Human CCR6+ Th Cells Show Both an Extended Stable Gradient of Th17 Activity and Imprinted Plasticity.

Th17 cells have been investigated in mice primarily for their contributions to autoimmune diseases. However, the pathways of differentiation of Th17 and related Th cells (type 17 cells) and the structure of the type 17 memory population in humans are not well understood; such understanding is critical for manipulating these cells in vivo. By exploiting differences in levels of surface CCR6, we found that human type 17 memory cells, including individual T cell clonotypes, form an elongated continuum of type 17 character along which cells can be driven by increasing RORγt. This continuum includes cells preserved within the memory pool with potentials that reflect the early preferential activation of multiple over single lineages. The phenotypes and epigenomes of CCR6+ cells are stable across cell divisions under noninflammatory conditions. Nonetheless, activation in polarizing and nonpolarizing conditions can yield additional functionalities, revealing, respectively, both environmentally induced and imprinted mechanisms that contribute differentially across the type 17 continuum to yield the unusual plasticity ascribed to type 17 cells.

Human CCR6 + Th cells show both an extended stable gradient of Th17 activity and imprinted plasticity.

Th17 cells have been investigated in mice primarily for their contributions to autoimmune diseases. However, the pathways of differentiation of Th17 and related (type 17) cells and the structure of the type 17 memory population in humans are not well understood; such understanding is critical for manipulating these cells in vivo . By exploiting differences in levels of surface CCR6, we found that human type 17 memory cells, including individual T cell clonotypes, form an elongated continuum of type 17 character along which cells can be driven by increasing RORγt. This continuum includes cells preserved within the memory pool with potentials that reflect the early preferential activation of multiple over single lineages. The CCR6 + cells' phenotypes and epigenomes are stable across cell divisions under homeostatic conditions. Nonetheless, activation in polarizing and non-polarizing conditions can yield additional functionalities, revealing, respectively, both environmentally induced and imprinted mechanisms that contribute differentially across the continuum to yield the unusual plasticity ascribed to type 17 cells.

Delayed boosting improves human antigen-specific Ig and B cell responses to the RH5.1/AS01B malaria vaccine.

Modifications to vaccine delivery that increase serum antibody longevity are of great interest for maximizing efficacy. We have previously shown that a delayed fractional (DFx) dosing schedule (0-1-6 month) - using AS01B-adjuvanted RH5.1 malaria antigen - substantially improves serum IgG durability as compared with monthly dosing (0-1-2 month; NCT02927145). However, the underlying mechanism and whether there are wider immunological changes with DFx dosing were unclear. Here, PfRH5-specific Ig and B cell responses were analyzed in depth through standardized ELISAs, flow cytometry, systems serology, and single-cell RNA-Seq (scRNA-Seq). Data indicate that DFx dosing increases the magnitude and durability of circulating PfRH5-specific B cells and serum IgG1. At the peak antibody magnitude, DFx dosing was distinguished by a systems serology feature set comprising increased FcRn binding, IgG avidity, and proportion of G2B and G2S2F IgG Fc glycans, alongside decreased IgG3, antibody-dependent complement deposition, and proportion of G1S1F IgG Fc glycan. Concomitantly, scRNA-Seq data show a higher CDR3 percentage of mutation from germline and decreased plasma cell gene expression in circulating PfRH5-specific B cells. Our data, therefore, reveal a profound impact of DFx dosing on the humoral response and suggest plausible mechanisms that could enhance antibody longevity, including improved FcRn binding by serum Ig and a potential shift in the underlying cellular response from circulating short-lived plasma cells to nonperipheral long-lived plasma cells.

Multiple modes of antigen exposure induce clonotypically diverse epitope-specific CD8+ T cells across multiple tissues in nonhuman primates.

