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Research Interests: Phylogeography, Gene Flow, Biology, Virology, Ecology, and 15 moreEnvironmental Monitoring, Rivers, Medicine, Biological Sciences, Humans, Animals, Animal migration, Mississippi, North America, influenza A virus, Genetic variation, Seasons, Anseriformes, Host Pathogen Interactions, and Medical and Health Sciences
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This thesis explores the evolution of avian influenza A viruses (IAV), as well as host-pathogen interactions between these viruses and their main reservoir host, the Mallard (Anas platyrhynchos). IAV is a genetically diverse, multi-host... more
This thesis explores the evolution of avian influenza A viruses (IAV), as well as host-pathogen interactions between these viruses and their main reservoir host, the Mallard (Anas platyrhynchos). IAV is a genetically diverse, multi-host virus and wild birds, particularly dabbling ducks, are the natural reservoir. At our study site, up to 30% of migratory Mallards are infected with IAV during an autumn season, and host a large number of virus subtypes. IAV diversity is driven by two main mechanisms: mutation, driving genetic drift; and reassortment following co-infection, resulting in genetic shift. Reassortment is pervasive within an autumn season, both across multiple subtypes and within a single subtype. It is a key genetic feature in long-term maintenance of common subtypes, as it allows for independent lineage turn-over, generating novel genetic constellations. I hypothesize that the decoupling of successful constellations and generation of novel annual constellations enables viruses to escape herd immunity; these genetic changes must confer antigenic change for the process to be favourable. Indeed, in an experiment utilizing vaccines, circulating viruses escaped homosubtypic immunity, resulting in the proliferation of infections with the same subtype as the vaccine. While the host plays an important role in shaping IAV evolutionary genetics, one must consider that Mallards are infected with a multitude of other microorganisms. Here, Mallards were infected with IAV, gamma coronaviruses, and avian paramyxovirus type 1 simultaneously, and we found a putative synergistic interaction between IAV and gamma coronaviruses. Mallards occupy the interface between humans, poultry, and wild birds, and are the reservoir of IAV diversity. New incursions of highly pathogenic H5 viruses to both Europe and North America reaffirms the role of wild birds, particularly waterfowl, in diffusion of viruses spatially. Using European low pathogenic viruses and Mallard model, this thesis contributes to aspects of epidemiology, ecology, and evolutionary dynamics of waterfowl viruses, particularly IAV
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Coinfection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and other viruses is inevitable as the COVID-19 pandemic continues. This study aimed to evaluate cell lines commonly used in virus diagnosis and isolation for... more
Coinfection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and other viruses is inevitable as the COVID-19 pandemic continues. This study aimed to evaluate cell lines commonly used in virus diagnosis and isolation for their susceptibility to SARS-CoV-2. While multiple kidney cell lines from monkeys were susceptible and permissive to SARS-CoV-2, many cell types derived from human, dog, mink, cat, mouse, or chicken were not. Analysis of MDCK cells, which are most commonly used for surveillance and study of influenza viruses, demonstrated that they were insusceptible to SARS-CoV-2 and that the cellular barrier to productive infection was due to low expression level of the angiotensin converting enzyme 2 (ACE2) receptor and lower receptor affinity to SARS-CoV-2 spike, which could be overcome by over-expression of canine ACE2 in trans. Moreover, SARS-CoV-2 cell tropism did not appear to be affected by a D614G mutation in the spike protein.
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Influenza viruses cause annual seasonal epidemics and sporadic pandemics; vaccination is the most effective countermeasure. Intranasal live attenuated influenza vaccines (LAIVs) are needle-free, mimic the natural route of infection, and... more
Influenza viruses cause annual seasonal epidemics and sporadic pandemics; vaccination is the most effective countermeasure. Intranasal live attenuated influenza vaccines (LAIVs) are needle-free, mimic the natural route of infection, and elicit robust immunity. However, some LAIVs require reconstitution and cold-chain requirements restrict storage and distribution of all influenza vaccines. We generated a dry-powder, thermostable LAIV (T-LAIV) using Preservation by Vaporization technology and assessed the stability, immunogenicity, and efficacy of T-LAIV alone or combined with delta inulin adjuvant (Advax™) in ferrets. Stability assays demonstrated minimal loss of T-LAIV titer when stored at 25 °C for 1 year. Vaccination of ferrets with T-LAIV alone or with delta inulin adjuvant elicited mucosal antibody and robust serum HI responses in ferrets, and was protective against homologous challenge. These results suggest that the Preservation by Vaporization-generated dry-powder vaccines c...
