octoFLUshow: an Interactive Tool Describing Spatial and Temporal Trends in the Genetic Diversity of Influenza A Virus in U.S. Swine.

Influenza A virus (IAV) is passively surveilled in swine in the United States through a U.S. Department of Agriculture administered surveillance system. We present an interactive Web tool to visualize and explore trends in the genetic and geographic diversity of IAV derived from the surveillance system.

Detection and Characterization of Swine Origin Influenza A(H1N1) Pandemic 2009 Viruses in Humans following Zoonotic Transmission.

Human-to-swine transmission of seasonal influenza viruses has led to sustained human-like influenza viruses circulating in the U.S. swine population. While some reverse zoonotic-origin viruses adapt and become enzootic in swine, nascent reverse zoonoses may result in virus detections that are difficult to classify as "swine-origin" or "human-origin" due to the genetic similarity of circulating viruses. This is the case for human-origin influenza A(H1N1) pandemic 2009 (pdm09) viruses detected in pigs following numerous reverse zoonosis events since the 2009 pandemic. We report the identification of two human infections with A(H1N1)pdm09 viruses originating from swine hosts and classify them as "swine-origin" variant influenza viruses based on phylogenetic analysis and sequence comparison methods. Phylogenetic analyses of viral genomes from two cases revealed these viruses were reassortants containing A(H1N1)pdm09 hemagglutinin (HA) and neuraminidase (NA) genes with genetic combinations derived from the triple reassortant internal gene cassette. Follow-up investigations determined that one individual had direct exposure to swine in the week preceding illness onset, while another did not report swine exposure. The swine-origin A(H1N1) variant cases were resolved by full genome sequence comparison of the variant viruses to swine influenza genomes. However, if reassortment does not result in the acquisition of swine-associated genes and swine virus genomic sequences are not available from the exposure source, future cases may not be discernible. We have developed a pipeline that performs maximum likelihood analyses, a k-mer-based set difference algorithm, and random forest algorithms to identify swine-associated sequences in the hemagglutinin gene to differentiate between human-origin and swine-origin A(H1N1)pdm09 viruses.IMPORTANCE Influenza virus infects a wide range of hosts, resulting in illnesses that vary from asymptomatic cases to severe pneumonia and death. Viral transfer can occur between human and nonhuman hosts, resulting in human and nonhuman origin viruses circulating in novel hosts. In this work, we have identified the first case of a swine-origin influenza A(H1N1)pdm09 virus resulting in a human infection. This shows that these viruses not only circulate in swine hosts, but are continuing to evolve and distinguish themselves from previously circulating human-origin influenza viruses. The development of techniques for distinguishing human-origin and swine-origin viruses are necessary for the continued surveillance of influenza viruses. We show that unique genetic signatures can differentiate circulating swine-associated strains from circulating human-associated strains of influenza A(H1N1)pdm09, and these signatures can be used to enhance surveillance of swine-origin influenza.

Aerosol Transmission from Infected Swine to Ferrets of an H3N2 Virus Collected from an Agricultural Fair and Associated with Human Variant Infections.

Influenza A viruses (IAV) sporadically transmit from swine to humans, typically associated with agricultural fairs in the United States. A human seasonal H3 virus from the 2010-2011 IAV season was introduced into the U.S. swine population and termed H3.2010.1 to differentiate it from the previous swine H3 virus. This H3N2 lineage became widespread in the U.S. commercial swine population, subsequently spilling over into exhibition swine, and caused a majority of H3N2 variant (H3N2v) cases in humans in 2016 and 2017. A cluster of human H3N2v cases were reported at an agricultural fair in 2017 in Ohio, where 2010.1 H3N2 IAV was concurrently detected in exhibition swine. Genomic analysis showed that the swine and human isolates were nearly identical. In this study, we evaluated the propensity of a 2010.1 H3N2 IAV (A/swine/Ohio/A01354299/2017 [sw/OH/2017]) isolated from a pig in the agricultural fair outbreak to replicate in ferrets and transmit from swine to ferret. sw/OH/2017 displayed robust replication in the ferret respiratory tract, causing slight fever and moderate weight loss. Further, sw/OH/2017 was capable of efficient respiratory droplet transmission from infected pigs to contact ferrets. These findings establish a model for evaluating the propensity of swine IAV to transmit from pig to ferret as a measure of risk to the human population. The identification of higher-risk swine strains can then be targeted for control measures to limit the dissemination at human-swine interfaces to reduce the risk of zoonotic infections and to inform pandemic planning.IMPORTANCE A recently emerged lineage of human-like H3N2 (H3.2010.1) influenza A virus (IAV) from swine has been frequently detected in commercial and exhibition swine in recent years and has been associated with H3N2 variant cases in humans from 2016 and 2017. To demonstrate a model for characterizing the potential for zoonotic transmission associated with swine IAV, we performed an in vivo study of transmission between pigs infected with an H3.2010.1 H3N2 IAV and aerosol contact ferrets. The efficient interspecies transmission demonstrated for the H3.2010.1 IAV in swine emphasizes the need for further characterization of viruses circulating at the swine-human interface for transmission potential prior to human spillover and the development and implementation of more robust vaccines and control strategies to mitigate human exposure to higher-risk swine strains.

