Spatiotemporal changes in influenza A virus prevalence among wild waterfowl inhabiting the continental United States throughout the annual cycle.

Avian influenza viruses can pose serious risks to agricultural production, human health, and wildlife. An understanding of viruses in wild reservoir species across time and space is important to informing surveillance programs, risk models, and potential population impacts for vulnerable species. Although it is recognized that influenza A virus prevalence peaks in reservoir waterfowl in late summer through autumn, temporal and spatial variation across species has not been fully characterized. We combined two large influenza databases for North America and applied spatiotemporal models to explore patterns in prevalence throughout the annual cycle and across the continental United States for 30 waterfowl species. Peaks in prevalence in late summer through autumn were pronounced for dabbling ducks in the genera Anas and Spatula, but not Mareca. Spatially, areas of high prevalence appeared to be related to regional duck density, with highest predicted prevalence found across the upper Midwest during early fall, though further study is needed. We documented elevated prevalence in late winter and early spring, particularly in the Mississippi Alluvial Valley. Our results suggest that spatiotemporal variation in prevalence outside autumn staging areas may also represent a dynamic parameter to be considered in IAV ecology and associated risks.

The Evolutionary Dynamics of Influenza A Viruses Circulating in Mallards in Duck Hunting Preserves in Maryland, USA.

Duck hunting preserves (DHP) have resident populations of farm-raised mallard ducks, which create potential foci for the evolution of novel influenza A viruses (IAVs). Through an eleven-year (2003-2013) IAV surveillance project in seven DHPs in Maryland, USA, we frequently identified IAVs in the resident, free-flying mallard ducks (5.8% of cloacal samples were IAV-positive). The IAV population had high genetic diversity, including 12 HA subtypes and 9 NA subtypes. By sequencing the complete genomes of 290 viruses, we determined that genetically diverse IAVs were introduced annually into DHP ducks, predominantly from wild birds in the Anatidae family that inhabit the Atlantic and Mississippi flyways. The relatively low viral gene flow observed out of DHPs suggests that raised mallards do not sustain long-term viral persistence nor do they serve as important sources of new viruses in wild birds. Overall, our findings indicate that DHPs offer reliable samples of the diversity of IAV subtypes, and could serve as regional sentinel sites that mimic the viral diversity found in local wild duck populations, which would provide a cost-efficient strategy for long-term IAV monitoring. Such monitoring could allow for early identification and characterization of viruses that threaten bird species of high economic and environmental interest.

Subtype Diversity of Influenza A Virus in North American Waterfowl: a Multidecade Study.

The discovery in 1976 of waterfowl as the primary reservoir of influenza A viruses (IAVs) has since spurred decades of waterfowl surveillance efforts by researchers dedicated to understanding the ecology of IAV and its subsequent threat to human and animal health. Here, we employed a multidecade, continental-scale approach of surveillance data to understand trends of seasonal IAV subtype diversity. Between 1976 and 2015, IAVs were detected in 8,427 (10.8%) of 77,969 samples from migratory waterfowl throughout the Central and Mississippi Migratory Flyways in the United States and Canada. A total of 96 hemagglutinin (HA)/neuraminidase (NA) subtype combinations were isolated, which included most HA (H1 to H14) and all 9 NA subtypes. We observed an annual trend of high influenza prevalence, involving a few dominant subtypes, on northern breeding grounds during summer with progressively lowered influenza prevalence, comprised of a highly diverse profile of subtypes, as waterfowl migrate toward southern wintering grounds. Isolates recovered during winter had the highest proportion of mixed and rare HA/NA combinations, indicating increased opportunity for reassortment of IAVs. In addition, 70% of H5 and 49% of H7 IAV isolates were recovered from samples collected during fall and spring, respectively; these are subtypes that can have significant implications for public health and agriculture sectors. Annual cyclical dominance of subtypes on northern breeding grounds is revealed through the longitudinal nature of this study. Our novel findings exhibit the unrealized potential for discovery using existing IAV surveillance data.IMPORTANCE Wild aquatic birds are the primary natural reservoir of influenza A viruses (IAVs) and are therefore responsible for the dispersal and maintenance of IAVs representing a broad range of antigenic and genetic diversity. The aims of IAV surveillance in waterfowl not only relate to understanding the risk of spillover risk to humans, but also to improving our understanding of basic questions related to IAV evolution and ecology. By evaluating several decades of surveillance data from wild aquatic birds sampled along North American migratory flyways, we discovered an annual trend of increasing subtype diversity during southbound migration, peaking on southern wintering grounds. Winter sampling revealed the highest proportion of mixed and rare infections that suggest higher opportunity for spillover. These findings allow improvements to surveillance efforts to robustly capture IAV diversity that will be used for vaccine development and cultivate a more thorough understanding of IAV evolution and persistence mechanisms.