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Swine are influenza virus reservoirs that have caused outbreaks and pandemics. Genomic characterization of these viruses enables pandemic risk assessment and vaccine comparisons, though this typically occurs after a novel swine virus... more
Swine are influenza virus reservoirs that have caused outbreaks and pandemics. Genomic characterization of these viruses enables pandemic risk assessment and vaccine comparisons, though this typically occurs after a novel swine virus jumps into humans. The greatest risk occurs where large groups of swine and humans comingle. At a large swine exhibition, we used Nanopore sequencing and on-site analytics to interpret 13 swine influenza virus genomes and identified an influenza virus cluster that was genetically highly varied to currently available vaccines. As part of the National Strategy for Pandemic Preparedness exercises, the sequences were emailed to colleagues at the CDC who initiated the development of a synthetically derived vaccine designed to match the viruses at the exhibition. Subsequently, this virus caused 14 infections in humans and was the dominant U.S. variant virus in 2018.
Research Interests: Biology, Virology, Medicine, Virus, Preparedness, and 3 moreinfluenza A virus, Outbreak, and Pandemic
Background Increased illness due to antigenically drifted A(H3N2) clade 3C.3a influenza viruses prompted concerns about vaccine effectiveness (VE) and vaccine strain selection. We used US virologic surveillance and US Influenza Vaccine... more
Background Increased illness due to antigenically drifted A(H3N2) clade 3C.3a influenza viruses prompted concerns about vaccine effectiveness (VE) and vaccine strain selection. We used US virologic surveillance and US Influenza Vaccine Effectiveness (Flu VE) Network data to evaluate consequences of this clade. Methods Distribution of influenza viruses was described using virologic surveillance data. The Flu VE Network enrolled ambulatory care patients aged ≥6 months with acute respiratory illness at 5 sites. Respiratory specimens were tested for influenza by means of reverse-transcriptase polymerase chain reaction and were sequenced. Using a test-negative design, we estimated VE, comparing the odds of influenza among vaccinated versus unvaccinated participants. Results During the 2018–2019 influenza season, A(H3N2) clade 3C.3a viruses caused an increasing proportion of influenza cases. Among 2763 Flu VE Network case patients, 1325 (48%) were infected with A(H1N1)pdm09 and 1350 (49%)...
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An outbreak of influenza A(H7N2) virus in cats in a shelter in New York, NY, USA, resulted in zoonotic transmission. Virus isolated from the infected human was closely related to virus isolated from a cat; both were related to low... more
An outbreak of influenza A(H7N2) virus in cats in a shelter in New York, NY, USA, resulted in zoonotic transmission. Virus isolated from the infected human was closely related to virus isolated from a cat; both were related to low pathogenicity avian influenza A(H7N2) viruses detected in the United States during the early 2000s.
Research Interests: Medical Microbiology, Biology, Virology, Polysaccharides, Medicine, and 15 moreEmerging Infectious Diseases, Cats, Disease Outbreaks, Humans, Virus, Birds, Animals, Clinical Sciences, New York, Public health systems and services research, influenza A virus, Pathogenicity, Protein Binding, Outbreak, and binding sites
BackgroundInfluenza vaccination aims to prevent infection by influenza virus and reduce associated morbidity and mortality; however, vaccine effectiveness (VE) can be modest, especially for subtype A/H3N2. Failure to achieve consistently... more
BackgroundInfluenza vaccination aims to prevent infection by influenza virus and reduce associated morbidity and mortality; however, vaccine effectiveness (VE) can be modest, especially for subtype A/H3N2. Failure to achieve consistently high VE has been attributed both to mismatches between the vaccine and circulating influenza strains and to the vaccine's elicitation of protective immunity in only a subset of the population. The low H3N2 VE in 2012-13 was attributed to egg-adaptive mutations that created antigenic mismatch between the intended (A/Victoria/361/2011) and actual vaccine strain (IVR-165).MethodsWe investigate the basis of the low VE in 2012-2013 by evaluating whether vaccinated and unvaccinated individuals were infected by different viral strains and assessing the serologic responses to A/Victoria/361/2011 and the IVR-165 vaccine strain in an adult cohort before and after vaccination.ResultsWe found no significant genetic differences between the strains that infec...
