ⫺3 ⫺4 ⫺1 ⫺4 ⫺3 0 0

⫺3.0 ⫺1.0 0.7

⫺5 ⫺1 ⫺1

b

HI ⫺2 ⫺4 ⫺6 0 ⫺3 ⫺2 ⫺1 ⫺2 ⫺1 ⫺6 ⫺5

OH/04_N15K⫹ R189Sc (r ⫽ 0.73)

a

SN, serum neutralization; HI, hemagglutination inhibition. Reference serum produced by delivering two doses of whole inactivated virus. c Serum from contact pigs exposed to OH/04_N145K/R189S that were boosted with one dose of whole inactivated virus. d r, Spearman’s rank correlation between the relative change in SN and HI titers. b

September 2016 Volume 90 Number 18

Journal of Virology

jvi.asm.org

8273

Abente et al.

FIG 2 Pathogenesis and transmissibility of wild-type OH/04 and OH/04_N145K⫹R189S. (A) Percentages of macroscopic lung pathology in the principal pigs. (B to E) Virus titers in the bronchoalveolar lavage fluid (BALF) (B) and nasal swabs (C to E) of principal pigs. (F) Virus titers in nasal swabs of contact pigs. In panels A to E, different lowercase letters within the same sampling day indicate statistically significant differences (P ⱕ 0.05). In panel F, an asterisk indicates statistically significant differences. NC, nonchallenged.

51%), as well as light green antigenic motif viruses (4/170; 2%). Clade IV-C, a minor clade with only 11 sequences, was composed of “other” (10/11; 91%) and a light green antigenic motif virus (1/11; 9%). Clade IV-D contained red antigenic motif viruses (13/ 26; 50%), “other” (11/26; 42%), and light green antigenic motif viruses (2/26; 8%). Clade IV-E had mostly “other” antigenic motif viruses (25/38; 66%), followed by red antigenic motif viruses (12/ 38; 32%), and a single light green antigenic motif virus (1/38; 2%). Lastly, in clade IV-F, we observed cyan antigenic motif viruses (48/72; 67%) and “other” antigenic motif viruses (24/72; 33%). In general, red antigenic motif viruses were more widely distributed throughout the phylogenetic tree, light green antigenic motif vi-

8274

jvi.asm.org

ruses were predominantly from clade IV-A (131/139; 94%), and cyan antigenic motif viruses were observed mostly in clade IV (141/190; 74%) and clade IV-F (48/190; 25%). DISCUSSION

Bioinformatic approaches have led to important advances in understanding the antigenic evolution of human H3 IAV (9, 36, 37), and substitutions at 7 amino acid positions, including the 6 observed for IAV-S, were experimentally shown to largely account for the antigenic evolution over a 36-year time span (14). These substitutions were associated with the emergence of antigenically distinct H3N2 in the human population with epidemiologic rele-

Journal of Virology

September 2016 Volume 90 Number 18

Antigenicity of H3 Swine Influenza Viruses

TABLE 6 Homologous and heterologous HI titers of contact pigs HI GMTa

Antigen

OH/04 antisera (n ⫽ 5; 17 dpc)b

OH/04_N145K⫹R189S antisera (n ⫽ 5; 17 dpc)

OH/04_N145K⫹R189S antisera (n ⫽ 3; boosted with WIV; 7 dpb)

OH/04 OH/04_N145K⫹R189S

279 20 (14.0)

13.2 (2.7) 35

50 (5.1) 253

a

GMT, geometric mean titer. Values in parentheses following the GMT are relative fold reductions of heterologous antigen compared to the homologous titer. Shaded values indicate homologous HI titers. WIV, whole inactivated virus. b The number of samples tested and the day postcontact (dpc) or postboost (dpb) when sera were collected are indicated in parentheses.