Identifying Gaps in Wild Waterfowl Influenza A Surveillance in Ohio, United States.

The Mississippi Flyway is of utmost importance in monitoring influenza A viral diversity in the natural reservoir, as it is used by approximately 40% of North American migratory waterfowl. In 2008, influenza A virus (IAV) surveillance was initiated in eight states within the flyway during annual southern migration, to gain better insight into the natural history of influenza A viruses in the natural reservoir. More than 45,000 samples have been collected and tested, resulting in hundreds of diverse influenza A viral isolates, but seasonal sampling may not be the best strategy to gain insight into the natural history of IAV. To investigate the progress of this sampling strategy toward understanding the ecology of IAV in wild waterfowl, data from mallard ducks (Anas platyrhynchos) sampled nearly year-round in Ohio were examined. Overall, 3,645 samples were collected from mallards in Ohio from 2008 to 2016, with IAV being recovered from 13.6% of all samples collected. However, when data from each month are examined individually, it becomes apparent that the aggregated summary may be providing a misleading view of IAV in Ohio mallards. For instance, in August the frequency of viral recovery is 29.8%, with isolates representing at least 47 hemagglutinin/ neuraminidase (HA/NA) combinations. In November, during the height of southern migration, IAV isolation drops to 6.2%, with only 25 HA/NA combinations being represented. Our biased sampling towards convenience and high IAV recovery has created gaps in the data set, which prohibit a full understanding of the IAV ecology in this waterfowl population.

Identificación de brechas en la vigilancia de la influenza aviar en aves acuáticas silvestres en Ohio, Estados Unidos. La ruta migratoria de Mississippi es de suma importancia para monitorear la diversidad del virus de la influenza A en los reservorios naturales, ya que es utilizada por aproximadamente el 40% de las aves acuáticas migratorias de América del Norte. En el año 2008, se inició la vigilancia del virus de la influenza A (IAV) en ocho estados dentro de dicha ruta migratoria durante la migración anual al sur, para obtener una mejor comprensión de la historia natural de los virus de la influenza A en el reservorio natural. Se han recolectado y analizado más de 45,000 muestras, lo que dio como resultado cientos de diversos aislados virales de influenza A, pero el muestreo estacional puede no ser la mejor estrategia para conocer la historia natural del virus de la influenza aviar. Para investigar el progreso de esta estrategia de muestreo para comprender la ecología del virus de la influenza aviar en aves acuáticas silvestres, se examinaron datos de patos de collar (Anas platyrhynchos) muestreados durante casi todo el año en Ohio. En total, se recolectaron 3,645 muestras de patos silvestres en Ohio desde el año 2008 hasta el 2016, y se recuperó al virus de la influenza aviar en el 13.6% de todas las muestras recolectadas. Sin embargo, cuando los datos de cada mes se examinaron individualmente, se hace evidente que el resumen agregado puede proporcionar un panorama engañoso del virus de la influenza aviar en los patos silvestres de Ohio. Por ejemplo, en agosto, la frecuencia de recuperación viral fue del 29.8%, con aislamientos que representan al menos 47 combinaciones de hemaglutininas (HA) y neuraminidasas (NA). En noviembre, durante el apogeo de la migración hacia el sur, el aislamiento del virus de la influenza se redujo a 6.2%, con solo 25 combinaciones de HA y NA representadas. El muestreo sesgado hacia la conveniencia y la alta recuperación del virus de influenza ha creado brechas en el conjunto de datos, que prohíbe una comprensión completa de. Abbreviations: HA = hemagglutinin; IAV = influenza A virus; NA = neuraminidase.

Feral Swine in the United States Have Been Exposed to both Avian and Swine Influenza A Viruses.