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In 2016, a total of 18 human infections with influenza A(H3N2) virus occurred after exposure to influenza-infected swine at 7 agricultural fairs. Sixteen of these cases were the result of infection by a reassorted virus with increasing... more
In 2016, a total of 18 human infections with influenza A(H3N2) virus occurred after exposure to influenza-infected swine at 7 agricultural fairs. Sixteen of these cases were the result of infection by a reassorted virus with increasing prevalence among US swine containing a hemagglutinin gene from 2010-11 human seasonal H3N2 strains.
Research Interests: Medical Microbiology, Biology, Virology, Agriculture, Medicine, and 15 moreAnimal Husbandry, Emerging Infectious Diseases, Phylogeny, Disease Outbreaks, Humans, Animals, Michigan, Clinical Sciences, Ohio, Swine, influenza A virus, Swine Diseases, Pandemic, Orthomyxoviridae Infections, and Epidemiological monitoring
In February 2016, three influenza B/Victoria/2/87 lineage viruses exhibiting 4- to 158-fold reduced inhibition by neuraminidase inhibitors were detected in Laos. These viruses had an H134N substitution in the neuraminidase and replicated... more
In February 2016, three influenza B/Victoria/2/87 lineage viruses exhibiting 4- to 158-fold reduced inhibition by neuraminidase inhibitors were detected in Laos. These viruses had an H134N substitution in the neuraminidase and replicated efficiently in vitro and in ferrets. Current antiviral drugs may be ineffective in controlling infections caused by viruses harboring this mutation.
Research Interests: Medical Microbiology, Biology, Adolescent, Medicine, Emerging Infectious Diseases, and 15 moreLAOS, Humans, Child, Female, Male, Infant, Neuraminidase, Clinical Sciences, Aged, Middle Aged, Adult, Public health systems and services research, Amino Acid Substitution Rates, Antiviral Agents, and Child preschool
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Influenza virus defective interfering (DI) particles are naturally occurring noninfectious virions typically generated during in vitro serial passages in cell culture of the virus at a high multiplicity of infection. DI particles are... more
Influenza virus defective interfering (DI) particles are naturally occurring noninfectious virions typically generated during in vitro serial passages in cell culture of the virus at a high multiplicity of infection. DI particles are recognized for the role they play in inhibiting viral replication and for the impact they have on the production of infectious virions. To date, influenza virus DI particles have been reported primarily as a phenomenon of cell culture and in experimentally infected embryonated chicken eggs. They have also been isolated from a respiratory infection of chickens. Using a sequencing approach, we characterize several subgenomic viral RNAs from human nasopharyngeal specimens infected with the influenza A(H1N1)pdm09 virus. The distribution of these in vivo -derived DI-like RNAs was similar to that of in vitro DIs, with the majority of the defective RNAs generated from the PB2 (segment 1) of the polymerase complex, followed by PB1 and PA. The lengths of the in ...