vance. The H3N2 component of the human vaccine for 2015-2016 was most recently recommended to be changed by the WHO vaccine strain selection process (38). The antigenic change that caused a significant loss in cross-reactivity with the previous H3 vaccine component was mapped to a single amino acid at position 159 (39), serving as a recent example that a single amino acid substitution can be associated with a loss in vaccine efficacy. The antigenic variation at these 7 sites was observed in the context of human seasonal IAV evolving under human population immune pressure, and evaluating and comparing evolution and host immune pressure on swine IAV have not previously been possible. Studies conducted by our group and others demonstrated that amino acids at positions 145, 155, 156, 158, 159, and 189 are associated with antigenic cross-reactivity of H3 IAV-S in the United States (8, 15). Unlike the human H3 antigenic evolution study that identified substitutions accounting for the sequential emergence of seasonal H3N2 viruses requiring vaccine strain updates that mirror the evolution observed at the genetic level (14, 40, 41), many putative antigenic variants of H3 viruses have cocirculated in U.S. pig populations since 2009. While there was no strong correlation between antigenic and genetic evolution as observed by phylogenetic analyses with IAV in U.S. swine, genetic analyses that incorporate phenotypically relevant changes will help refine genetic predictors of antigenic evolution and allow investigation of the relative prevalence and temporal-spatial dynamics of emerging antigenic variants. However, since we used an isogenic backbone for all mutations, as opposed to retracing the sequential emergence of antigenically distinct variants from a common ancestor, further work based on naturally emerging antigenic variants remains to be explored. The antigenic effect of genetic variation at additional sites other than those studied, and determining whether there is intergene epistasis, has yet to be determined but may also affect antigenic drift. Positions 145, 155, 156, 158, 159, and 189 included in the putative antigenic motif are located adjacent to the RBS, and others have reported that modifications of HA antigenicity can cause changes in sialic acid binding properties (42–46). Even though the substitutions introduced in the OH/04 mutants match amino acids known to exist in circulating strains, additional mutations in the HA, as well as epistatic mutations in the NA, can arise to compensate for loss of receptor avidity and/or affinity (46, 47). However, the strength of the study reported here demonstrates that combinations of substitutions at these discrete positions in the HA caused significant changes in the antigenic phenotype of a clade IV prototype cyan virus. As surveillance methods improve and sequence data become more readily available, extrapolating potential antigenicity based more heavily on the HA sequence has important vaccine implications.

September 2016 Volume 90 Number 18

In contrast to influenza monitoring in humans, an extensive and concerted surveillance effort for IAV-S in the United States only began in 2009 (16, 48). H3 IAVs circulating in swine in the United States since 2005 have been almost exclusively from clade IV, which then diversified into clades A to F around 2010 (16, 32). Based on previous experimental data, the antigenic phenotype was not strongly correlated with the phylogenetic clade (8). Extending on this analysis, we extrapolated the potential antigenic phenotype based on the antigenic motif of publically available contemporary U.S. IAV-S H3 sequences (Fig. 3). According to these data, there were at least three major antigenic clusters: cyan, red, and light green. Putative cyan-like viruses like OH/04 were predominant in 2009, and the cyan antigenic motif was the most common antigenic motif of clades IV and IV-F. However, detection of viruses with the cyan antigenic motif was greatly reduced after 2009, and this motif has been rarely detected since 2013. Commercial vaccines produced and tailored to the epidemiology of IAV-S prior to 2009 would have likely targeted cyan motif viruses, potentially having reduced efficacy against the red antigenic cluster viruses that emerged in 2010 and continue to circulate at a high frequency. Viruses with “other” antigenic motifs represented the greatest proportion of swine IAVs in the United States in 2015. Based on the antigenic distances we have documented, it is likely that a vaccine formulated to protect against cyan or red antigenic clusters would not protect against these outlier motif antigenic variants. Experimental data suggest that the protection provided by commercial killed vaccines against contemporary IAV-S is limited (49, 50). Importantly, the USDA Center for Veterinary Biologics implemented a new licensure policy in 2007 that allows vaccine manufacturers to update vaccine strains more rapidly under a currently approved licensing agreement (51). However, manufacturers are not required to inform or include strains contained in the vaccine on the label, so we cannot speculate on the potential coverage of commercial vaccines. Additionally, autogenous vaccines are commonly used (52, 53), accounting for approximately 50% of vaccines produced in 2008 to 2011 (53). All of these factors complicate our understanding of which antigens are being routinely used for vaccination and obscure large-scale objective measures of vaccine efficacy in the field. We found it remarkable that OH/04 tolerated many mutations at sites known to affect antigenic determinants, but total HA antigenic plasticity has not yet been determined. Of note, reversions were observed in 12 of 37 mutants, suggesting that tolerability of amino acid changes in the antigenic motif is context dependent and likely involves compensatory mutations elsewhere in the HA. Deep sequencing of viruses during outbreaks in vaccinated populations may help elucidate the level of plasticity within the anti-

Journal of Virology

jvi.asm.org

8275

FIG 3 Temporal and phylogenetic distribution of antigenic motifs. (A) All IAV-S H3 HA sequences available from the Influenza Research Database as of 9 July 2015 were obtained and analyzed to determine their antigenic motifs (amino acids at positions 145, 155, 156, 158, 159, and 189). Shown are the percentages of virus strains that encode antigenic motifs matching the red, light green, or cyan antigenic cluster or none of those (“other”) over time. (B) Percentages of “other” virus strains that encode antigenic motifs matching outlier antigen motifs. Virus strains encoding an antigenic motif that has not been previously characterized are designated “unknown.” (C) Maximum-likelihood phylogeny of the HA of 1,341 swine influenza viruses, 3 turkey viruses, and 12 human viruses. H3N2 genetic clades are colored: clade IV is brown, clade IV-A is red, clade IV-B is blue, clade IV-C is green, clade IV-D is yellow, clade IV-E is gray, clade IV-F is dark yellow, and human and recent human-like swine viruses are pink. The tree is midpoint rooted for clarity, and all branch lengths are drawn to scale; the scale bar indicates nucleotide substitutions per site. Pie charts to the right of the phylogeny show distribution of predicted antigenic phenotypes for each clade. The number of HA sequences corresponding to each clade is indicated adjacent to the pie chart.