Antigen-specific CD8+ T cells play a key role in the host's antiviral response. T cells recognize viral epitopes via the T cell receptor (TCR), which contains the complementarity-determining region-3 (CDR3), comprising the variable, diversity and joining regions of the TCRß gene. During chronic simian immunodeficiency virus (SIV) infection of Asian macaque nonhuman primates, tissue-specific clonotypes are identifiable among SIV-specific CD8+ T cells. Here, we sought to determine level of antigen exposure responsible for the tissue-specific clonotypic structure. We examined whether the priming event and/or chronic antigen exposure is response for tissue-specific TCR repertoires. We evaluated the TCR repertoire of SIV-specific CD8+ T cells after acute antigen exposure following inoculation with a SIV DNA vaccine, longitudinally during the acute and chronic phases of SIV, and after administration of antiretrovirals (ARVs). Finally, we assessed the TCR repertoire of cytomegalovirus (CMV)-specific CD8+ T cells to establish if TCR tissue-specificity is shared among viruses that chronically replicate. TCR sequences unique to anatomical sites were identified after acute antigen exposure via vaccination and upon acute SIV infection. Tissue-specific clones also persisted into chronic infection and the clonotypic structure continued to evolve after ARV administration. Finally, tissue-specific clones were also observed in CMV-specific CD8+ T cells. Together, these data suggest that acute antigen priming is sufficient to induce tissue-specific clones and that this clonal hierarchy can persist when antigen loads are naturally or therapeutically reduced, providing mechanistic insight into tissue-residency.

Broad coverage of neutralization-resistant SIV strains by second-generation SIV-specific antibodies targeting the region involved in binding CD4.

Both SIV and SHIV are powerful tools for evaluating antibody-mediated prevention and treatment of HIV-1. However, owing to a lack of rhesus-derived SIV broadly neutralizing antibodies (bnAbs), testing of bnAbs for HIV-1 prevention or treatment has thus far been performed exclusively in the SHIV NHP model using bnAbs from HIV-1-infected individuals. Here we describe the isolation and characterization of multiple rhesus-derived SIV bnAbs capable of neutralizing most isolates of SIV. Eight antibodies belonging to two clonal families, ITS102 and ITS103, which target unique epitopes in the CD4 binding site (CD4bs) region, were found to be broadly neutralizing and together neutralized all SIV strains tested. A rare feature of these bnAbs and two additional antibody families, ITS92 and ITS101, which mediate strain-specific neutralizing activity against SIV from sooty mangabeys (SIVsm), was their ability to achieve near complete (i.e. 100%) neutralization of moderately and highly neutralization-resistant SIV. Overall, these newly identified SIV bnAbs highlight the potential for evaluating HIV-1 prophylactic and therapeutic interventions using fully simian, rhesus-derived bnAbs in the SIV NHP model, thereby circumventing issues related to rapid antibody clearance of human-derived antibodies, Fc mismatch and limited genetic diversity of SHIV compared to SIV.

Translocated microbiome composition determines immunological outcome in treated HIV infection.

The impact of the microbiome on HIV disease is widely acknowledged although the mechanisms downstream of fluctuations in microbial composition remain speculative. We detected rapid, dynamic changes in translocated microbial constituents during two years after cART initiation. An unbiased systems biology approach revealed two distinct pathways driven by changes in the abundance ratio of Serratia to other bacterial genera. Increased CD4 T cell numbers over the first year were associated with high Serratia abundance, pro-inflammatory innate cytokines, and metabolites that drive Th17 gene expression signatures and restoration of mucosal integrity. Subsequently, decreased Serratia abundance and downregulation of innate cytokines allowed re-establishment of systemic T cell homeostasis promoting restoration of Th1 and Th2 gene expression signatures. Analyses of three other geographically distinct cohorts of treated HIV infection established a more generalized principle that changes in diversity and composition of translocated microbial species influence systemic inflammation and consequently CD4 T cell recovery.

Vaccination with prefusion-stabilized respiratory syncytial virus fusion protein induces genetically and antigenically diverse antibody responses.

An effective vaccine for respiratory syncytial virus (RSV) is an unrealized public health goal. A single dose of the prefusion-stabilized fusion (F) glycoprotein subunit vaccine (DS-Cav1) substantially increases serum-neutralizing activity in healthy adults. We sought to determine whether DS-Cav1 vaccination induces a repertoire mirroring the pre-existing diversity from natural infection or whether antibody lineages targeting specific epitopes predominate. We evaluated RSV F-specific B cell responses before and after vaccination in six participants using complementary B cell sequencing methodologies and identified 555 clonal lineages. DS-Cav1-induced lineages recognized the prefusion conformation of F (pre-F) and were genetically diverse. Expressed antibodies recognized all six antigenic sites on the pre-F trimer. We identified 34 public clonotypes, and structural analysis of two antibodies from a predominant clonotype revealed a common mode of recognition. Thus, vaccination with DS-Cav1 generates a diverse polyclonal response targeting the antigenic sites on pre-F, supporting the development and advanced testing of pre-F-based vaccines against RSV.