Research Interests: RNA, Computational Biology, Biology, Virology, Medicine, and 14 moreBiological Sciences, Humans, Sequence alignment, Virus, Molecular cloning, In Vivo, influenza A virus, Rc, Base Sequence, Viral Replication, Molecular Sequence Data, Viral Interference, Orthomyxoviridae, and Medical and Health Sciences
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A(H9N2) influenza viruses are widespread in poultry in many parts of the world and for over 20 years have sporadically jumped species barriers to cause human infection. As these viruses continue to diversify genetically and antigenically,... more
A(H9N2) influenza viruses are widespread in poultry in many parts of the world and for over 20 years have sporadically jumped species barriers to cause human infection. As these viruses continue to diversify genetically and antigenically, it is critical to closely monitor viruses responsible for human infections, to ascertain if A(H9N2) viruses are acquiring properties that make them better suited to infect and spread among humans. In this study, we describe an active poultry surveillance system established in Vietnam to identify the scope of influenza viruses present in live bird markets and the threat they pose to human health. Assessment of a recent A(H9N2) virus isolated from an individual in China in 2018 is also reported, and it was found to exhibit properties of adaptation to humans and, importantly, it shows similarities to strains isolated from the live bird markets of Vietnam.
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Limited effective reassortment shapes influenza dynamics in a human host Ashley Sobel Leonard, Micah T. McClain, Gavin J. D. Smith, David E. Wentworth, Rebecca A. Halpin, Xudong Lin, Amy Ransier, Timothy B. Stockwell, Suman R. Das,... more
Limited effective reassortment shapes influenza dynamics in a human host Ashley Sobel Leonard, Micah T. McClain, Gavin J. D. Smith, David E. Wentworth, Rebecca A. Halpin, Xudong Lin, Amy Ransier, Timothy B. Stockwell, Suman R. Das, Anthony S. Gilbert, Rob Lambkin-Williams, Geoffrey S. Ginsburg, Christopher W. Woods, Katia Koelle, Christopher J. R. Illingworth6,7∗ 1 Department of Biology, Duke University, Durham, USA 2 Duke Center for Applied Genomics and Precision Medicine, Durham, NC, USA 3 Programme in Emerging Infectious Diseases, Duke-NUS Medical School, 8 College Road, 169857, Singapore 4 J. Craig Venter Institute, Rockville, Maryland, USA 5 hVivo PLC, The QMB Innovation Centre, Queen Mary, University of London, 42 New Road, London, UK 6 Department of Genetics, University of Cambridge, Cambridge, UK 7 Department of Applied Maths and Theoretical Physics, Centre for Mathematical Sciences, Wilberforce Road, University of Cambridge, Cambridge, UK 8 Current address: Centers for Dise...
January 16, 2018 at 1:00 p.m. (ET)Note: January 16 CDC Public Health Grand Rounds topic change to: \u201cPublic Health Response to Severe Influenza\u201dThe topic for CDC\u2019s Public Health Grand Rounds on January 16 will now focus on... more
January 16, 2018 at 1:00 p.m. (ET)Note: January 16 CDC Public Health Grand Rounds topic change to: \u201cPublic Health Response to Severe Influenza\u201dThe topic for CDC\u2019s Public Health Grand Rounds on January 16 will now focus on influenza. With the spike in flu cases around the country, this Grand Rounds will provide key and timely information for public health professionals on how to reduce the spread of seasonal flu in communities and adjust to spot shortages in antiviral drugs because of high influenza activity in some areas.To date, this influenza season is notable for the sheer volume of flu that most of the United States is seeing at the same time which can stress health systems. The vast majority of this activity has been caused by influenza A H3N2, associated with severe illness in young children and people 65 years and older.Presented by: Anne Schuchat, MD (RADM, USPHS), Principal Deputy Director, Centers for Disease Control and Prevention [\u201cIntroduction\u201d]; Dan Jernigan, MD, MPH, Captain, USPHS, Director, Influenza Division, National Center for Immunization and Respiratory Diseases, Centers for Disease Control and Prevention [\u201cChasing Flu\u201d]; David Wentworth, PhD, Chief, Virology, Surveillance, and Diagnosis Branch, Influenza Division, National Center for Immunization and Respiratory Diseases. Centers for Disease Control and Prevention [\u201cThe Problem of H3N2\u201d]; Alicia Fry, MD, MPH, Captain, USPHS, Chief, Epidemiology and Prevention Branch, Influenza Division, National Center for Immunization and Respiratory Diseases, Centers for Disease Control and Prevention [\u201cMitigating Influenza with Vaccines and Antivirals\u201d].Chasing Flu [PDF version of the PowerPoint presentation by Dan Jernigan, p. 2-15] -- The Problem of H3N2 [PDF version of the PowerPoint presentation by David Wentworth, p. 16-30] -- Mitigating Influenza with Vaccines and Antivirals [PDF version of the PowerPoint presentation by Alicia Fry, p. 31-45]
