The Role of Human Transportation Networks in Mediating the Genetic Structure of Seasonal Influenza in the United States

Recent studies have demonstrated the importance of accounting for human mobility networks when modeling epidemics in order to accurately predict spatial dynamics. However, little is known about the impact these movement networks have on the genetic structure of pathogen populations and whether these effects are scale-dependent. We investigated how human movement along the aviation and commuter networks contributed to intra-seasonal genetic structure of influenza A epidemics in the continental United States using spatially-referenced hemagglutinin nucleotide sequences collected from 2003–2013 for both the H3N2 and H1N1 subtypes. Comparative analysis of these transportation networks revealed that the commuter network is highly spatially-organized and more heavily traveled than the aviation network, which instead is characterized by high connectivity between all state pairs. We found that genetic distance between sequences often correlated with distance based on interstate commuter network connectivity for the H1N1 subtype, and that this correlation was not as prevalent when geographic distance or aviation network connectivity distance was assessed against genetic distance. However, these patterns were not as apparent for the H3N2 subtype at the scale of the continental United States. Finally, although sequences were spatially referenced at the level of the US state of collection, a community analysis based on county to county commuter connections revealed that commuting communities did not consistently align with state geographic boundaries, emphasizing the need for the greater availability of more specific sequence location data. Our results highlight the importance of utilizing host movement data in characterizing the underlying genetic structure of pathogen populations and demonstrate a need for a greater understanding of the differential effects of host movement networks on pathogen transmission at various spatial scales.     

Molecular epidemiology of A/H3N2 and A/H1N1 influenza virus during a single epidemic season in the United States

To determine the spatial and temporal dynamics of influenza A virus during a single epidemic, we examined whole-genome sequences of 284 A/H1N1 and 69 A/H3N2 viruses collected across the continental United States during the 2006-2007 influenza season, representing the largest study of its kind undertaken to date. A phylogenetic analysis revealed that multiple clades of both A/H1N1 and A/H3N2 entered and co-circulated in the United States during this season, even in localities that are distant from major metropolitan areas, and with no clear pattern of spatial spread. In addition, co-circulating clades of the same subtype exchanged genome segments through reassortment, producing both a minor clade of A/H3N2 viruses that appears to have re-acquired sensitivity to the adamantane class of antiviral drugs, as well as a likely antigenically distinct A/H1N1 clade that became globally dominant following this season. Overall, the co-circulation of multiple viral clades during the 2006-2007 epidemic season revealed patterns of spatial spread that are far more complex than observed previously, and suggests a major role for both migration and reassortment in shaping the epidemiological dynamics of human influenza A virus.     

Contrasting the epidemiological and evolutionary dynamics of influenza spatial transmission

In the past decade, rapid increases in the availability of high-resolution molecular and epidemiological data, combined with developments in statistical and computational methods to simulate and infer migration patterns, have provided key insights into the spatial dynamics of influenza A viruses in humans. In this review, we contrast findings from epidemiological and molecular studies of influenza virus transmission at different spatial scales. We show that findings are broadly consistent in large-scale studies of inter-regional or inter-hemispheric spread in temperate regions, revealing intense epidemics associated with multiple viral introductions, followed by deep troughs driven by seasonal bottlenecks. However, aspects of the global transmission dynamics of influenza viruses are still debated, especially with respect to the existence of tropical source populations experiencing high levels of genetic diversity and the extent of prolonged viral persistence between epidemics. At the scale of a country or community, epidemiological studies have revealed spatially structured diffusion patterns in seasonal and pandemic outbreaks, which were not identified in molecular studies. We discuss the role of sampling issues in generating these conflicting results, and suggest strategies for future research that may help to fully integrate the epidemiological and evolutionary dynamics of influenza virus over space and time.     

Landscape attributes governing local transmission of an endemic zoonosis: Rabies virus in domestic dogs

Landscape heterogeneity plays an important role in disease spread and persistence, but quantifying landscape influences and their scale dependence is challenging. Studies have focused on how environmental features or global transport networks influence pathogen invasion and spread, but their influence on local transmission dynamics that underpin the persistence of endemic diseases remains unexplored. Bayesian phylogeographic frameworks that incorporate spatial heterogeneities are promising tools for analysing linked epidemiological, environmental and genetic data. Here, we extend these methodological approaches to decipher the relative contribution and scale‐dependent effects of landscape influences on the transmission of endemic rabies virus in Serengeti district, Tanzania (area ~4,900 km²). Utilizing detailed epidemiological data and 152 complete viral genomes collected between 2004 and 2013, we show that the localized presence of dogs but not their density is the most important determinant of diffusion, implying that culling will be ineffective for rabies control. Rivers and roads acted as barriers and facilitators to viral spread, respectively, and vaccination impeded diffusion despite variable annual coverage. Notably, we found that landscape effects were scale‐dependent: rivers were barriers and roads facilitators on larger scales, whereas the distribution of dogs was important for rabies dispersal across multiple scales. This nuanced understanding of the spatial processes that underpin rabies transmission can be exploited for targeted control at the scale where it will have the greatest impact. Moreover, this research demonstrates how current phylogeographic frameworks can be adapted to improve our understanding of endemic disease dynamics at different spatial scales.     