Influenza A viruses (IAVs) in swine can cause sporadic infections and pandemic outbreaks among humans, but how avian IAV emerges in swine is still unclear. Unlike domestic swine, feral swine are free ranging and have many opportunities for IAV exposure through contacts with various habitats and animals, including migratory waterfowl, a natural reservoir for IAVs. During the period from 2010 to 2013, 8,239 serum samples were collected from feral swine across 35 U.S. states and tested against 45 contemporary antigenic variants of avian, swine, and human IAVs; of these, 406 (4.9%) samples were IAV antibody positive. Among 294 serum samples selected for antigenic characterization, 271 cross-reacted with ≥1 tested virus, whereas the other 23 did not cross-react with any tested virus. Of the 271 IAV-positive samples, 236 cross-reacted with swine IAVs, 1 with avian IAVs, and 16 with avian and swine IAVs, indicating that feral swine had been exposed to both swine and avian IAVs but predominantly to swine IAVs. Our findings suggest that feral swine could potentially be infected with both avian and swine IAVs, generating novel IAVs by hosting and reassorting IAVs from wild birds and domestic swine and facilitating adaptation of avian IAVs to other hosts, including humans, before their spillover. Continued surveillance to monitor the distribution and antigenic diversities of IAVs in feral swine is necessary to increase our understanding of the natural history of IAVs.IMPORTANCE There are more than 5 million feral swine distributed across at least 35 states in the United States. In contrast to domestic swine, feral swine are free ranging and have unique opportunities for contact with wildlife, livestock, and their habitats. Our serological results indicate that feral swine in the United States have been exposed to influenza A viruses (IAVs) consistent with those found in both domestic swine and wild birds, with the predominant infections consisting of swine-adapted IAVs. Our findings suggest that feral swine have been infected with IAVs at low levels and could serve as hosts for the generation of novel IAVs at the interface of feral swine, wild birds, domestic swine, and humans.

Development of Multilocus Sequence Typing (MLST) for Mycoplasma synoviae.

Mycoplasma synoviae (MS) is a poultry pathogen that has had an increasing incidence and economic impact over the past few years. Strain identification is necessary for outbreak investigation, infection source identification, and facilitating prevention and control as well as eradication efforts. Currently, a segment of the variable lipoprotein hemagglutinin A (vlhA) gene (420 bp) is the only target that is used for MS strain identification. A major limitation of this assay is that colonality of typed samples can only be inferred if their vlhA sequences are identical; however, if their sequences are different, the degree of relatedness is uncertain. In this study we propose a multilocus sequence typing (MLST) assay to further refine MS strain identification. After initial screening of 24 housekeeping genes as potential targets, seven genes were selected for the MLST assay. An internal segment (450-711 bp) from each of the seven genes was successfully amplified and sequenced from 58 different MS strains and field isolates (n = 30) or positive clinical samples (n = 28). The collective sequence of all seven gene segments (3960 bp total) was used for MS sequence typing. The 58 tested MS samples were typed into 30 different sequence types using the MLST assay and, coincidentally, all the samples were typed into 30 sequence types using the vlhA assay. However, the phylogenetic tree generated using the MLST data was more congruent to the epidemiologic information than was the tree generated by the vlhA assay. We suggest that the newly developed MLST assay and the vlhA assay could be used in tandem for MS typing. The MLST assay will be a valuable and more reliable tool for MS sequence typing, providing better understanding of the epidemiology of MS infection. This in turn will aid disease prevention, control, and eradication efforts.

The enigma of the apparent disappearance of Eurasian highly pathogenic H5 clade 2.3.4.4 influenza A viruses in North American waterfowl.

One of the major unresolved questions in influenza A virus (IAV) ecology is exemplified by the apparent disappearance of highly pathogenic (HP) H5N1, H5N2, and H5N8 (H5Nx) viruses containing the Eurasian hemagglutinin 2.3.4.4 clade from wild bird populations in North America. The introduction of Eurasian lineage HP H5 clade 2.3.4.4 H5N8 IAV and subsequent reassortment with low-pathogenic H?N2 and H?N1 North American wild bird-origin IAVs in late 2014 resulted in widespread HP H5Nx IAV infections and outbreaks in poultry and wild birds across two-thirds of North America starting in November 2014 and continuing through June 2015. Although the stamping out strategies adopted by the poultry industry and animal health authorities in Canada and the United States-which included culling, quarantining, increased biosecurity, and abstention from vaccine use-were successful in eradicating the HP H5Nx viruses from poultry, these activities do not explain the apparent disappearance of these viruses from migratory waterfowl. Here we examine current and historical aquatic bird IAV surveillance and outbreaks of HP H5Nx in poultry in the United States and Canada, providing additional evidence of unresolved mechanisms that restrict the emergence and perpetuation of HP avian influenza viruses in these natural reservoirs.