Antibodies with potent and broad neutralizing activity against antigenically diverse and highly transmissible SARS-CoV-2 variants.

The emergence of highly transmissible SARS-CoV-2 variants of concern (VOC) that are resistant to therapeutic antibodies highlights the need for continuing discovery of broadly reactive antibodies. We identify four receptor-binding domain targeting antibodies from three early-outbreak convalescent donors with potent neutralizing activity against 12 variants including the B.1.1.7 and B.1.351 VOCs. Two of them are ultrapotent, with sub-nanomolar neutralization titers (IC50 <0.0006 to 0.0102 µ g/mL; IC80 < 0.0006 to 0.0251 µ g/mL). We define the structural and functional determinants of binding for all four VOC-targeting antibodies, and show that combinations of two antibodies decrease the in vitro generation of escape mutants, suggesting potential means to mitigate resistance development. These results define the basis of therapeutic cocktails against VOCs and suggest that targeted boosting of existing immunity may increase vaccine breadth against VOCs.

Benchmarking of T cell receptor repertoire profiling methods reveals large systematic biases.

Monitoring the T cell receptor (TCR) repertoire in health and disease can provide key insights into adaptive immune responses, but the accuracy of current TCR sequencing (TCRseq) methods is unclear. In this study, we systematically compared the results of nine commercial and academic TCRseq methods, including six rapid amplification of complementary DNA ends (RACE)-polymerase chain reaction (PCR) and three multiplex-PCR approaches, when applied to the same T cell sample. We found marked differences in accuracy and intra- and inter-method reproducibility for T cell receptor α (TRA) and T cell receptor ß (TRB) TCR chains. Most methods showed a lower ability to capture TRA than TRB diversity. Low RNA input generated non-representative repertoires. Results from the 5' RACE-PCR methods were consistent among themselves but differed from the RNA-based multiplex-PCR results. Using an in silico meta-repertoire generated from 108 replicates, we found that one genomic DNA-based method and two non-unique molecular identifier (UMI) RNA-based methods were more sensitive than UMI methods in detecting rare clonotypes, despite the better clonotype quantification accuracy of the latter.

The Identity of Human Tissue-Emigrant CD8+ T Cells.

Lymphocyte migration is essential for adaptive immune surveillance. However, our current understanding of this process is rudimentary, because most human studies have been restricted to immunological analyses of blood and various tissues. To address this knowledge gap, we used an integrated approach to characterize tissue-emigrant lineages in thoracic duct lymph (TDL). The most prevalent immune cells in human and non-human primate efferent lymph were T cells. Cytolytic CD8+ T cell subsets with effector-like epigenetic and transcriptional signatures were clonotypically skewed and selectively confined to the intravascular circulation, whereas non-cytolytic CD8+ T cell subsets with stem-like epigenetic and transcriptional signatures predominated in tissues and TDL. Moreover, these anatomically distinct gene expression profiles were recapitulated within individual clonotypes, suggesting parallel differentiation programs independent of the expressed antigen receptor. Our collective dataset provides an atlas of the migratory immune system and defines the nature of tissue-emigrant CD8+ T cells that recirculate via TDL.

Epigenetic silencing of CD4 expression in nonpathogenic SIV infection in African green monkeys.

African green monkeys (AGMs) are natural hosts of SIV that postthymically downregulate CD4 to maintain a large population of CD4-CD8aa+ virus-resistant cells with Th functionality, which can result in AGMs becoming apparently cured of SIVagm infection. To understand the mechanisms of this process, we performed genome-wide transcriptional analysis on T cells induced to downregulate CD4 in vitro from AGMs and closely related patas monkeys and T cells that maintain CD4 expression from rhesus macaques. In T cells that downregulated CD4, pathway analysis revealed an atypical regulation of the DNA methylation machinery, which was reversible when pharmacologically targeted with 5-aza-2 deoxycytidine. This signature was driven largely by the dioxygenase TET3, which became downregulated with loss of CD4 expression. CpG motifs within the AGM CD4 promoter region became methylated during CD4 downregulation in vitro and were stably imprinted in AGM CD4-CD8aa+ T cells sorted directly ex vivo. These results suggest that AGMs use epigenetic mechanisms to durably silence the CD4 gene. Manipulation of these mechanisms could provide avenues for modulating SIV and HIV-1 entry receptor expression in hosts that become progressively infected with SIV, which could lead to novel therapeutic interventions aimed to reduce HIV viremia in vivo.