Network analysis of global influenza spread

Although vaccines pose the best means of preventing influenza infection, strain selection and optimal implementation remain difficult due to antigenic drift and a lack of understanding global spread. Detecting viral movement by sequence analysis is complicated by skewed geographic and seasonal distributions in viral isolates. We propose a probabilistic method that accounts for sampling bias through spatiotemporal clustering and modeling regional and seasonal transmission as a binomial process. Analysis of H3N2 not only confirmed East-Southeast Asia as a source of new seasonal variants, but also increased the resolution of observed transmission to a country level. H1N1 data revealed similar viral spread from the tropics. Network analysis suggested China and Hong Kong as the origins of new seasonal H3N2 strains and the United States as a region where increased vaccination would maximally disrupt global spread of the virus. These techniques provide a promising methodology for the analysis of any seasonal virus, as well as for the continued surveillance of influenza.     

Virus evolution and transmission in an ever more connected world

The frequency and global impact of infectious disease outbreaks, particularly those caused by emerging viruses, demonstrate the need for a better understanding of how spatial ecology and pathogen evolution jointly shape epidemic dynamics. Advances in computational techniques and the increasing availability of genetic and geospatial data are helping to address this problem, particularly when both information sources are combined. Here, we review research at the intersection of evolutionary biology, human geography and epidemiology that is working towards an integrated view of spatial incidence, host mobility and viral genetic diversity. We first discuss how empirical studies have combined viral spatial and genetic data, focusing particularly on the contribution of evolutionary analyses to epidemiology and disease control. Second, we explore the interplay between virus evolution and global dispersal in more depth for two pathogens: human influenza A virus and chikungunya virus. We discuss the opportunities for future research arising from new analyses of human transportation and trade networks, as well as the associated challenges in accessing and sharing relevant spatial and genetic data.     

Spatial dynamics of human-origin H1 influenza A virus in North American swine

The emergence and rapid global spread of the swine-origin H1N1/09 pandemic influenza A virus in humans underscores the importance of swine populations as reservoirs for genetically diverse influenza viruses with the potential to infect humans. However, despite their significance for animal and human health, relatively little is known about the phylogeography of swine influenza viruses in the United States. This study utilizes an expansive data set of hemagglutinin (HA1) sequences (n = 1516) from swine influenza viruses collected in North America during the period 2003-2010. With these data we investigate the spatial dissemination of a novel influenza virus of the H1 subtype that was introduced into the North American swine population via two separate human-to-swine transmission events around 2003. Bayesian phylogeographic analysis reveals that the spatial dissemination of this influenza virus in the US swine population follows long-distance swine movements from the Southern US to the Midwest, a corn-rich commercial center that imports millions of swine annually. Hence, multiple genetically diverse influenza viruses are introduced and co-circulate in the Midwest, providing the opportunity for genomic reassortment. Overall, the Midwest serves primarily as an ecological sink for swine influenza in the US, with sources of virus genetic diversity instead located in the Southeast (mainly North Carolina) and South-central (mainly Oklahoma) regions. Understanding the importance of long-distance pig transportation in the evolution and spatial dissemination of the influenza virus in swine may inform future strategies for the surveillance and control of influenza, and perhaps other swine pathogens.     

Exploring the Molecular Epidemiology and Evolutionary Dynamics of Influenza A Virus in Taiwan