Assessment of Molecular, Antigenic, and Pathological Features of Canine Influenza A(H3N2) Viruses That Emerged in the United States.

Background: A single subtype of canine influenza virus (CIV), A(H3N8), was circulating in the United States until a new subtype, A(H3N2), was detected in Illinois in spring 2015. Since then, this CIV has caused thousands of infections in dogs in multiple states. Methods: In this study, genetic and antigenic properties of the new CIV were evaluated. In addition, structural and glycan array binding features of the recombinant hemagglutinin were determined. Replication kinetics in human airway cells and pathogenesis and transmissibility in animal models were also assessed. Results: A(H3N2) CIVs maintained molecular and antigenic features related to low pathogenicity avian influenza A(H3N2) viruses and were distinct from A(H3N8) CIVs. The structural and glycan array binding profile confirmed these findings and revealed avian-like receptor-binding specificity. While replication kinetics in human airway epithelial cells was on par with that of seasonal influenza viruses, mild-to-moderate disease was observed in infected mice and ferrets, and the virus was inefficiently transmitted among cohoused ferrets. Conclusions: Further adaptation is needed for A(H3N2) CIVs to present a likely threat to humans. However, the potential for coinfection of dogs and possible reassortment of human and other animal influenza A viruses presents an ongoing risk to public health.

The genomic evolution of H1 influenza A viruses from swine detected in the United States between 2009 and 2016.

Transmission of influenza A virus (IAV) from humans to swine occurs with relative frequency and is a critical contributor to swine IAV diversity. Subsequent to the introduction of these human seasonal lineages, there is often reassortment with endemic viruses and antigenic drift. To address whether particular genome constellations contributed to viral persistence following the introduction of the 2009 H1N1 human pandemic virus to swine in the USA, we collated and analysed 616 whole genomes of swine H1 isolates. For each gene, sequences were aligned, the best-known maximum likelihood phylogeny was inferred, and each virus was assigned a clade based upon its evolutionary history. A time-scaled Bayesian approach was implemented for the haemagglutinin (HA) gene to determine the patterns of genetic diversity over time. From these analyses, we observed an increase in genome diversity across all H1 lineages and clades, with the H1-γ and H1-δ1 genetic clades containing the greatest number of unique genome patterns. We documented 74 genome patterns from 2009 to 2016, of which 3 genome patterns were consistently detected at a significantly higher level than others across the entire time period. Eight genome patterns increased significantly, while five genome patterns were shown to decline in detection over time. Viruses with genome patterns identified as persisting in the US swine population may possess a greater capacity to infect and transmit in swine. This study highlights the emerging genetic diversity of US swine IAV from 2009 to 2016, with implications for swine and public health and vaccine control efforts.

Reassortment between Swine H3N2 and 2009 Pandemic H1N1 in the United States Resulted in Influenza A Viruses with Diverse Genetic Constellations with Variable Virulence in Pigs.

Repeated spillovers of the H1N1 pandemic virus (H1N1pdm09) from humans to pigs resulted in substantial evolution of influenza A viruses infecting swine, contributing to the genetic and antigenic diversity of influenza A viruses (IAV) currently circulating in swine. The reassortment with endemic swine viruses and maintenance of some of the H1N1pdm09 internal genes resulted in the circulation of different genomic constellations in pigs. Here, we performed a whole-genome phylogenetic analysis of 368 IAV circulating in swine from 2009 to 2016 in the United States. We identified 44 different genotypes, with the most common genotype (32.33%) containing a clade IV-A HA gene, a 2002-lineage NA gene, an M-pdm09 gene, and remaining gene segments of triple reassortant internal gene (TRIG) origin. To understand how different genetic constellations may relate to viral fitness, we compared the pathogenesis and transmission in pigs of six representative genotypes. Although all six genotypes efficiently infected pigs, they resulted in different degrees of pathology and viral shedding. These results highlight the vast H3N2 genetic diversity circulating in U.S. swine after 2009. This diversity has important implications in the control of this disease by the swine industry, as well as a potential risk for public health if swine-adapted viruses with H1N1pdm09 genes have an increased risk to humans, as occurred in the 2011-2012 and 2016 human variant H3N2v cases associated with exhibition swine. IMPORTANCE: People continue to spread the 2009 H1N1 pandemic (H1N1pdm09) IAV to pigs, allowing H1N1pdm09 to reassort with endemic swine IAV. In this study, we determined the 8 gene combinations of swine H3N2 IAV detected from 2009 to 2016. We identified 44 different genotypes of H3N2, the majority of which contained at least one H1N1pdm09 gene segment. We compared six representative genotypes of H3N2 in pigs. All six genotypes efficiently infected pigs, but they resulted in different degrees of lung damage and viral shedding. These results highlight the vast genetic diversity of H3N2 circulating in U.S. swine after 2009, with important implications for the control of IAV for the swine industry. Because H1N1pdm09 is also highly adapted to humans, these swine viruses pose a potential risk to public health if swine-adapted viruses with H1N1pdm09 genes also have an increased risk for human infection.

