Author response: The global antigenic diversity of swine influenza A viruses (2016)

Full text Figures and data Side by side Abstract eLife digest Introduction Results Discussion Materials and methods Data availability References Decision letter Author response Article and author information Metrics Abstract Swine influenza presents a substantial disease burden for pig populations worldwide and poses a potential pandemic threat to humans. There is considerable diversity in both H1 and H3 influenza viruses circulating in swine due to the frequent introductions of viruses from humans and birds coupled with geographic segregation of global swine populations. Much of this diversity is characterized genetically but the antigenic diversity of these viruses is poorly understood. Critically, the antigenic diversity shapes the risk profile of swine influenza viruses in terms of their epizootic and pandemic potential. Here, using the most comprehensive set of swine influenza virus antigenic data compiled to date, we quantify the antigenic diversity of swine influenza viruses on a multi-continental scale. The substantial antigenic diversity of recently circulating viruses in different parts of the world adds complexity to the risk profiles for the movement of swine and the potential for swine-derived infections in humans. https://doi.org/10.7554/eLife.12217.001 eLife digest Influenza viruses, commonly called flu, infect millions of people and animals every year and occasionally causes pandemics in humans. The immune system can neutralise flu viruses by recognising the proteins on the virus surface, generically referred to as antigens. These antigens change as flu viruses evolve to escape detection by the immune system. These changes tend to be relatively small such that exposure to one flu virus generates immunity that is still effective against other related flu viruses. However, over time, the accumulation of these small changes can result in larger differences such that prior infections no longer provide protection against the new virus. Influenza A viruses infect a wide variety of birds and mammals. Viruses can also transmit from one species to another, which may result in the introduction of viruses with antigens that are new to the recipient species and which have the potential to cause substantial outbreaks. Pig flu viruses have long been considered to be a potential risk for human pandemic viruses and were the source of the 2009 pandemic H1N1 virus. Importantly, humans often transmit flu viruses to pigs. Understanding the dynamics and consequences of this two-way transmission is important for designing effective strategies to detect and respond to new strains of flu. Influenza A viruses of the H1 and H3 subtypes circulate widely in pigs. However, it was poorly understood how closely related swine and human viruses circulating in different regions were to one another and how much the antigens varied between the different viruses. Lewis, Russell et al. have now analysed the antigenic variation of hundreds of H1 and H3 viruses from pigs on multiple continents. The antigenic diversity of recent swine flu viruses resembles the diversity of H1 and H3 viruses observed in humans over the last 40 years. A key factor driving the diversity of the H1 and H3 viruses in pigs is the frequent introduction of human viruses to pigs. In contrast, only one flu virus from a bird had contributed to the observed antigenic diversity in pigs in a substantial way. Once in pigs, human-derived flu viruses continue to evolve their antigens. This results in a tremendous diversity of flu viruses that can be transmitted to other pigs and also to humans. These flu viruses could pose a serious risk to public health because they are no longer similar to the current human flu strains. These findings have important implications not only for developing flu vaccines for pigs but also for informing the development of more-effective surveillance and disease-control strategies to prevent the spread of new flu variants. https://doi.org/10.7554/eLife.12217.002 Introduction Swine have been hypothesized to be a mixing vessel for influenza viruses and were the source of the 2009 H1N1 human pandemic virus (Garten et al., 2009; Smith et al., 2009). The directionality and relative threat of transmission is generally assumed to be from swine into humans with attention only recently turning to introductions in the opposite direction – from humans into swine – and the role that such anthroponotic introductions play in influenza virus evolution (Nelson et al., 2012; Terebuh et al., 2010). Understanding the dynamics of influenza viruses at the interface