The evolution and population dynamics of human influenza in Taiwan is a microcosm of the viruses circulating worldwide, which has not yet been studied in detail. We collected 343 representative full genome sequences of human influenza A viruses isolated in Taiwan between 1979 and 2009. Phylogenetic and antigenic data analysis revealed that H1N1 and H3N2 viruses consistently co-circulated in Taiwan, although they were characterized by different temporal dynamics and degrees of genetic diversity. Moreover, influenza A viruses of both subtypes underwent internal gene reassortment involving all eight segments of the viral genome, some of which also occurred during non-epidemic periods. The patterns of gene reassortment were different in the two subtypes. The internal genes of H1N1 viruses moved as a unit, separately from the co-evolving HA and NA genes. On the other hand, the HA and NA genes of H3N2 viruses tended to segregate consistently with different sets of internal gene segments. In particular, as reassortment occurred, H3HA always segregated as a group with the PB1, PA and M genes, while N2NA consistently segregated with PB2 and NP. Finally, the analysis showed that new phylogenetic lineages and antigenic variants emerging in summer were likely to be the progenitors of the epidemic strains in the following season. The synchronized seasonal patterns and high genetic diversity of influenza A viruses observed in Taiwan make possible to capture the evolutionary dynamic and epidemiological rules governing antigenic drift and reassortment and may serve as a “warning” system that recapitulates the global epidemic.     

Allele dynamics plots for the study of evolutionary dynamics in viral populations

Phylodynamic techniques combine epidemiological and genetic information to analyze the evolutionary and spatiotemporal dynamics of rapidly evolving pathogens, such as influenza A or human immunodeficiency viruses. We introduce 'allele dynamics plots' (AD plots) as a method for visualizing the evolutionary dynamics of a gene in a population. Using AD plots, we propose how to identify the alleles that are likely to be subject to directional selection. We analyze the method's merits with a detailed study of the evolutionary dynamics of seasonal influenza A viruses. AD plots for the major surface protein of seasonal influenza A (H3N2) and the 2009 swine-origin influenza A (H1N1) viruses show the succession of substitutions that became fixed in the evolution of the two viral populations. They also allow the early identification of those viral strains that later rise to predominance, which is important for the problem of vaccine strain selection. In summary, we describe a technique that reveals the evolutionary dynamics of a rapidly evolving population and allows us to identify alleles and associated genetic changes that might be under directional selection. The method can be applied for the study of influenza A viruses and other rapidly evolving species or viruses.     

Human avian influenza A (H5N1) virus infection in China

Highly pathogenic influenza A (H5N1) virus causes a widespread poultry deaths worldwide. The first human H5N1 infected case was reported in Hong Kong Special Administrative Region of China in 1997. Since then, the virus re-emerged in 2003 and continues to infect people worldwide. Currently, over 400 human infections have been reported in more than 15 countries and mortality rate is greater than 60%. H5N1 viruses still pose a potential pandemic threat in the future because of the continuing global spread and evolution. Here, we summarize the epidemiological, clinical and virological characteristics of human H5N1 infection in China monitored and identified by our national surveillance systems. Chinese Nature Science Foundation Key Project (Grant No. 30599433), Chinese Basic Science Research Program (973)Key Project (Grant No. 2005CB523006)     

Phylogeography and evolutionary history of reassortant H9N2 viruses with potential human health implications

Avian influenza viruses of the H9N2 subtype have seriously affected the poultry industry of the Far and Middle East since the mid-1990s and are considered one of the most likely candidates to cause a new influenza pandemic in humans. To understand the genesis and epidemiology of these viruses, we investigated the spatial and evolutionary dynamics of complete genome sequences of H9N2 viruses circulating in nine Middle Eastern and Central Asian countries from 1998 to 2010. We identified four distinct and cocirculating groups (A, B, C, and D), each of which has undergone widespread inter- and intrasubtype reassortments, leading to the generation of viruses with unknown biological properties. Our analysis also suggested that eastern Asia served as the major source for H9N2 gene segments in the Middle East and Central Asia and that in this geographic region within-country evolution played a more important role in shaping viral genetic diversity than migration between countries. The genetic variability identified among the H9N2 viruses was associated with specific amino acid substitutions that are believed to result in increased transmissibility in mammals, as well as resistance to antiviral drugs. Our study highlights the need to constantly monitor the evolution of H9N2 viruses in poultry to better understand the potential risk to human health posed by these viruses.     

Spatiotemporal Structure of Molecular Evolution of H5N1 Highly Pathogenic Avian Influenza Viruses in Vietnam

Background

Vietnam is one of the countries most affected by outbreaks of H5N1 highly pathogenic avian influenza viruses. First identified in Vietnam in poultry in 2001 and in humans in 2004, the virus has since caused 111 cases and 56 deaths in humans. In 2003/2004 H5N1 outbreaks, nearly the entire poultry population of Vietnam was culled. Our earlier study (Wan et al., 2008, PLoS ONE, 3(10): e3462) demonstrated that there have been at least six independent H5N1 introductions into Vietnam and there were nine newly emerged reassortants from 2001 to 2007 in Vietnam. H5N1 viruses in Vietnam cluster distinctly around Hanoi and Ho Chi Minh City. However, the nature of the relationship between genetic divergence and geographic patterns is still unclear.