8276

jvi.asm.org

Journal of Virology

September 2016 Volume 90 Number 18

Antigenicity of H3 Swine Influenza Viruses

TABLE 7 Frequency of antigenic motifs from 271 virus strains circulating from 2009 to 2015 that do not match previously characterized antigenic motifs (designated “unknown” in Fig. 3B)

TABLE 8 Amino acid identity of the hemagglutinin subdomain HA1 of wild-type OH/04 and reference antigenic cluster/outlier viruses

Antigenic motif

No. of virus strains (% of total) with motif

NYSNYK SYKNYK KTHNFK KHNNHK KYHNYK KHHNNK KYNNSK NHNDYK NHSNYK KYHNNK KYTNHK NHNNHK KTHNSK RYNNYK NHSNYM NYKNYK KHNNYR KHSNFK KYKNYK KYNDYK KYNNDK KYNNHK NHKNYR NHNGYR NYNNNK NYNNYR KHKNFS KHSNYK NHHNHM NHKDYK NHSGYK NYHNYK NYKNYN KHAEYR KHKNYN KHNDYK KHNDYR KHSEYK KHSNSR KYHNHK KYHNNN KYHNNR KYNNYQ KYQSHR KYSDYK KYSNYK NHHGHE NHKTNS NHNNDR NHNNHR NHSDYR NYHNNR NYKDYK NYKDYM NYNNYN NYNNYT RYKNYK RYSNYK SNNNYK SYKDYK

38 (14.02) 22 (8.12) 20 (7.38) 18 (6.64) 15 (5.54) 14 (5.17) 13 (4.80) 11 (4.06) 11 (4.06) 8 (2.95) 6 (2.21) 6 (2.21) 5 (1.85) 5 (1.85) 4 (1.48) 4 (1.48) 3 (1.11) 3 (1.11) 3 (1.11) 3 (1.11) 3 (1.11) 3 (1.11) 3 (1.11) 3 (1.11) 3 (1.11) 3 (1.11) 2 (0.74) 2 (0.74) 2 (0.74) 2 (0.74) 2 (0.74) 2 (0.74) 2 (0.74) 1 (0.37) 1 (0.37) 1 (0.37) 1 (0.37) 1 (0.37) 1 (0.37) 1 (0.37) 1 (0.37) 1 (0.37) 1 (0.37) 1 (0.37) 1 (0.37) 1 (0.37) 1 (0.37) 1 (0.37) 1 (0.37) 1 (0.37) 1 (0.37) 1 (0.37) 1 (0.37) 1 (0.37) 1 (0.37) 1 (0.37) 1 (0.37) 1 (0.37) 1 (0.37) 1 (0.37)

September 2016 Volume 90 Number 18

Reference virus (antigenic cluster/outlier classification)

% amino acid identity with wt OH/04

A/Swine/Minnesota/01146/2006 (cyan) A/Swine/Pennsylvania/A01076777/2010 (red) A/Swine/Iowa/A01203196/2012 (orange) A/Swine/Wyoming/A01444562/2013 (light green) A/Swine/North_Carolina/A01432566/2013 (dark green) A/Swine/Iowa/A01432500/2013 (purple) A/Swine/Michigan/A01203498/2012 (blue) A/Swine/Nebraska/A01271549/2012 (purple)