Continual Reintroduction of Human Pandemic H1N1 Influenza A Viruses into Swine in the United States, 2009 to 2014.

UNLABELLED: The diversity of influenza A viruses in swine (swIAVs) presents an important pandemic threat. Knowledge of the human-swine interface is particularly important for understanding how viruses with pandemic potential evolve in swine hosts. Through phylogenetic analysis of contemporary swIAVs in the United States, we demonstrate that human-to-swine transmission of pandemic H1N1 (pH1N1) viruses has occurred continuously in the years following the 2009 H1N1 pandemic and has been an important contributor to the genetic diversity of U.S. swIAVs. Although pandemic H1 and N1 segments had been largely removed from the U.S. swine population by 2013 via reassortment with other swIAVs, these antigens reemerged following multiple human-to-swine transmission events during the 2013-2014 seasonal epidemic. These findings indicate that the six internal gene segments from pH1N1 viruses are likely to be sustained long term in the U.S. swine population, with periodic reemergence of pandemic hemagglutinin (HA) and neuraminidase (NA) segments in association with seasonal pH1N1 epidemics in humans. Vaccinating U.S. swine workers may reduce infection of both humans and swine and in turn limit the role of humans as sources of influenza virus diversity in pigs. IMPORTANCE: Swine are important hosts in the evolution of influenza A viruses with pandemic potential. Here, we analyze influenza virus sequence data generated by the U.S. Department of Agriculture's national surveillance system to identify the central role of humans in the reemergence of pandemic H1N1 (pH1N1) influenza viruses in U.S. swine herds in 2014. These findings emphasize the important role of humans as continuous sources of influenza virus diversity in swine and indicate that influenza viruses with pandemic HA and NA segments are likely to continue to reemerge in U.S. swine in association with seasonal pH1N1 epidemics in humans.

Characterization of co-circulating swine influenza A viruses in North America and the identification of a novel H1 genetic clade with antigenic significance.

Multiple genetically and antigenically distinct hemagglutinin genes of the H1 and H3 influenza A virus (IAV) subtypes co-circulate in North American swine. This diversity has evolved by repeated transmission of IAVs from humans to swine and subsequent antigenic drift in swine. To understand the evolutionary dynamics of these diverse HA lineages in North American swine, we undertook a phylogenetic analysis of 1576 H1 and 607 H3 HA gene segments, as well as 834 N1 and 1293 N2 NA gene segments, and 2126 M gene segments. These data revealed yearly co-circulation of H1N1, H1N2, and H3N2 viruses, with three HA clades representing the majority of the HA sequences: of the H1 viruses, 42% were classified as H1δ1 and 40.6% were classified as H1γ; and of the H3 viruses 53% were classified as cluster IV-A H3N2. We detected a genetically distinct minor clade consisting of 37 H1 viruses isolated between 2003 and 2013, which we classified as H1γ-2. We estimated that this clade circulated in swine since approximately 1995, but it was not detected in swine until 2003. Though this clade only represents 1.07% of swine H1 sequences reported over the past 10 years, hemagglutination inhibition (HI) assays demonstrated that representatives of this clade of viruses are antigenically distinct, and, when measured using antigenic cartography, were as many as 7 antigenic units from other H1γ viruses. Therefore vaccines against the contemporary H1γ viruses are not likely to cross-protect against γ-2 viruses. The long-term circulation of these γ-2 viruses suggests that minor populations of viruses may be underreported in the national dataset given the long branch lengths and gaps in detections. The identification of these γ-2 viruses demonstrates the need for robust surveillance to capture the full diversity IAVs in swine in the USA and the importance of antigenic drift in the diversification and emergence of new antigenic variants in swine, which complicates vaccine design.