Methodology/Principal Findings

In this study, we hypothesized that genetic distances between H5N1 viruses in Vietnam are correlated with geographic distances, as the result of distinct population and environment patterns along Vietnam''s long north to south longitudinal extent. Based on this hypothesis, we combined spatial statistical methods with genetic analytic techniques and explicitly used geographic space to explore genetic evolution of H5N1 highly pathogenic avian influenza viruses at the sub-national scale in Vietnam. Our dataset consisted of 125 influenza viruses (with whole genome sets) isolated in Vietnam from 2003 to 2007. Our results document the significant effect of space and time on genetic evolution and the rise of two regional centers of genetic mixing by 2007. These findings give insight into processes underlying viral evolution and suggest that genetic differentiation is associated with the distance between concentrations of human and poultry populations around Hanoi and Ho Chi Minh City.

Conclusions/Significance

The results show that genetic evolution of H5N1 viruses in Vietnamese domestic poultry is highly correlated with the location and spread of those viruses in geographic space. This correlation varies by scale, time, and gene, though a classic isolation by distance pattern is observed. This study is the first to characterize the geographic structure of influenza viral evolution at the sub-national scale in Vietnam and can shed light on how H5N1 HPAIVs evolve in certain geographic settings.     

Tmprss2 Is Essential for Influenza H1N1 Virus Pathogenesis in Mice

Annual influenza epidemics and occasional pandemics pose a severe threat to human health. Host cell factors required for viral spread but not for cellular survival are attractive targets for novel approaches to antiviral intervention. The cleavage activation of the influenza virus hemagglutinin (HA) by host cell proteases is essential for viral infectivity. However, it is unknown which proteases activate influenza viruses in mammals. Several candidates have been identified in cell culture studies, leading to the concept that influenza viruses can employ multiple enzymes to ensure their cleavage activation in the host. Here, we show that deletion of a single HA-activating protease gene, Tmprss2, in mice inhibits spread of mono-basic H1N1 influenza viruses, including the pandemic 2009 swine influenza virus. Lung pathology was strongly reduced and mutant mice were protected from weight loss, death and impairment of lung function. Also, after infection with mono-basic H3N2 influenza A virus body weight loss and survival was less severe in Tmprss2 mutant compared to wild type mice. As expected, Tmprss2-deficient mice were not protected from viral spread and pathology after infection with multi-basic H7N7 influenza A virus. In conclusion, these results identify TMPRSS2 as a host cell factor essential for viral spread and pathogenesis of mono-basic H1N1 and H3N2 influenza A viruses.     

Influenza A: From highly pathogenic H5N1 to pandemic 2009 H1N1. Epidemiology and clinical features

The last decade has seen the emergence of two new influenza A subtypes and they have become a cause of concern for the global community. These are the highly pathogenic H5N1 influenza A virus (H5N1) and the Pandemic 2009 influenza H1N1 virus. Since 2003 the H5N1 virus has caused widespread disease and death in poultry, mainly in south East Asia and Africa. In humans the number of cases infected with this virus is few but the mortality has been about 60%. Most patients have presented with severe pneumonia and acute respiratory distress syndrome. The second influenza virus, the pandemic H1N1 2009, emerged in Mexico in March this year. This virus acquired the ability for sustained human to human spread and within a few months spread throughout the world and infected over 4 lakh individuals. The symptoms of infection with this virus are similar to seasonal influenza but it currently affecting younger individuals more often. Fortunately the mortality has been low. Both these new influenza viruses are currently circulating and have different clinical and epidemiological characteristics.     

Testing spatiotemporal hypothesis of bacterial evolution using methicillin-resistant Staphylococcus aureus ST239 genome-wide data within a bayesian framework

Staphylococcus aureus is a common cause of infections that has undergone rapid global spread over recent decades. Formal phylogeographic methods have not yet been applied to the molecular epidemiology of bacterial pathogens because the limited genetic diversity of data sets based on individual genes usually results in poor phylogenetic resolution. Here, we investigated a whole-genome single nucleotide polymorphism (SNP) data set of health care-associated Methicillin-resistant S. aureus sequence type 239 (HA-MRSA ST239) strains, which we analyzed using Markov spatial models that incorporate geographical sampling distributions. The reconstructed timescale indicated a temporal origin of this strain shortly after the introduction of Methicillin, followed by global pandemic spread. The estimate of the temporal origin was robust to the molecular clock, coalescent prior, full/intergenic/synonymous SNP inclusion, and correction for excluded invariant site patterns. Finally, phylogeographic analyses statistically supported the role of human movement in the global dissemination of HA-MRSA ST239, although it was unable to conclusively resolve the location of the root. This study demonstrates that bacterial genomes can indeed contain sufficient evolutionary information to elucidate the temporal and spatial dynamics of transmission. Future applications of this approach to other bacterial strains may provide valuable epidemiological insights that may justify the cost of genome-wide typing.     