between humans and swine is key for designing optimal surveillance and control strategies. Currently there are three dominant subtypes of influenza A viruses circulating enzootically in swine: H1N1, H1N2, and H3N2. The evolutionary history of these viruses reflects multiple introductions of influenza viruses into swine from other species (Nelson et al., 2012; Vincent et al., 2014; Grøntvedt et al., 2013; Cappuccio et al., 2011; Ottis et al., 1982; Pereda, 2010; Njabo et al., 2012; Karasin et al., 2006; Hofshagen, 2009; Holyoake et al., 2011; Forgie et al., 2011; Pensaert et al., 1981). Much of the work on characterizing swine influenza viruses and their ancestry has been carried out in the U.S and in Europe (Vincent et al., 2014; Marozin et al., 2002; Nelson et al., 2014; Simon et al., 2014; Brown et al., 1993; Brown et al., 1997; Brown et al., 1998; Webby et al., 2000; Webby et al., 2004; Vincent et al., 2009a; Vincent et al., 2009b; Kyriakis et al., 2011; Watson et al., 2015) with evidence for numerous introductions of human seasonal H1 and H3 influenza viruses into swine since 1990. This pattern continued with the 2009 human pandemic H1N1 virus (H1N1pdm09) with at least 49 separate human-to-swine transmission events, some of which appear to have become established in pig populations (Nelson et al., 2012). In addition to viruses from humans, avian influenza viruses also occasionally infect swine. Notably, in 1979, epidemics of an avian H1N1 influenza virus lineage were reported in Belgian swine leading to the establishment of an 'avian-like' H1N1 virus lineage in Europe. This Eurasian avian-like 1C lineage (Zell et al., 2013) has continued to circulate in swine until the present day, and been introduced into other geographic areas through international movement of swine (Pensaert et al., 1981; Zhu et al., 2013; Nelson et al., 2015c). Unsurprisingly, genetic diversity and multi-species origins of influenza viruses in swine are not restricted solely to pig populations in the U.S. and Europe. China has the largest swine population of any country and shows similar patterns of frequent introductions of both H1 and H3 influenza viruses from both avian and human hosts as well as introductions of swine lineages from other geographic regions (Chen et al., 2014; Vijaykrishna et al., 2011; Zhu et al., 2011). Once established in swine, influenza viruses of human or avian origin pose a substantial threat to swine populations' health and may spread to other geographically segregated populations (Nelson et al., 2015c). Similarly, these viruses also pose a threat for introduction or re-introduction into the human population (Nelson et al., 2014), with associated outbreak and pandemic risks. This risk is exemplified by the re-introduction of the H3N2v (variant) virus with surface glycoprotein genes of human origin to over 300 people in 2011–12 from an H3N2 virus that has been endemic in swine in the U.S. since the late 1990s (Centers for Disease Control and Prevention, 2011; Bowman et al., 2014). The influenza virus hemagglutinin (HA) surface glycoprotein is the primary target of the humoral immune response, undergoes antigenic drift over time, and is the major antigenic constituent in influenza vaccines used in both humans and livestock. The genetic heterogeneity among the HAs of influenza viruses circulating in swine in certain geographic regions has been relatively well studied and characterized (Smith et al., 2009; Nelson et al., 2012; Cappuccio et al., 2011; Nelson et al., 2015c; Vijaykrishna et al., 2011; Kitikoon et al., 2013; Anderson et al., 2015; Takemae et al., 2008; 2013; Perera et al., 2013; Pereda et al., 2011). However, the relationships between genetic diversity and antigenic diversity among contemporary swine influenza viruses and their antigenic similarity to human seasonal influenza strains are unknown, largely owing to a lack of antigenic data. Such antigenic data are critical in assessing the phenotype of the virus, any potential cross-immunity with viruses circulating in humans and in swine, and the risk of re-emergence from these hosts. These analyses are also critical in the design of antigenically well-matched vaccines. In order to help fill this knowledge gap, we compiled the largest and most geographically comprehensive antigenic dataset of swine influenza viruses to date. We then used a Bayesian framework (Bedford et al., 2014) to quantify the antigenic diversity of influenza viruses circulating in humans and swine thus generating a framework for exploring the antigenic component of risk of introduction of influenza A viruses from swine into humans and to swine in other geographic