VRC34-Antibody Lineage Development Reveals How a Required Rare Mutation Shapes the Maturation of a Broad HIV-Neutralizing Lineage.

Rare mutations have been proposed to restrict the development of broadly neutralizing antibodies against HIV-1, but this has not been explicitly demonstrated. We hypothesized that such rare mutations might be identified by comparing broadly neutralizing and non-broadly neutralizing branches of an antibody-developmental tree. Because sequences of antibodies isolated from the fusion peptide (FP)-targeting VRC34-antibody lineage suggested it might be suitable for such rare mutation analysis, we carried out next-generation sequencing (NGS) on B cell transcripts from donor N123, the source of the VRC34 lineage, and functionally and structurally characterized inferred intermediates along broadly neutralizing and poorly neutralizing developmental branches. The broadly neutralizing VRC34.01 branch required the rare heavy-chain mutation Y33P to bind FP, whereas the early bifurcated VRC34.05 branch did not require this rare mutation and evolved less breadth. Our results demonstrate how a required rare mutation can restrict development and shape the maturation of a broad HIV-1-neutralizing antibody lineage.

Elite control of HIV is associated with distinct functional and transcriptional signatures in lymphoid tissue CD8+ T cells.

The functional properties of circulating CD8+ T cells have been associated with immune control of HIV. However, viral replication occurs predominantly in secondary lymphoid tissues, such as lymph nodes (LNs). We used an integrated single-cell approach to characterize effective HIV-specific CD8+ T cell responses in the LNs of elite controllers (ECs), defined as individuals who suppress viral replication in the absence of antiretroviral therapy (ART). Higher frequencies of total memory and follicle-homing HIV-specific CD8+ T cells were detected in the LNs of ECs compared with the LNs of chronic progressors (CPs) who were not receiving ART. Moreover, HIV-specific CD8+ T cells potently suppressed viral replication without demonstrable cytolytic activity in the LNs of ECs, which harbored substantially lower amounts of CD4+ T cell-associated HIV DNA and RNA compared with the LNs of CPs. Single-cell RNA sequencing analyses further revealed a distinct transcriptional signature among HIV-specific CD8+ T cells from the LNs of ECs, typified by the down-regulation of inhibitory receptors and cytolytic molecules and the up-regulation of multiple cytokines, predicted secreted factors, and components of the protein translation machinery. Collectively, these results provide a mechanistic framework to expedite the identification of novel antiviral factors, highlighting a potential role for the localized deployment of noncytolytic functions as a determinant of immune efficacy against HIV.

Activation Dynamics and Immunoglobulin Evolution of Pre-existing and Newly Generated Human Memory B cell Responses to Influenza Hemagglutinin.

Vaccine-induced memory B cell responses to evolving viruses like influenza A involve activation of pre-existing immunity and generation of new responses. To define the contribution of these two types of responses, we analyzed the response to H7N9 vaccination in H7N9-naive adults. We performed comprehensive comparisons at the single-cell level of the kinetics, Ig repertoire, and activation phenotype of established pre-existing memory B cells recognizing conserved epitopes and the newly generated memory B cells directed toward H7 strain-specific epitopes. The recall response to conserved epitopes on H7 HA involved a transient expansion of memory B cells with little observed adaptation. However, the B cell response to newly encountered epitopes was phenotypically distinct and generated a sustained memory population that evolved and affinity matured months after vaccination. These findings establish clear differences between newly generated and pre-existing memory B cells, highlighting the challenges in achieving long-lasting, broad protection against an ever-evolving virus.

T-cell receptor sequencing demonstrates persistence of virus-specific T cells after antiviral immunotherapy.