Prospective of Genomics in Revealing Transmission, Reassortment and Evolution of Wildlife-Borne Avian Influenza A (H5N1) Viruses

The outbreak of highly pathogenic avian influenza (HPAI) H5N1 disease has led to significant loss of poultry and wild life and case fatality rates in humans of 60%. Wild birds are natural hosts for all avian influenza virus subtypes and over120 bird species have been reported with evidence of H5N1 infection. Influenza A viruses possess a segmented RNA genome and are characterized by frequently occurring genetic reassortment events, which play a very important role in virus evolution and the spread of novel gene constellations in immunologically naïve human and animal populations. Phylogenetic analysis of whole genome or sub-genomic sequences is a standard means for delineating genetic variation, novel reassortment events, and surveillance to trace the global transmission pathways. In this paper, special emphasis is given to the transmission and circulation of H5N1 among wild life populations, and to the reassortment events that are associated with inter-host transmission of the H5N1 viruses when they infect different hosts, such as birds, pigs and humans. In addition, we review the inter-subtype reassortment of the viral segments encoding inner proteins between the H5N1 viruses and viruses of other subtypes, such as H9N2 and H6N1. Finally, we highlight the usefulness of genomic sequences in molecular epidemiological analysis of HPAI H5N1 and the technical limitations in existing analytical methods that hinder them from playing a greater role in virological research.     

Genome-scale evolution and phylodynamics of equine H3N8 influenza A virus

Equine influenza viruses (EIVs) of the H3N8 and H7N7 subtypes are the causative agents of an important disease of horses. While EIV H7N7 apparently is extinct, H3N8 viruses have circulated for more than 50 years. Like human influenza viruses, EIV H3N8 caused a transcontinental pandemic followed by further outbreaks and epidemics, even in populations with high vaccination coverage. Recently, EIV H3N8 jumped the species barrier to infect dogs. Despite its importance as an agent of infectious disease, the mechanisms that underpin the evolutionary and epidemiological dynamics of EIV are poorly understood, particularly at a genomic scale. To determine the evolutionary history and phylodynamics of EIV H3N8, we conducted an extensive analysis of 82 complete viral genomes sampled during a 45-year span. We show that both intra- and intersubtype reassortment have played a major role in the evolution of EIV, and we suggest that intrasubtype reassortment resulted in enhanced virulence while heterosubtypic reassortment contributed to the extinction of EIV H7N7. We also show that EIV evolves at a slower rate than other influenza viruses, even though it seems to be subject to similar immune selection pressures. However, a relatively high rate of amino acid replacement is observed in the polymerase acidic (PA) segment, with some evidence for adaptive evolution. Most notably, an analysis of viral population dynamics provided evidence for a major population bottleneck of EIV H3N8 during the 1980s, which we suggest resulted from changes in herd immunity due to an increase in vaccination coverage.     

Spatial Transmission of 2009 Pandemic Influenza in the US

The 2009 H1N1 influenza pandemic provides a unique opportunity for detailed examination of the spatial dynamics of an emerging pathogen. In the US, the pandemic was characterized by substantial geographical heterogeneity: the 2009 spring wave was limited mainly to northeastern cities while the larger fall wave affected the whole country. Here we use finely resolved spatial and temporal influenza disease data based on electronic medical claims to explore the spread of the fall pandemic wave across 271 US cities and associated suburban areas. We document a clear spatial pattern in the timing of onset of the fall wave, starting in southeastern cities and spreading outwards over a period of three months. We use mechanistic models to tease apart the external factors associated with the timing of the fall wave arrival: differential seeding events linked to demographic factors, school opening dates, absolute humidity, prior immunity from the spring wave, spatial diffusion, and their interactions. Although the onset of the fall wave was correlated with school openings as previously reported, models including spatial spread alone resulted in better fit. The best model had a combination of the two. Absolute humidity or prior exposure during the spring wave did not improve the fit and population size only played a weak role. In conclusion, the protracted spread of pandemic influenza in fall 2009 in the US was dominated by short-distance spatial spread partially catalysed by school openings rather than long-distance transmission events. This is in contrast to the rapid hierarchical transmission patterns previously described for seasonal influenza. The findings underline the critical role that school-age children play in facilitating the geographic spread of pandemic influenza and highlight the need for further information on the movement and mixing patterns of this age group.