Influenza A Viruses from Overwintering and Spring-Migrating Waterfowl in the Lake Erie Basin, United States.

Influenza A virus (IAV) surveillance in migratory waterfowl in the United States has primarily occurred during late summer and the autumn southern migration. Data concerning the presence and ecology of IAVs in waterfowl during winter and spring seasons in the U.S. northern latitudes have been limited, mainly due to limited access to waterfowl for sampling. The southwestern Lake Erie Basin is an important stopover site for waterfowl during migration periods, and over the past 28 years, 8.72% of waterfowl sampled in this geographic location have been positive for IAV recovery during summer and autumn (June-December). To gain a better understanding of influenza A viral dynamics in waterfowl populations during winter and spring migration (February through April), cloacal swabs were collected from overwintering and spring-migrating waterfowl in Ohio and Michigan in 2006, 2007, 2013, and 2014. A total of 740 cloacal swabs were collected and tested using virus isolation in embryonating chicken eggs, resulting in the recovery of 33 (4.5%) IAV isolates. The influenza A isolates were recovered from eight waterfowl species in the order Anseriformes. Antigenically, the IAV isolates represent 15 distinct hemagglutinin (HA) and neuraminidase (NA) combinations, with seven (21%) of the isolates reported as mixed infections based on antigenic HA subtyping, NA subtyping, or both. This effort demonstrates the presence of antigenically diverse IAV in waterfowl during overwintering and spring migration at northern latitudes in the United States, thereby contributing to the understanding of the maintenance of diversity among waterfowl-origin IAVs.

Antigenic Characterization of H3 Subtypes of Avian Influenza A Viruses from North America.

Besides humans, H3 subtypes of influenza A viruses (IAVs) can infect various animal hosts, including avian, swine, equine, canine, and sea mammal species. These H3 viruses are both antigenically and genetically diverse. Here, we characterized the antigenic diversity of contemporary H3 avian IAVs recovered from migratory birds in North America. Hemagglutination inhibition (HI) assays were performed on 37 H3 isolates of avian IAVs recovered from 2007 to 2011 using generated reference chicken sera. These isolates were recovered from samples taken in the Atlantic, Mississippi, Central, and Pacific waterfowl migration flyways. Antisera to all the tested H3 isolates cross-reacted with each other and, to a lesser extent, with those to H3 canine and H3 equine IAVs. Antigenic cartography showed that the largest antigenic distance among the 37 avian IAVs is about four units, and each unit corresponds to a 2 log 2 difference in the HI titer. However, none of the tested H3 IAVs cross-reacted with ferret sera derived from contemporary swine and human IAVs. Our results showed that the H3 avian IAVs we tested lacked significant antigenic diversity, and these viruses were antigenically different from those circulating in swine and human populations. This suggests that H3 avian IAVs in North American waterfowl are antigenically relatively stable.

Evolutionary Dynamics of Influenza A Viruses in US Exhibition Swine.

The role of exhibition swine in influenza A virus transmission was recently demonstrated by >300 infections with influenza A(H3N2) variant viruses among individuals who attended agricultural fairs. Through active influenza A virus surveillance in US exhibition swine and whole-genome sequencing of 380 isolates, we demonstrate that exhibition swine are actively involved in the evolution of influenza A viruses, including zoonotic strains. First, frequent introduction of influenza A viruses from commercial swine populations provides new genetic diversity in exhibition pigs each year locally. Second, genomic reassortment between viruses cocirculating in exhibition swine increases viral diversity. Third, viral migration between exhibition swine in neighboring states demonstrates that movements of exhibition pigs contributes to the spread of genetic diversity. The unexpected frequency of viral exchange between commercial and exhibition swine raises questions about the understudied interface between these populations. Overall, the complexity of viral evolution in exhibition swine indicates that novel viruses are likely to continually reemerge, presenting threats to humans.

Influenza A Virus Surveillance in Waterfowl in Missouri, USA, 2005-2013.