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Influenza A(H3N2) viruses evade human immunity primarily by acquiring antigenic changes in the haemagglutinin (HA). HA receptor-binding features of contemporary A(H3N2) viruses hinder traditional antigenic characterization using... more
Influenza A(H3N2) viruses evade human immunity primarily by acquiring antigenic changes in the haemagglutinin (HA). HA receptor-binding features of contemporary A(H3N2) viruses hinder traditional antigenic characterization using haemagglutination inhibition and promote selection of HA mutants. Thus, alternative approaches are needed to reliably assess antigenic relatedness between circulating viruses and vaccines. We developed a high content imaging-based neutralization test (HINT) to reduce antigenic mischaracterization resulting from virus adaptation to cell culture. Ferret reference antisera were raised using clinical specimens containing viruses representing recent vaccine strains. Analysis of viruses circulating during 2011–2018 showed that gain of an N158-linked glycosylation in HA was a molecular determinant of antigenic distancing between A/Hong Kong/4801/2014-like (clade 3C.2a) and A/Texas/50/2012-like viruses (clade 3C.1), while multiple evolutionary HA F193S substitution ...
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Swine are susceptible to infection by both avian and human influenza viruses, and this feature is thought to contribute to novel reassortant influenza viruses. In this study, the influenza virus reassortment rate in swine and human cells... more
Swine are susceptible to infection by both avian and human influenza viruses, and this feature is thought to contribute to novel reassortant influenza viruses. In this study, the influenza virus reassortment rate in swine and human cells was determined. Swine cells co-infected with 2009 pandemic H1N1 (huH1N1), and an endemic swine H1N2 (A/swine/Illinois/02860/09) virus (swH1N2), resulted in a 23% reassortment rate that was independent of alpha 2,3 or 2,6 sialic acid distribution on the cells. The reassortants had altered pathogenic phenotypes linked to introduction of the swine PA and NA into huH1N1. In mice, the huH1N1 PA and NA mediated increased MIP-2 expression early post-infection resulting in substantial pulmonary neutrophilia with enhanced lung pathology and disease. The findings support the notion that swine are a mixing vessel for influenza reassortants independent of sialic acid distribution. These results show the potential for continued reassortment of the 2009 pandemic ...
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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... more
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.
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During the evolution of SARS-CoV-2 in humans a D614G substitution in the spike (S) protein emerged and became the predominant circulating variant (S-614G) of the COVID-19 pandemic1. However, whether the increasing prevalence of the S-614G... more
During the evolution of SARS-CoV-2 in humans a D614G substitution in the spike (S) protein emerged and became the predominant circulating variant (S-614G) of the COVID-19 pandemic1. However, whether the increasing prevalence of the S-614G variant represents a fitness advantage that improves replication and/or transmission in humans or is merely due to founder effects remains elusive. Here, we generated isogenic SARS-CoV-2 variants and demonstrate that the S-614G variant has (i) enhanced binding to human ACE2, (ii) increased replication in primary human bronchial and nasal airway epithelial cultures as well as in a novel human ACE2 knock-in mouse model, and (iii) markedly increased replication and transmissibility in hamster and ferret models of SARS-CoV-2 infection. Collectively, our data show that while the S-614G substitution results in subtle increases in binding and replication in vitro, it provides a real competitive advantage in vivo, particularly during the transmission bottl...