regions based on the antigenic characteristics of swine influenza viruses circulating in different parts of the world. Results Dimensionality of influenza virus antigenic evolution in swine We used hemagglutination inhibition (HI) assays to antigenically characterize a geographically diverse collection of H1 viruses from swine and humans from 1930–2013 (n=194) and H3 viruses from swine and humans from 1968–2013 (n=379) and genetically sequenced all antigenically characterized viruses. Of these, 101 H1 and 73 H3 swine influenza viruses were newly characterized for this study. In order to accurately quantify the antigenic variation of influenza viruses from swine we first determined the appropriate number of dimensions for accurately representing their antigenic relationships. Previous work on quantifying the antigenic evolution of seasonal influenza viruses showed that the evolution of H3 viruses in humans, as measured in HI assays, could be accurately visualized in two dimensions (2D) using antigenic cartography (Smith et al., 2004). In contrast, previous work on swine and equine H3 influenza viruses found that three dimensions (3D) were necessary to accurately visualize antigenic evolution using antigenic cartography (Lewis et al., 2011; 2014; de Jong et al., 2007; Lorusso et al., 2011). In this study we extensively tested the dimensionality of H1 and H3 influenza virus antigenic maps and found that the antigenic variation of both virus subtypes was most accurately represented in three dimensions. Antigenic diversity of H1 influenza viruses in swine To investigate the combined antigenic and genetic evolution of our study viruses we implemented a Bayesian multi-dimensional scaling (BMDS) method for inferring antigenic and genetic relationships (Bedford et al., 2014). As of 2013, three major H1 lineages circulated in swine: 1. the classical 1A lineage – descended from the ancestors of the 1918 human influenza pandemic, was first detected in swine in the 1930's, and gave rise to the human H1N1pdm09 viruses and swine H1N1pdm09-like sub-lineage viruses; 2. the human seasonal-like 1B lineage – resulting from multiple introductions from human seasonal H1 viruses; 3. the Eurasian avian-like 1C lineage – arising from an introduction from wild birds into swine and first detected in Europe in the 1980's (Figure 1A, Zell et al., 2013). Figure 1 with 7 supplements see all Download asset Open asset Evolutionary relationships of H1 (A, B) and H3 (C, D) influenza viruses circulating in swine and humans inferred by Bayesian Multi-dimensional scaling (BMDS). Each colored ball represents a single virus. Viruses are colored by lineage (A,C) and by geography (B,D). Lines connecting each virus represent inferred phylogenetic relationships. Distances for antigenic dimensions are measured in antigenic units (AU) and each unit is equivalent to a two-fold dilution in HI assay data. Antigenic distance can be interpreted as a measure of antigenic similarity – viruses close to one another are more antigenically similar than viruses further apart. Interactive visualizations are available at https://phylogeography.github.io/influenzaH1/ and https://phylogeography.github.io/influenzaH3/. Source data and GIF files for rotational views of 3D antigenic maps in Figure 1 have been deposited in Dryad (Lewis et al., 2016). https://doi.org/10.7554/eLife.12217.003 Our combined antigenic and genetic analyses of H1 viruses in swine showed an inconsistent relationship between antigenic clustering and genetic lineage through time with occasional emergence of new antigenic variants. In all geographic regions for which we had antigenic data we found unique patterns of antigenic evolution within individual lineages. In Asia, we observed co-circulation of classical 1A, human seasonal-like 1B, and Eurasian avian-like 1C lineage viruses. However all three lineages were not detected in any one country. In Hong Kong – where samples were derived at slaughter from swine sourced from China – classical swine 1A lineage including the H1N1pdm09 sub-lineage, and Eurasian avian-like 1C lineage viruses were detected (Figure 1A, light red). For the viruses from Hong Kong, the mean pairwise antigenic distance (MPD) between the H1N1pdm09 sub-lineage viruses and the two classical 1A lineage clades was 5.0 and 6.0 AU away (Figure 1—figure supplement 1: Figure 1—figure supplement 2, Supplementary file 2). The Classical swine 1A lineage viruses collected in Japan (Figure 1A, dark red) differed from the Hong Kong H1 viruses with a MPD of 5.6 AU. Within the Hong Kong Eurasian avian-like 1C lineage viruses there were two distinct phylogenetic groups – group 1 and group 