97.3 96.1 93.3 93.9 93.0 92.1 94.5 93.0

genic motif and further identify compensatory substitutions that may arise. Though still undetermined, additional phenotypic advantages for viruses with specific antigenic motifs may exist, as viruses with the red antigenic motif have been predominant for the last 5 years whereas viruses with a cyan antigenic motif were predominant in 2009 and earlier. Antigenic motifs classified as “other” may also represent intra-antigenic cluster diversity or may be novel antigenic clusters poised to emerge as the next dominant antigenic cluster. Individual substitutions within the antigenic motif caused moderate changes (0.4 to 2.0 AU), whereas multiple substitutions that replaced the cyan antigenic motif with motifs matching other antigenic clusters or outliers resulted in significant changes of 2.4 to 6.9 AU (Fig. 1 and Tables 2 and 3; see Fig. S1 in the supplemental material). Introducing either the red, light green, or dark green antigenic motif into OH/04 not only yielded a 2.4-, 3.8-, and 6.9-AU change relative to wt OH/04, respectively, but also relocated the mutant OH/04 antigens to within 1.9 AU of the reference outlier viruses (Table 3). While residues within the antigenic motif were shown to be major contributors to antigenic drift, other positions also likely affect antibody recognition of the HA and changes at other positions may also influence antigenic phenotype. Indeed, here we observed that mutants generated in the OH/04 background did not always map close to the outlier when the sequence identity at the amino acid level between the OH/04generated mutant and the observed wild-type virus outlier was low. Thus, some of these additional amino acid changes also likely influenced the observed antigenic phenotype (Table 8; see Fig. S1). Further work is required to determine additional positions that may contribute to the antigenic evolution of H3 viruses in swine. Antigenic changes can come at a cost of fitness, sometimes due to lower avidity to sialic acids, and substitutions outside the antigenic motif may be compensatory mutations or simply hitchhiker mutations (43, 44, 54, 55). We selected one OH/04 mutant with large antigenic changes to assess in vivo other virus properties and found that its replication in the upper and lower respiratory tract in directly inoculated pigs was comparable to that of wt OH/04 but caused significantly lower levels of lung lesions (Fig. 2). The implications of the differences in macroscopic lung lesions have yet to be explored. Despite the similar virus titers between wt and mutant OH/04 viruses in the primary infected pigs, there was an apparent impact on the kinetics of transmission and replication in the contact pigs exposed to the double mutant. This observation also deserves further investigation. Consistent with the data generated from the antigenic map, heterologous HI tests with antisera

Journal of Virology

jvi.asm.org

8277

Abente et al.

from naturally exposed pigs with wt and double mutant OH/04 viruses also showed reduced cross-reactivity (Table 6). The fold change between homologous and heterologous reactions was remarkable considering that only 2 amino acid changes in the HA were introduced into the isogenic OH/04 backbone. OH/ 04_N145K⫹R189S had a receptor binding avidity similar to that of the wt (Table 4), but further in vitro and in vivo studies with additional mutants are required to better understand other properties of the HA that may be affected due to the significant antigenic changes observed in the OH/04 mutants. Since the putative antigenic motif (positions 145, 155, 156, 158, 159, and 189) caused significant changes in the antigenic phenotype, ongoing comprehensive analyses of contemporary field isolates that combine phylogeny with antigenic motif is warranted to make assessments of the overall antigenic diversity of H3 in the U.S. swine population and to inform vaccine selection. HI analysis and antigenic maps of circulating field isolates that differ in the antigenic motif region would further validate the results observed in the present study and provide useful information to vaccine manufacturers and swine producers. As sequencing is common in veterinary diagnostic labs and in surveillance efforts, screening the antigenic motif of virus isolates may serve as a rational approach to select viruses of interest for antigenic analysis and potential vaccine seed strains. Screening of sequences of human H3 IAV through a user-friendly website that allows for real-time tracking of antigenic motifs from circulating strains is available (56) and may also be applicable for IAV-S as we better understand the antigenic determinants of H3 IAV in the context of the swine host. Continued research into the antigenic evolution of IAV-S is a necessary step to help guide swine vaccine manufacturers to develop more efficacious vaccines and has a dual benefit of improving health and production in swine and of mitigating the risk of sporadic zoonotic infections and/or the emergence of swine IAV with pandemic potential. ACKNOWLEDGMENTS We thank Michelle Harland and Gwen Nordholm for technical assistance and Jason Huegel and Tyler Standley for assistance with animal studies. The mention of trade names or commercial products in this article is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the USDA, DOE, or ORISE/ ORAU. This study was supported by USDA-ARS, by USDA-APHIS, and by an NIH-National Institute of Allergy and Infectious Diseases (NIAID) interagency agreement (AA14006) associated with CRIP (Center of Research in Influenza Pathogenesis), an NIAID-funded Center of Excellence in Influenza Research and Surveillance (CEIRS; HHSN272201400008C). N.S.L. was supported in part by USDA-ARS agreement 58-3625-4-071F. E.J.A. and T.K.A. were supported in part by an appointment to the ARS-USDA Research Participation Program administered by the Oak Ridge Institute for Science and Education (ORISE) through an interagency agreement between the U.S. Department of Energy (DOE) and USDA. ORISE is managed by ORAU under DOE contract number DEAC05-06OR23100.