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Seasonal influenza virus A/H3N2 is a major cause of death globally. Vaccination remains the most effective preventative. Rapid mutation of hemagglutinin allows viruses to escape adaptive immunity. This antigenic drift necessitates regular... more
Seasonal influenza virus A/H3N2 is a major cause of death globally. Vaccination remains the most effective preventative. Rapid mutation of hemagglutinin allows viruses to escape adaptive immunity. This antigenic drift necessitates regular vaccine updates. Effective vaccine strains need to represent H3N2 populations circulating one year after strain selection. Experts select strains based on experimental measurements of antigenic drift and predictions made by models from hemagglutinin sequences. We developed a novel influenza forecasting framework that integrates phenotypic measures of antigenic drift and functional constraint with previously published sequence-only fitness estimates. Forecasts informed by phenotypic measures of antigenic drift consistently outperformed previous sequence-only estimates, while sequence-only estimates of functional constraint surpassed more comprehensive experimentally-informed estimates. Importantly, the best models integrated estimates of both functi...
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In July 1991, an influenza A virus, designated A/Maryland/12/91 (A/MD), was isolated from the bronchial secretions of a 27-year-old animal caretaker. He had been admitted to the hospital with bilateral pneumonia and died of acute... more
In July 1991, an influenza A virus, designated A/Maryland/12/91 (A/MD), was isolated from the bronchial secretions of a 27-year-old animal caretaker. He had been admitted to the hospital with bilateral pneumonia and died of acute respiratory distress syndrome 13 days later. Antigenic analyses with postinfection ferret antisera and monoclonal antibodies to recent H1 swine hemagglutinins indicated that the hemagglutinin of this virus was antigenically related to, but distinguishable from, those of other influenza A (H1N1) viruses currently circulating in swine. Oligonucleotide mapping of total viral RNAs revealed differences between A/MD and other contemporary swine viruses. However, partial sequencing of each RNA segment of A/MD demonstrated that all segments were related to those of currently circulating swine viruses. Sequence analysis of the entire hemagglutinin, nucleoprotein, and matrix genes of A/MD revealed a high level of identity with other contemporary swine viruses. Our st...
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The complex and unresolved evolutionary origins of the 2009 H1N1 influenza pandemic exposed major gaps in our knowledge of the global spatial ecology and evolution of influenza A viruses in swine (swIAVs). Here we undertake an expansive... more
The complex and unresolved evolutionary origins of the 2009 H1N1 influenza pandemic exposed major gaps in our knowledge of the global spatial ecology and evolution of influenza A viruses in swine (swIAVs). Here we undertake an expansive phylogenetic analysis of swIAV sequence data and demonstrate that the global live swine trade strongly predicts the spatial dissemination of swIAVs, with Europe and North America acting as sources of viruses in Asian countries. In contrast, China has the world's largest swine population but is not a major exporter of live swine, and is not an important source of swIAVs in neighbouring Asian countries or globally. A meta-population simulation model incorporating trade data predicts that the global ecology of swIAVs is more complex than previously thought, and the United States and China's large swine populations are unlikely to be representative of swIAV diversity in their respective geographic regions, requiring independent surveillance effor...
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Despite evidence for avian influenza A virus (AIV) transmission between wild and domestic ecosystems, the roles of bird migration and poultry trade in the spread of viruses remain enigmatic. In this study, we integrate ecosystem... more
Despite evidence for avian influenza A virus (AIV) transmission between wild and domestic ecosystems, the roles of bird migration and poultry trade in the spread of viruses remain enigmatic. In this study, we integrate ecosystem interactions into a phylogeographic model to assess the contribution of wild and domestic hosts to AIV distribution and persistence. Analysis of globally sampled AIV datasets shows frequent two-way transmission between wild and domestic ecosystems. In general, viral flow from domestic to wild bird populations was restricted to within a geographic region. In contrast, spillover from wild to domestic populations occurred both within and between regions. Wild birds mediated long-distance dispersal at intercontinental scales whereas viral spread among poultry populations was a major driver of regional spread. Viral spread between poultry flocks frequently originated from persistent lineages circulating in regions of intensive poultry production. Our analysis of long-term surveillance data demonstrates that meaningful insights can be inferred from integrating ecosystem into phylogeographic reconstructions that may be consequential for pandemic preparedness and livestock protection. It is assumed that AIV outbreaks in poultry are introduced from wild birds. To test this, we incorporated ecosystem and location of isolation into a comparative genetic analysis. We show high rates of viral transmission from domestic to wild birds within a region and,