Missouri, United States, is located within the Mississippi Migratory Bird Flyway where wild waterfowl stop to feed and rest during migration and, weather permitting, to overwinter. Historically, Missouri has experienced sporadic influenza A virus (IAV) outbreaks in poultry and commercial swine. The introduction of IAVs from wild, migratory waterfowl is one possible source for the IAV, IAV genomic segments, or both involved in these outbreaks in key agricultural species. During 2005 through 2013, 3984 cloacal swabs were collected from hunter-harvested waterfowl in Missouri as part of an active IAV surveillance effort. Twenty-four avian species were represented in the sample population and 108 (2.7%) of the samples tested positive for IAV recovery. These IAV isolates represented 12 HA and nine NA subtypes and at least 27 distinct HA-NA combinations. An H14 IAV isolate recovered in Missouri during the sample period provided evidence for further establishment of the H14 subtype in North American wild waterfowl and gave proof that the previously rare subtype is more genetically diverse than previously detected. The present surveillance effort also produced IAV isolates that were genomically linked to the highly pathogenic H7N3 IAV strain that emerged in 2012 and caused severe disease in Mexico's domestic poultry. The presence of antigenically diverse IAV's circulating in wild waterfowl in the vicinity of commercial poultry and swine, along with the association of several wild-bird-lineage IAV genomic segments in viruses infecting poultry in North America, justifies continued attention to biosecurity efforts in food animal production systems and ongoing active IAV surveillance in wild birds.

Spread and persistence of influenza A viruses in waterfowl hosts in the North American Mississippi migratory flyway.

UNLABELLED: While geographic distance often restricts the spread of pathogens via hosts, this barrier may be compromised when host species are mobile. Migratory waterfowl in the order Anseriformes are important reservoir hosts for diverse populations of avian-origin influenza A viruses (AIVs) and are assumed to spread AIVs during their annual continental-scale migrations. However, support for this hypothesis is limited, and it is rarely tested using data from comprehensive surveillance efforts incorporating both the temporal and spatial aspects of host migratory patterns. We conducted intensive AIV surveillance of waterfowl using the North American Mississippi Migratory Flyway (MMF) over three autumn migratory seasons. Viral isolates (n = 297) from multiple host species were sequenced and analyzed for patterns of gene dispersal between northern staging and southern wintering locations. Using a phylogenetic and nucleotide identity framework, we observed a larger amount of gene dispersal within this flyway rather than between the other three longitudinally identified North American flyways. Across seasons, we observed patterns of regional persistence of diversity for each genomic segment, along with limited survival of dispersed AIV gene lineages. Reassortment increased with both time and distance, resulting in transient AIV constellations. This study shows that within the MMF, AIV gene flow favors spread along the migratory corridor within a season, and also that intensive surveillance during bird migration is important for identifying virus dispersal on time scales relevant to pandemic responsiveness. In addition, this study indicates that comprehensive monitoring programs to capture AIV diversity are critical for providing insight into AIV evolution and ecology in a major natural reservoir. IMPORTANCE: Migratory birds are a reservoir for antigenic and genetic diversity of influenza A viruses (AIVs) and are implicated in the spread of virus diversity that has contributed to previous pandemic events. Evidence for dispersal of avian-origin AIVs by migratory birds is rarely examined on temporal scales relevant to pandemic or panzootic threats. Therefore, characterizing AIV movement by hosts within a migratory season is important for implementing effective surveillance strategies. We conducted surveillance following birds along a major North American migratory route and observed that within a migratory season, AIVs rapidly reassorted and gene lineages were dispersed primarily within the migratory corridor. Patterns of regional persistence were observed across seasons for each gene segment. We show that dispersal of AIV gene lineages by migratory birds occurs quickly along migratory routes and that surveillance for AIVs threatening human and animal health should focus attention on these routes.

Contemporary avian influenza A virus subtype H1, H6, H7, H10, and H15 hemagglutinin genes encode a mammalian virulence factor similar to the 1918 pandemic virus H1 hemagglutinin.