FUNDING INFORMATION This work, including the efforts of Amy L. Vincent, was funded by HHS | NIH | National Institute of Allergy and Infectious Diseases (NIAID) (HHSN272201400008C). The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

8278

jvi.asm.org

REFERENCES 1. Huber VC. 2014. Influenza vaccines: from whole virus preparations to recombinant protein technology. Expert Rev Vaccines 13:31– 42. http: //dx.doi.org/10.1586/14760584.2014.852476. 2. U.S. Department of Agriculture. 2007. Swine 2006. Part II: reference of swine health and health management practices in the United States, 2006. U.S. Department of Agriculture, Fort Collins, CO. 3. Ma W, Richt JA. 2010. Swine influenza vaccines: current status and future perspectives. Anim Health Res Rev 11:81–96. http://dx.doi.org/10.1017 /S146625231000006X. 4. Krammer F, Palese P. 2015. Advances in the development of influenza virus vaccines. Nat Rev Drug Discov 14:167–182. http://dx.doi.org/10 .1038/nrd4529. 5. Wiley DC, Wilson IA, Skehel JJ. 1981. Structural identification of the antibody-binding sites of Hong Kong influenza haemagglutinin and their involvement in antigenic variation. Nature 289:373–378. http://dx.doi .org/10.1038/289373a0. 6. Bush RM, Bender CA, Subbarao K, Cox NJ, Fitch WM. 1999. Predicting the evolution of human influenza A. Science 286:1921–1925. http://dx.doi .org/10.1126/science.286.5446.1921. 7. Lapedes A, Farber R. 2001. The geometry of shape space: application to influenza. J Theor Biol 212:57– 69. http://dx.doi.org/10.1006/jtbi.2001 .2347. 8. Lewis NS, Anderson TK, Kitikoon P, Skepner E, Burke DF, Vincent AL. 2014. Substitutions near the hemagglutinin receptor-binding site determine the antigenic evolution of influenza A H3N2 viruses in U.S. swine. J Virol 88:4752– 4763. http://dx.doi.org/10.1128/JVI.03805-13. 9. Smith DJ, Lapedes AS, de Jong JC, Bestebroer TM, Rimmelzwaan GF, Osterhaus AD, Fouchier RA. 2004. Mapping the antigenic and genetic evolution of influenza virus. Science 305:371–376. http://dx.doi.org/10 .1126/science.1097211. 10. Lewis NS, Daly JM, Russell CA, Horton DL, Skepner E, Bryant NA, Burke DF, Rash AS, Wood JL, Chambers TM, Fouchier RA, Mumford JA, Elton DM, Smith DJ. 2011. Antigenic and genetic evolution of equine influenza A (H3N8) virus from 1968 to 2007. J Virol 85:12742–12749. http://dx.doi.org/10.1128/JVI.05319-11. 11. Ampofo WK, Azziz-Baumgartner E, Bashir U, Cox NJ, Fasce R, Giovanni M, Grohmann G, Huang S, Katz J, Mironenko A, Mokhtari-Azad T, Sasono PM, Rahman M, Sawanpanyalert P, Siqueira M, Waddell AL, Waiboci L, Wood J, Zhang W, Ziegler T, WHO Writing Group. 2015. Strengthening the influenza vaccine virus selection and development process: report of the 3rd WHO Informal Consultation for Improving Influenza Vaccine Virus Selection held at WHO headquarters, Geneva, Switzerland, 1–3 April 2014. Vaccine 33:4368 – 4382. http://dx.doi.org/10 .1016/j.vaccine.2015.06.090. 12. Barr IG, Russell C, Besselaar TG, Cox NJ, Daniels RS, Donis R, Engelhardt OG, Grohmann G, Itamura S, Kelso A, McCauley J, Odagiri T, Schultz-Cherry S, Shu Y, Smith D, Tashiro M, Wang D, Webby R, Xu X, Ye Z, Zhang W, Writing Committee of the World Health Organization Consultation on Northern Hemisphere Influenza Vaccine Composition for 2013–2014. 2014. WHO recommendations for the viruses used in the 2013-2014 Northern Hemisphere influenza vaccine: epidemiology, antigenic and genetic characteristics of influenza A(H1N1)pdm09, A(H3N2) and B influenza viruses collected from October 2012 to January 2013. Vaccine 32:4713– 4725. http://dx.doi.org/10 .1016/j.vaccine.2014.02.014. 13. Russell CA, Jones TC, Barr IG, Cox NJ, Garten RJ, Gregory V, Gust ID, Hampson AW, Hay AJ, Hurt AC, de Jong JC, Kelso A, Klimov AI, Kageyama T, Komadina N, Lapedes AS, Lin YP, Mosterin A, Obuchi M, Odagiri T, Osterhaus AD, Rimmelzwaan GF, Shaw MW, Skepner E, Stohr K, Tashiro M, Fouchier RA, Smith DJ. 2008. Influenza vaccine strain selection and recent studies on the global migration of seasonal influenza viruses. Vaccine 26(Suppl 4):D31–D34. http://dx.doi.org/10 .1016/j.vaccine.2008.07.078. 14. Koel BF, Burke DF, Bestebroer TM, van der Vliet S, Zondag GC, Vervaet G, Skepner E, Lewis NS, Spronken MI, Russell CA, Eropkin MY, Hurt AC, Barr IG, de Jong JC, Rimmelzwaan GF, Osterhaus AD, Fouchier RA, Smith DJ. 2013. Substitutions near the receptor binding site determine major antigenic change during influenza virus evolution. Science 342:976 –979. http://dx.doi.org/10.1126/science.1244730. 15. Feng Z, Gomez J, Bowman AS, Ye J, Long LP, Nelson SW, Yang J, Martin B, Jia K, Nolting JM, Cunningham F, Cardona C, Zhang J, Yoon

Journal of Virology

September 2016 Volume 90 Number 18

Antigenicity of H3 Swine Influenza Viruses

16.