Viral infections are a serious cause of morbidity and mortality following haematopoietic stem cell transplantation (HSCT). Adoptive cellular therapy with virus-specific T cells (VSTs) has been successful in preventing or treating targeted viruses in prior studies, but the composition of ex vivo expanded VST and the critical cell populations that mediate antiviral activity in vivo are not well defined. We utilized deep sequencing of the T-cell receptor beta chain (TCRB) in order to classify and track VST populations in 12 patients who received VSTs following HSCT to prevent or treat viral infections. TCRB sequencing was performed on sorted VST products and patient peripheral blood mononuclear cells samples. TCRB diversity was gauged using the Shannon entropy index, and repertoire similarity determined using the Morisita-Horn index. Similarity indices reflected an early change in TCRB diversity in eight patients, and TCRB clonotypes corresponding to targeted viral epitopes expanded in eight patients. TCRB repertoire diversity increased in nine patients, and correlated with cytomegalovirus (CMV) viral load following VST infusion (P = 0·0071). These findings demonstrate that allogeneic VSTs can be tracked via TCRB sequencing, and suggests that T-cell receptor repertoire diversity may be critical for the control of CMV reactivation after HSCT.

Longitudinal Analysis Reveals Early Development of Three MPER-Directed Neutralizing Antibody Lineages from an HIV-1-Infected Individual.

Lineage-based vaccine design is an attractive approach for eliciting broadly neutralizing antibodies (bNAbs) against HIV-1. However, most bNAb lineages studied to date have features indicative of unusual recombination and/or development. From an individual in the prospective RV217 cohort, we identified three lineages of bNAbs targeting the membrane-proximal external region (MPER) of the HIV-1 envelope. Antibodies RV217-VRC42.01, -VRC43.01, and -VRC46.01 used distinct modes of recognition and neutralized 96%, 62%, and 30%, respectively, of a 208-strain virus panel. All three lineages had modest levels of somatic hypermutation and normal antibody-loop lengths and were initiated by the founder virus MPER. The broadest lineage, VRC42, was similar to the known bNAb 4E10. A multimeric immunogen based on the founder MPER activated B cells bearing the unmutated common ancestor of VRC42, with modest maturation of early VRC42 intermediates imparting neutralization breadth. These features suggest that VRC42 may be a promising template for lineage-based vaccine design.

A high throughput lentivirus sieving assay identifies neutralization resistant Envelope sequences and predicts in vivo sieving.

An effective prophylactic vaccine against human immunodeficiency virus (HIV) will likely require a potent antibody response that can neutralize the virus at the mucosal portal of entry. The elicitation of potent broadly-neutralizing anti-sera will be an iterative process, optimizing candidates that only block a fraction of potential viral strains. This effect, termed "sieving", is evidence of a partially efficacious vaccine. Understanding the mechanisms of resistance of the breakthrough viruses is important for improving vaccines. We developed a high-throughput assay that can be used on vaccine-elicited antisera or monoclonal antibodies. Using the SIVsmE660 swarm stock and sera from a large NHP vaccine/challenge study, our in vitro sieving assay identified the same viral subspecies as in the animal study-those with a canonical C1 amino acid variants conferring global neutralization resistance to antibodies. Using a genetically divergent swarm stock, we identified five other amino acid variants that confer global resistance; the C1 mutations in this stock were not selected, also in agreement with in vivo challenge studies. Thus, the in vitro sieving assay can be used with genetically diverse challenge stocks to predict the coverage of a vaccine-elicited sera and possibly inform candidate vaccine development efforts.

Type I IFN signaling blockade by a PASylated antagonist during chronic SIV infection suppresses specific inflammatory pathways but does not alter T cell activation or virus replication.

Chronic activation of the immune system in HIV infection is one of the strongest predictors of morbidity and mortality. As such, approaches that reduce immune activation have received considerable interest. Previously, we demonstrated that administration of a type I interferon receptor antagonist (IFN-1ant) during acute SIV infection of rhesus macaques results in increased virus replication and accelerated disease progression. Here, we administered a long half-life PASylated IFN-1ant to ART-treated and ART-naïve macaques during chronic SIV infection and measured expression of interferon stimulated genes (ISG) by RNA sequencing, plasma viremia, plasma cytokines, T cell activation and exhaustion as well as cell-associated virus in CD4 T cell subsets sorted from peripheral blood and lymph nodes. Our study shows that IFN-1ant administration in both ART-suppressed and ART-untreated chronically SIV-infected animals successfully results in reduction of IFN-I-mediated inflammation as defined by reduced expression of ISGs but had no effect on plasma levels of IL-1ß, IL-1ra, IL-6 and IL-8. Unlike in acute SIV infection, we observed no significant increase in plasma viremia up to 25 weeks after IFN-1ant administration or up to 15 weeks after ART interruption. Likewise, cell-associated virus measured by SIV gag DNA copies was similar between IFN-1ant and placebo groups. In addition, evaluation of T cell activation and exhaustion by surface expression of CD38, HLA-DR, Ki67, LAG-3, PD-1 and TIGIT, as well as transcriptome analysis showed no effect of IFN-I blockade. Thus, our data show that blocking IFN-I signaling during chronic SIV infection suppresses IFN-I-related inflammatory pathways without increasing virus replication, and thus may constitute a safe therapeutic intervention in chronic HIV infection.