2 (as previously reported in Yang et al. (2016) and Figure 1—figure supplement 1, Supplementary file 2). The Group 1 and Group 2 viruses were 6.5 AU and 4.4 AU (MPD) away from the H1N1pdm09 sub-lineage viruses. The MPD between all Asian and European Eurasian avian-like 1C lineage strains was 5.2 AU. All of these distances are sufficiently large that it would be surprising if prior infection or un-adjuvanted vaccines would provide any protection among these virus groups. In Europe, the Eurasian avian-like 1C lineage co-circulated with the H1N1pdm09 sub-lineage of classical 1A viruses and the human seasonal-like 1B lineage. The human seasonal-like 1B lineage viruses were first detected in European swine in the mid-1990's but have a putative human seasonal ancestor from the 1980's. The human seasonal-like 1B viruses differed substantially from the Eurasian avian-like swine 1C viruses and the H1N1pdm09 sub-lineage viruses (MPD: 6.7 AU and 10.4 AU respectively). The H1N1pdm2009 sub-lineage viruses also differed substantially from the human seasonal-like 1B lineage viruses (MPD:10.6 AU). In Canada, only the classical swine 1A lineages viruses and H1N1pdm09 sub-lineage viruses were detected. Most of the viruses within the classical swine 1A lineage were antigenically similar to each other but differed from H1N1pdm09 sublineages viruses with a MPD of 6.5 AU. However, the alpha sub-lineage of classical 1A lineage viruses (Figure 1B green, Supplementary file 2) antigenically diverged in Canadian swine to form a separate antigenic group with a MPD of 7.0 AU from the H1N1pdm09 strains and 4.2 AU from the other Canadian classical swine 1A lineage viruses. In the USA, the classical swine 1A lineage viruses co-circulated with the H1N1pdm09 sub-lineage and the human seasonal-like 1B lineage viruses. In the USA the human seasonal-like 1B lineage viruses evolved two sub-lineages – Delta 1 and Delta 2 (Figure 1—figure supplement 1, Supplementary file 2) – likely as the result of two separate but nearly contemporaneous introductions into swine first detected in the early 2000's. The Delta 1 and 2 sub-lineage viruses antigenically differed from the H1N1pdm2009 lineage viruses with an MPD of 7.8 AU. Representatives of alpha sub-lineage of the classical swine 1A lineage in the USA were 7.0 AU (MPD) from the H1N1pdm2009 sub-lineage viruses. The beta and gamma sub-lineages of the classical swine 1A lineage were 6.6 and 4.7 AU (MPD) respectively from the H1N1pdm2009 viruses. To assess the risk of virus introduction associated with moving live swine between continents we compared the MPDs of swine influenza H1 viruses circulating in different parts of the world. The human seasonal-like 1B viruses circulating in European swine were on average 11.7 AU (MPD) from the human seasonal-like 1B viruses in USA swine. The Eurasian avian-like 1C lineage viruses were 6.8 AU (MPD) from the human seasonal-like 1B lineage viruses in the USA. Antigenic diversity of H3 influenza viruses in swine Just as for the H1 viruses, we used BMDS (Bedford et al., 2014) to investigate the antigenic and genetic evolution of H3 viruses (Figure 1C,D). In European swine there were four H3 introductions included in our antigenic characterization (Figure 1—figure supplement 3: Figure 1—figure supplement 4). Of these, the European 3A lineage (A/Port Chalmers/1973-like) introduction was the most commonly detected lineage, showing evidence for three sequential antigenic clusters and evolving on a markedly different path from the putative ancestor in humans (Figure 1C,D [blue]). A separate introduction from humans into swine of an antigenically distinct 1970's-like human seasonal virus also circulated in European swine in the 1980s and 1990s (Figure 1—figure supplement 1 [Europe 2 Introduction]). The earliest strain in the European 3A lineage was 6.5 AU from the A/Port Chalmers/1973 reference strain (Figure 1C large grey) and the most recent strain was 9.8 AU (Supplementary file 2). The distance from our representative of recent human seasonal H3 viruses (A/Victoria/361/2011) to the H3 European virus in swine and was 14.9 AU (MPD). In the USA, we found evidence for three H3 virus introduction events from humans into swine in the mid-1990's, one of which resulted in the establishment of the 3B lineage (Figure 1C,D) which became the most frequently detected lineage in swine through 2013, and gave rise to the H3N2v viruses which infected >300 humans in the US from 2011–2012 (Centers for Disease Control and Prevention, 2012). We only antigenically characterizes a single representative of the H3N2v viruses – A/Indiana/8/2011 – and found that it differed from