17.

18.

19.

20.

21.

22.

23.

24.

25.

26.

27. 28.

29. 30.

31.

KJ, Slemons RD, Wan XF. 2013. Antigenic characterization of H3N2 influenza A viruses from Ohio agricultural fairs. J Virol 87:7655–7667. http://dx.doi.org/10.1128/JVI.00804-13. Anderson TK, Nelson MI, Kitikoon P, Swenson SL, Korslund JA, Vincent AL. 2013. Population dynamics of cocirculating swine influenza A viruses in the United States from 2009 to 2012. Influenza Other Respir Viruses 7(Suppl 4):S42–S51. Tang Y, Lee CW, Zhang Y, Senne DA, Dearth R, Byrum B, Perez DR, Suarez DL, Saif YM. 2005. Isolation and characterization of H3N2 influenza A virus from turkeys. Avian Dis 49:207–213. http://dx.doi.org/10 .1637/7288-101304R. Pena L, Vincent AL, Ye J, Ciacci-Zanella JR, Angel M, Lorusso A, Gauger PC, Janke BH, Loving CL, Perez DR. 2011. Modifications in the polymerase genes of a swine-like triple-reassortant influenza virus to generate live attenuated vaccines against 2009 pandemic H1N1 viruses. J Virol 85:456 – 469. http://dx.doi.org/10.1128/JVI.01503-10. Chen H, Ye J, Xu K, Angel M, Shao H, Ferrero A, Sutton T, Perez DR. 2012. Partial and full PCR-based reverse genetics strategy for influenza viruses. PLoS One 7:e46378. http://dx.doi.org/10.1371/journal.pone .0046378. de Jong JC, Smith DJ, Lapedes AS, Donatelli I, Campitelli L, Barigazzi G, Van Reeth K, Jones TC, Rimmelzwaan GF, Osterhaus AD, Fouchier RA. 2007. Antigenic and genetic evolution of swine influenza A (H3N2) viruses in Europe. J Virol 81:4315– 4322. http://dx.doi.org/10.1128/JVI .02458-06. Lorusso A, Vincent AL, Harland ML, Alt D, Bayles DO, Swenson SL, Gramer MR, Russell CA, Smith DJ, Lager KM, Lewis NS. 2011. Genetic and antigenic characterization of H1 influenza viruses from United States swine from 2008. J Gen Virol 92:919 –930. http://dx.doi.org/10.1099/vir.0 .027557-0. Nfon CK, Berhane Y, Hisanaga T, Zhang S, Handel K, Kehler H, Labrecque O, Lewis NS, Vincent AL, Copps J, Alexandersen S, Pasick J. 2011. Characterization of H1N1 swine influenza viruses circulating in Canadian pigs in 2009. J Virol 85:8667– 8679. http://dx.doi.org/10.1128 /JVI.00801-11. Kitikoon P, Nilubol D, Erickson BJ, Janke BH, Hoover TC, Sornsen SA, Thacker EL. 2006. The immune response and maternal antibody interference to a heterologous H1N1 swine influenza virus infection following vaccination. Vet Immunol Immunopathol 112:117–128. http://dx.doi.org /10.1016/j.vetimm.2006.02.008. Gauger PC, Vincent AL, Loving CL, Henningson JN, Lager KM, Janke BH, Kehrli ME, Jr, Roth JA. 2012. Kinetics of lung lesion development and pro-inflammatory cytokine response in pigs with vaccine-associated enhanced respiratory disease induced by challenge with pandemic (2009) A/H1N1 influenza virus. Vet Pathol 49:900 –912. http://dx.doi.org/10 .1177/0300985812439724. Halbur PG, Paul PS, Frey ML, Landgraf J, Eernisse K, Meng XJ, Lum MA, Andrews JJ, Rathje JA. 1995. Comparison of the pathogenicity of two US porcine reproductive and respiratory syndrome virus isolates with that of the Lelystad virus. Vet Pathol 32:648 – 660. http://dx.doi.org/10 .1177/030098589503200606. Khurana S, Loving CL, Manischewitz J, King LR, Gauger PC, Henningson J, Vincent AL, Golding H. 2013. Vaccine-induced anti-HA2 antibodies promote virus fusion and enhance influenza virus respiratory disease. Sci Transl Med 5:200ra114. http://dx.doi.org/10.1126/scitranslmed .3006366. Edgar RC. 2004. MUSCLE: multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Res 32:1792–1797. http://dx.doi .org/10.1093/nar/gkh340. Kearse M, Moir R, Wilson A, Stones-Havas S, Cheung M, Sturrock S, Buxton S, Cooper A, Markowitz S, Duran C, Thierer T, Ashton B, Meintjes P, Drummond A. 2012. Geneious Basic: an integrated and extendable desktop software platform for the organization and analysis of sequence data. Bioinformatics 28:1647–1649. http://dx.doi.org/10.1093 /bioinformatics/bts199. Stamatakis A. 2014. RAxML version 8: a tool for phylogenetic analysis and post-analysis of large phylogenies. Bioinformatics 30:1312–1313. http: //dx.doi.org/10.1093/bioinformatics/btu033. Miller M, Wayne Pfeiffer, and Terri Schwartz. 2010. Creating the CIPRES Science Gateway for inference of large phylogenetic trees, p 1– 8. In Proceedings of the Gateway Computing Environments Workshop, New Orleans, LA, 14 November 2010. Pattengale ND, Alipour M, Bininda-Emonds OR, Moret BM, Stamata-