Identification and characterization of HIV-specific resident memory CD8+ T cells in human lymphoid tissue.

Current paradigms of CD8+ T cell-mediated protection in HIV infection center almost exclusively on studies of peripheral blood, which is thought to provide a window into immune activity at the predominant sites of viral replication in lymphoid tissues (LTs). Through extensive comparison of blood, thoracic duct lymph (TDL), and LTs in different species, we show that many LT memory CD8+ T cells bear phenotypic, transcriptional, and epigenetic signatures of resident memory T cells (TRMs). Unlike their circulating counterparts in blood or TDL, most of the total and follicular HIV-specific CD8+ T cells in LTs also resemble TRMs Moreover, high frequencies of HIV-specific CD8+ TRMs with skewed clonotypic profiles relative to matched blood samples are present in LTs of individuals who spontaneously control HIV replication in the absence of antiretroviral therapy (elite controllers). Single-cell RNA sequencing analysis confirmed that HIV-specific TRMs are enriched for effector-related immune genes and signatures compared with HIV-specific non-TRMs in elite controllers. Together, these data indicate that previous studies in blood have largely failed to capture the major component of HIV-specific CD8+ T cell responses resident within LTs.

Avian Influenza Viruses in Wild Birds: Virus Evolution in a Multihost Ecosystem.

Wild ducks and gulls are the major reservoirs for avian influenza A viruses (AIVs). The mechanisms that drive AIV evolution are complex at sites where various duck and gull species from multiple flyways breed, winter, or stage. The Republic of Georgia is located at the intersection of three migratory flyways: the Central Asian flyway, the East Africa/West Asia flyway, and the Black Sea/Mediterranean flyway. For six complete study years (2010 to 2016), we collected AIV samples from various duck and gull species that breed, migrate, and overwinter in Georgia. We found a substantial subtype diversity of viruses that varied in prevalence from year to year. Low-pathogenic AIV (LPAIV) subtypes included H1N1, H2N3, H2N5, H2N7, H3N8, H4N2, H6N2, H7N3, H7N7, H9N1, H9N3, H10N4, H10N7, H11N1, H13N2, H13N6, H13N8, and H16N3, and two highly pathogenic AIVs (HPAIVs) belonging to clade 2.3.4.4, H5N5 and H5N8, were found. Whole-genome phylogenetic trees showed significant host species lineage restriction for nearly all gene segments and significant differences in observed reassortment rates, as defined by quantification of phylogenetic incongruence, and in nucleotide sequence diversity for LPAIVs among different host species. Hemagglutinin clade 2.3.4.4 H5N8 viruses, which circulated in Eurasia during 2014 and 2015, did not reassort, but analysis after their subsequent dissemination during 2016 and 2017 revealed reassortment in all gene segments except NP and NS. Some virus lineages appeared to be unrelated to AIVs in wild bird populations in other regions, with maintenance of local AIVs in Georgia, whereas other lineages showed considerable genetic interrelationships with viruses circulating in other parts of Eurasia and Africa, despite relative undersampling in the area.IMPORTANCE Waterbirds (e.g., gulls and ducks) are natural reservoirs of avian influenza viruses (AIVs) and have been shown to mediate the dispersal of AIVs at intercontinental scales during seasonal migration. The segmented genome of influenza viruses enables viral RNA from different lineages to mix or reassort when two viruses infect the same host. Such reassortant viruses have been identified in most major human influenza pandemics and several poultry outbreaks. Despite their importance, we have only recently begun to understand AIV evolution and reassortment in their natural host reservoirs. This comprehensive study illustrates AIV evolutionary dynamics within a multihost ecosystem at a stopover site where three major migratory flyways intersect. Our analysis of this ecosystem over a 6-year period provides a snapshot of how these viruses are linked to global AIV populations. Understanding the evolution of AIVs in the natural host is imperative to mitigating both the risk of incursion into domestic poultry and the potential risk to mammalian hosts, including humans.