A/Victoria/361/2011 by 6.4 AU (Figure 1 pink). The MPD between the swine H3 viruses circulating in the USA and in Europe was 12.6 AU. In Asia we found evidence for multiple introductions of H3 viruses and subsequent antigenic drift from the six representative viruses in our dataset including an introduction likely mediated by translocation of a virus circulating in USA swine to Asian swine (Nelson et al., 2015d). Two separate introductions from humans were detected in Japan that resulted in circulation in swine beyond the time of circulation of the ancestral strain in humans. The introductions were ~5.2 AU from A/Victoria/361/2011 and 10.9 AU (MPD) from the H3 strains circulating in European swine. The most recent seasonal human H3 introduction detected in Asia was from Vietnam and this virus was 3.8 AU away from A/Victoria/361/2011 (Supplementary file 2). Rates of antigenic drift in swine To investigate the rates of swine influenza virus antigenic drift away from the most recent common ancestor for each genetic lineage in each geographic region we used TimeSlicer available in SPREAD (Bielejec et al., 2011). For H1 viruses in swine, the classical 1A lineage evolved at a mean rate of 0.15 AU per year from the earliest antigenically characterized representative in the dataset (A/swine/Iowa/15/1930) (Figure 2A, Supplementary file 3). The human seasonal-like 1B viruses introduced into European swine evolved at a mean rate of 0.17 AU per year (Figure 2B, Supplementary file 3). The introductions of human seasonal H1 viruses into USA swine that gave rise to the Delta-1 and Delta-2 lineages showed slightly different rates of antigenic drift – 0.63 for Delta-1 and 0.85 for Delta-2 – both significantly faster than the human seasonal introduction in European swine (Figure 2B, Supplementary file 3) but they were only observed over a relatively short time period. The Eurasian avian-like swine 1C lineage evolved at a mean rate of 0.15 AU per year (Figure 2C, Supplementary file 3). With the exception of the human seasonal-like 1B lineage viruses in the USA, the overall antigenic diversity of H1 viruses in swine remained remarkably constant over the study period (Figure 2A,B,C). The antigenic diversity of the Delta-1 and Delta-2 sublineages in the USA began to expand rapidly around 2009 for reasons that remain to be elucidated (Figure 2B). Figure 2 Download asset Open asset Time series of year-to-year rates of antigenic drift distance and antigenic diversity of H1 and H3 viruses in swine by genetic lineage. Solid colored lines represent year-to-year antigenic drift distance, where drift for year i is measured as the mean of Euclidean distances among strains in a phylogenetic lineage in year i compared to the mean of Euclidean distances among strains of that phylogenetic lineage from the previous year (i–1). The dotted line represents antigenic diversity among H1 and H3 strains by lineage through time. For the solid and dotted lines, the shaded region represents the range of the highest posterior density estimates. Multiple introductions which circulate for >5 years of the human seasonal-like swine H1 lineage in European (purple) and USA (gold) swine were calculated separately. Source data for Figure 2 has been deposited in Dryad (Lewis et al., 2016). https://doi.org/10.7554/eLife.12217.011 For H3 viruses, we found that the 3B lineages viruses in USA swine evolved antigenically at a faster rate than the H3 viruses introduced into European swine (0.49 AU per year vs 0.28 AU per year [Figure 2D,E, Supplementary file 3]). Additionally, the antigenic diversity of H3 viruses in USA swine, particularly since 2010, has increased at a faster rate than that of H3 viruses in Europeans swine. Thus sustained circulation of antigenic drift variants within US swine has substantially increased the observed antigenic diversity in this lineage relative to the observed antigenic diversity in other geographic regions. Additional antigenic diversity can be inferred from genetic sequence data In addition to the BMDS analyses of viruses for which we had both antigenic and genetic data, we also performed large-scale phylogenetic analyses of publicly available swine influenza virus sequence. Through these analyses we inferred potentially important gaps in the global antigenic data that suggest our antigenic dataset underrepresents the true global antigenic diversity of swine influenza viruses. For H1 viruses that circulated in swine as of 2013, we find that the major genetic lineages have been captured in this study (Figure 1—figure supplement 5: Figure 1—figure supplement 6). However, new genetic evidence recently became available to demonstrate