September 2016 Volume 90 Number 18

32.

33. 34.

35.

36. 37. 38. 39.

40. 41. 42.

43.

44.

45.

46.

47. 48.

49.

kis A. 2010. How many bootstrap replicates are necessary? J Comput Biol 17:337–354. http://dx.doi.org/10.1089/cmb.2009.0179. Kitikoon P, Nelson MI, Killian ML, Anderson TK, Koster L, Culhane MR, Vincent AL. 2013. Genotype patterns of contemporary reassorted H3N2 virus in U.S. swine. J Gen Virol 94:1236 –1241. http://dx.doi.org/10 .1099/vir.0.051839-0. Yassine HM, Al-Natour MQ, Lee CW, Saif YM. 2007. Interspecies and intraspecies transmission of triple reassortant H3N2 influenza A viruses. Virol J 4:129. http://dx.doi.org/10.1186/1743-422X-4-129. Monto AS, Petrie JG, Cross RT, Johnson E, Liu M, Zhong W, Levine M, Katz JM, Ohmit SE. 2015. Antibody to influenza virus neuraminidase: an independent correlate of protection. J Infect Dis 212:1191–1199. http://dx .doi.org/10.1093/infdis/jiv195. Veguilla V, Hancock K, Schiffer J, Gargiullo P, Lu X, Aranio D, Branch A, Dong L, Holiday C, Liu F, Steward-Clark E, Sun H, Tsang B, Wang D, Whaley M, Bai Y, Cronin L, Browning P, Dababneh H, Noland H, Thomas L, Foster L, Quinn CP, Soroka SD, Katz JM. 2011. Sensitivity and specificity of serologic assays for detection of human infection with 2009 pandemic H1N1 virus in U.S. populations. J Clin Microbiol 49: 2210 –2215. http://dx.doi.org/10.1128/JCM.00229-11. Luksza M, Lassig M. 2014. A predictive fitness model for influenza. Nature 507:57– 61. http://dx.doi.org/10.1038/nature13087. Neher RA, Russell CA, Shraiman BI. 11 November 2014. Predicting evolution from the shape of genealogical trees. eLife 3. http://dx.doi.org /10.7554/eLife.03568. Anonymous. 2015. Recommended composition of influenza virus vaccines for use in the 2015-2016 northern hemisphere influenza season. Wkly Epidemiol Rec 90:97–108. Chambers BS, Parkhouse K, Ross TM, Alby K, Hensley SE. 2015. Identification of hemagglutinin residues responsible for H3N2 antigenic drift during the 2014-2015 influenza season. Cell Rep 12:1– 6. http://dx .doi.org/10.1016/j.celrep.2015.06.005. Volz EM, Koelle K, Bedford T. 2013. Viral phylodynamics. PLoS Comput Biol 9:e1002947. http://dx.doi.org/10.1371/journal.pcbi.1002947. Koelle K, Cobey S, Grenfell B, Pascual M. 2006. Epochal evolution shapes the phylodynamics of interpandemic influenza A (H3N2) in humans. Science 314:1898 –1903. http://dx.doi.org/10.1126/science.1132745. Gulati S, Smith DF, Cummings RD, Couch RB, Griesemer SB, St George K, Webster RG, Air GM. 2013. Human H3N2 influenza viruses isolated from 1968 to 2012 show varying preference for receptor substructures with no apparent consequences for disease or spread. PLoS One 8:e66325. http://dx.doi.org/10.1371/journal.pone.0066325. Hensley SE, Das SR, Bailey AL, Schmidt LM, Hickman HD, Jayaraman A, Viswanathan K, Raman R, Sasisekharan R, Bennink JR, Yewdell JW. 2009. Hemagglutinin receptor binding avidity drives influenza A virus antigenic drift. Science 326:734 –736. http://dx.doi.org/10.1126/science .1178258. Li Y, Bostick DL, Sullivan CB, Myers JL, Griesemer SB, Stgeorge K, Plotkin JB, Hensley SE. 2013. Single hemagglutinin mutations that alter both antigenicity and receptor binding avidity influence influenza virus antigenic clustering. J Virol 87:9904 –9910. http://dx.doi.org/10.1128/JVI .01023-13. Das SR, Hensley SE, David A, Schmidt L, Gibbs JS, Puigbo P, Ince WL, Bennink JR, Yewdell JW. 2011. Fitness costs limit influenza A virus hemagglutinin glycosylation as an immune evasion strategy. Proc Natl Acad Sci U S A 108:E1417–E1422. http://dx.doi.org/10.1073/pnas .1108754108. Das SR, Hensley SE, Ince WL, Brooke CB, Subba A, Delboy MG, Russ G, Gibbs JS, Bennink JR, Yewdell JW. 2013. Defining influenza A virus hemagglutinin antigenic drift by sequential monoclonal antibody selection. Cell Host Microbe 13:314 –323. http://dx.doi.org/10.1016/j.chom .2013.02.008. Kryazhimskiy S, Dushoff J, Bazykin GA, Plotkin JB. 2011. Prevalence of epistasis in the evolution of influenza A surface proteins. PLoS Genet 7:e1001301. http://dx.doi.org/10.1371/journal.pgen.1001301. Corzo CA, Culhane M, Juleen K, Stigger-Rosser E, Ducatez MF, Webby RJ, Lowe JF. 2013. Active surveillance for influenza A virus among swine, midwestern United States, 2009-2011. Emerg Infect Dis 19:954 –960. http: //dx.doi.org/10.3201/eid1906.121637. Kitikoon P, Gauger PC, Anderson TK, Culhane MR, Swenson S, Loving CL, Perez DR, Vincent AL. 2013. Swine influenza virus vaccine serologic cross-reactivity to contemporary US swine H3N2 and efficacy in pigs in-