UNLABELLED: Zoonotic avian influenza virus infections may lead to epidemics or pandemics. The 1918 pandemic influenza virus has an avian influenza virus-like genome, and its H1 hemagglutinin was identified as a key mammalian virulence factor. A chimeric 1918 virus expressing a contemporary avian H1 hemagglutinin, however, displayed murine pathogenicity indistinguishable from that of the 1918 virus. Here, isogenic chimeric avian influenza viruses were constructed on an avian influenza virus backbone, differing only by hemagglutinin subtype expressed. Viruses expressing the avian H1, H6, H7, H10, and H15 subtypes were pathogenic in mice and cytopathic in normal human bronchial epithelial cells, in contrast to H2-, H3-, H5-, H9-, H11-, H13-, H14-, and H16-expressing viruses. Mouse pathogenicity was associated with pulmonary macrophage and neutrophil recruitment. These data suggest that avian influenza virus hemagglutinins H1, H6, H7, H10, and H15 contain inherent mammalian virulence factors and likely share a key virulence property of the 1918 virus. Consequently, zoonotic infections with avian influenza viruses bearing one of these hemagglutinins may cause enhanced disease in mammals. IMPORTANCE: Influenza viruses from birds can cause outbreaks in humans and may contribute to the development of pandemics. The 1918 pandemic influenza virus has an avian influenza virus-like genome, and its main surface protein, an H1 subtype hemagglutinin, was identified as a key mammalian virulence factor. In a previous study, a 1918 virus expressing an avian H1 gene was as virulent in mice as the reconstructed 1918 virus. Here, a set of avian influenza viruses was constructed, differing only by hemagglutinin subtype. Viruses with the avian H1, H6, H7, H10, and H15 subtypes caused severe disease in mice and damaged human lung cells. Consequently, infections with avian influenza viruses bearing one of these hemagglutinins may cause enhanced disease in mammals, and therefore surveillance for human infections with these subtypes may be important in controlling future outbreaks.

Swine-to-human transmission of influenza A(H3N2) virus at agricultural fairs, Ohio, USA, 2012.

Agricultural fairs provide an opportunity for bidirectional transmission of influenza A viruses. We sought to determine influenza A virus activity among swine at fairs in the United States. As part of an ongoing active influenza A virus surveillance project, nasal swab samples were collected from exhibition swine at 40 selected Ohio agricultural fairs during 2012. Influenza A(H3N2) virus was isolated from swine at 10 of the fairs. According to a concurrent public health investigation, 7 of the 10 fairs were epidemiologically linked to confirmed human infections with influenza A(H3N2) variant virus. Comparison of genome sequences of the subtype H3N2 isolates recovered from humans and swine from each fair revealed nucleotide identities of >99.7%, confirming zoonotic transmission between swine and humans. All influenza A(H3N2) viruses isolated in this study, regardless of host species or fair, were >99.5% identical, indicating that 1 virus strain was widely circulating among exhibition swine in Ohio during 2012.

Utility of snout wipe samples for influenza A virus surveillance in exhibition swine populations.

BACKGROUND: Sporadic influenza A virus (IAV) outbreaks in humans and swine have resulted from commingling of large numbers of people and pigs at agricultural fairs in the United States. Current antemortem IAV surveillance strategies in swine require collecting nasal swabs, which entails restraining pigs with snares. Restraint is labor-intensive for samplers, stressful for pigs, and displeasing to onlookers because pigs often resist and vocalize. OBJECTIVE: To evaluate the utility of snout wipes in exhibition swine as a method to make IAV surveillance efforts less intrusive, less labor-intensive, and more widely accepted among pig owners and exhibition officials. METHODS: Three materials (rayon/polyester gauze, cotton gauze, and Swiffer(®) Sweeper dry cloths) were inoculated with IAV, and viral recoveries from these materials were quantified using qRT-PCR and TCID50 assays. In a field trial, paired cotton gauze snout wipes and gold standard polyester-tipped nasal swabs were collected from 553 pigs representing 29 agricultural fairs and the qualitative results of rRT-PCR and viral isolation were compared. RESULTS AND CONCLUSIONS: Viral recoveries from potential snout wipe materials ranged from 0.26 to 1.59 log10 TCID50 /ml less than that of the positive control in which no substrate was included; rayon/polyester gauze performed significantly worse than the other materials. In the field, snout wipes and nasal swabs had high levels of agreement for both rRT-PCR detection and virus isolation. Although further investigation and refinement of the sampling method is needed, results indicate that snout wipes will facilitate convenient and undisruptive IAV surveillance in pigs at agricultural fairs.

Genomic analyses detect Eurasian-lineage H10 and additional H14 influenza A viruses recovered from waterfowl in the Central United States.

The accurate and timely characterization of influenza A viruses (IAV) from natural reservoirs is essential for responses to animal and public health threats. Differences between antigenic and genetic subtyping results for 161 IAV isolates recovered from migratory birds in the central United States during 2010-2011 delayed the recognition of four isolates of interest. Genomic sequencing identified the first reported Eurasian-origin H10 subtype in North America and three additional H14 isolates showing divergence from previously reported H14 isolates. Genomic analyses revealed additional diversity among IAV isolates not detected by antigenic subtyping and provided further insight into interhemispheric spread of avian-origin IAVs.