additional human seasonal introductions into swine in previously regions such as and (Nelson et al., et al., 2013; Nelson et al., (Figure 1—figure supplement genetic lineage not antigenic we any their in where we have the long associated with some introductions could antigenic drift away from the characterized The markedly different evolutionary patterns in the H3 lineages characterized in swine and the inferred antigenic profile of the viruses introduced to swine, that the true diversity of H3 viruses circulating in swine is likely to be substantially than the diversity found in our BMDS In we additional of introductions of seasonal H3 viruses from humans in (Figure 1—figure supplement – Figure 1—figure supplement on the likely time of these introductions from humans into swine we that the introduced viruses represent at least distinct antigenic variants based on the antigenic variation of their human seasonal influenza to the H1 genetic lineages these additional H3 lineages also antigenic to the of these introductions on the global antigenic diversity of in swine. by swine influenza viruses to the humans In addition to the the observed antigenic diversity poses to swine the antigenic diversity of swine influenza viruses also substantial for human populations. The outbreak and pandemic potential of swine influenza viruses in humans is at least determined by human population immunity against swine viruses. This component of risk can be inferred from the antigenic distance of swine influenza viruses to seasonal viruses or strains in humans to which humans are likely to have Importantly, are to have immunity to viruses that only circulated they were et al., 2014). most of the current swine influenza viruses are the of human seasonal influenza virus introductions into swine, we at least some immunity in the human population that could potentially with re-introduction of these viruses. For the H1N1pdm09 viruses circulating in both humans and swine are similar antigenically and likely at least some cross-immunity in both hosts (Figure However, for the H1 H3 and H3 3B human seasonal lineages in swine, the risk of re-introduction into the human population with the of the human population that were the human virus circulated in humans and is by antigenic evolution of these viruses in swine. in terms of antigenic similarity and likely prior introduced lineages of human H1 and H3 viruses – particularly with antigenically similar to the H3 pandemic strain – pose the current risk to humans because of the or of immunity in since the In the H3N2v viruses, which from a human seasonal virus introduction in the have infected people the Discussion In this study we characterized the antigenic diversity H1 and H3 viruses circulating in both swine and humans on a multi-continental scale. The observed diversity was largely by frequent introductions of variants from humans into swine and subsequent antigenic evolution within swine combined with geographic segregation of multiple virus lineages. Much of the antigenic evolution of viruses in swine was long and evolutionary from observed in humans. These for the re-introduction of viruses into humans as well as the for antigenically strains in humans to be introduced into swine. the introduction of viruses from birds to swine has only one major to the antigenic diversity of influenza viruses in swine. This could be at least due to the antigenic similarity of the avian-like and the classical swine 1A lineages found an of antigenic similarity between avian and swine H1 and H3 viruses could not be performed as of this study and to the of our knowledge has not been reported introduction of avian influenza viruses into swine also likely be by other including for transmission et al., 2012; et al., and other and In addition to multiple introduction events and subsequent antigenic the patterns of antigenic diversity and heterogeneity among geographic regions are also by the relative geographic of swine populations resulting in patterns of virus evolution and The observed patterns of antigenic heterogeneity among geographic regions are also likely by system particularly differences in the movement of swine within and between USA commonly swine among geographically areas at different in the there is or movement of animals a single Such movement patterns have been to the evolution of influenza viruses in swine in the USA, with the as an for swine influenza virus with the source of genetic diversity in the and for some lineages (Nelson et al., 2011). is used extensively as a to control influenza in swine in the USA and Control strategies by region with some no swine influenza for i