Journal of Virology

jvi.asm.org

8279

Abente et al.

fected with an H3N2 similar to 2011-2012 H3N2v. Influenza Other Respir Viruses 7(Suppl 4):S32–S41. 50. Loving CL, Lager KM, Vincent AL, Brockmeier SL, Gauger PC, Anderson TK, Kitikoon P, Perez DR, Kehrli ME, Jr. 2013. Efficacy in pigs of inactivated and live attenuated influenza virus vaccines against infection and transmission of an emerging H3N2 similar to the 20112012 H3N2v. J Virol 87:9895–9903. http://dx.doi.org/10.1128/JVI .01038-13. 51. Anonymous. 2007. Center for Veterinary Biologics notice no. 07-17: labeling of equine influenza and swine influenza vaccines. USDA Animal and Plant Health Inspection Service, Ames, IA. 52. Rajao DS, Anderson TK, Gauger PC, Vincent AL. 2014. Pathogenesis and vaccination of influenza A virus in swine. Curr Top Microbiol Immunol 385:307–326. http://dx.doi.org/10.1007/82_2014_391.

8280

jvi.asm.org

53. Sandbulte MR, Spickler AR, Zaabel PK, Roth JA. 2015. Optimal use of vaccines for control of influenza A virus in swine. Vaccines 3:22–73. http: //dx.doi.org/10.3390/vaccines3010022. 54. Myers JL, Wetzel KS, Linderman SL, Li Y, Sullivan CB, Hensley SE. 2013. Compensatory hemagglutinin mutations alter antigenic properties of influenza viruses. J Virol 87:11168 –11172. http://dx.doi.org/10.1128 /JVI.01414-13. 55. Fonville JM. 24 June 2015. The expected effect of deleterious mutations on within-host adaptation of pathogens. J Virol http://dx.doi.org/10.1128 /JVI.00832-15. 56. Neher RA, Bedford T. 26 June 2015. nextflu: real-time tracking of seasonal influenza virus evolution in humans. Bioinformatics http://dx.doi .org/10.1093/bioinformatics/btv381.

Journal of Virology

September 2016 Volume 90 Number 18

The Molecular Determinants of Antibody Recognition and Antigenic Drift in the H3 Hemagglutinin of Swine Influenza A Virus.

Influenza A virus (IAV) of the H3 subtype is an important respiratory pathogen that affects both humans and swine. Vaccination to induce neutralizing ...
2MB Sizes 1 Downloads 9 Views