Exploration of risk factors contributing to the presence of influenza A virus in swine at agricultural fairs.

Influenza A virus infections occurring in exhibition swine populations at agricultural fairs during 2012 served as a source of H3N2 variant influenza A viruses transmitted to humans resulting in more than 300 documented cases. Prior to the outbreak, this investigation was initiated to identify fair-level risk factors contributing to influenza A virus infections in pigs at agricultural fairs. As part of an ongoing active surveillance program, nasal swabs and associated fair-level metadata were collected from pigs at 40 junior fair market swine shows held in Ohio during the 2012 fair season. Analyses of the data show that the adjusted odds of having influenza A virus-infected pigs at a fair were 1.27 (95% confidential interval (CI): 1.04-1.66) higher for every 20 pig increase in the size of the swine show. Additionally, four of the five fairs that hosted breeding swine shows in addition to their junior fair market swine shows had pigs test positive for influenza A virus. While the current study was limited to 40 fairs within one state, the findings provided insight for veterinary and public health officials developing mitigation strategies to decrease the intra- and inter-species transmission of influenza A virus at fairs.

Low pathogenic influenza A virus activity at avian interfaces in Ohio zoos, 2006-2009.

This investigation to examine influenza A virus activity in avian species at four Ohio zoos was initiated to better understand the ecology of avian-origin influenza A (AIV) virus in wild aquatic birds and the possibility of spill-over of such viruses into captive zoo birds, both native and foreign species. Virus isolation efforts resulted in the recovery of three low pathogenic (LP) AIV isolates (one H7N3 and two H3N6) from oral-pharyngeal or cloacal swabs collected from over 1000 zoo birds representing 94 species. In addition, 21 LPAIV isolates possessing H3N6, H4N6, or H7N3 subtype combinations were recovered from 627 (3.3%) environmental fecal samples collected from outdoor habitats accessible to zoo and wild birds. Analysis of oral-pharyngeal and cloacal swabs collected from free-ranging mallards (Anas platyrhynchos) live-trapped at one zoo in 2007 resulted in the recovery of 164 LPAIV isolates (48% of samples) representing five HA and six NA subtypes and at least nine HA-NA combinations. The high frequency of isolate recovery is undoubtedly due to the capture and holding of wild ducks in a common pen before relocation. Serologic analyses using an agar gel immune diffusion assay detected antibodies to the influenza A virus type-specific antigen in 147 of 1237 (11.9%) zoo bird sera and in 14 of 154 (9%) wild mallard sera. Additional analyses of a limited number of zoo bird sera demonstrated HA- and NA-inhibition activity to 15 HA and nine NA subtypes. The spectrum of HA antibodies indicate antibody diversity of AIV infecting zoo birds; however, the contribution of heterologous cross-reactions and steric interference was not ruled out. This proactive investigation documented that antigenically diverse LPAIVs were active in all three components of the avian zoologic-wild bird interfaces at Ohio zoos (zoo birds, the environment, and wild birds). The resulting baseline data provides insight and justification for preventive medicine strategies for zoo birds.

Mutation from arginine to lysine at the position 189 of hemagglutinin contributes to the antigenic drift in H3N2 swine influenza viruses.

Two distinct antigenic clusters were previously identified among the H3N2 swine influenza A viruses (IAVs) and were designated H3N2SIV-alpha and H3N2SIV-beta (Feng et al., 2013. Journal of Virology 87 (13), 7655-7667). A consistent mutation was observed at the position 189 of hemagglutinin (R189K) between H3N2SIV-alpha and H3N2SIV-beta fair isolates. To evaluate the contribution of R189K mutation to the antigenic drift from H3N2SIV-alpha to H3N2SIV-beta, four reassortant viruses with 189R or 189K were generated. The antigenic cartography demonstrated that the R189K mutation in the hemagglutinin of H3N2 IAV contributed to the antigenic drift, separating these viruses into H3N2SIV-alpha to H3N2SIV-beta. This R189K mutation was also found to contribute to the cross-reaction with several ferret sera raised against historical human IAVs with hemagglutinin carrying 189K. This study suggests that the R189K mutation plays a vital role in the antigenicity of swine and human H3N2 IAVs and identification of this antigenic determinant will help us rapidly identify antigenic variants in influenza surveillance.