The roles and regulation of MDM2 and MDMX: it is not just about p53.

Most well studied as proteins that restrain the p53 tumor suppressor protein, MDM2 and MDMX have rich lives outside of their relationship to p53. There is much to learn about how these two proteins are regulated and how they can function in cells that lack p53. Regulation of MDM2 and MDMX, which takes place at the level of transcription, post-transcription, and protein modification, can be very intricate and is context-dependent. Equally complex are the myriad roles that these two proteins play in cells that lack wild-type p53; while many of these independent outcomes are consistent with oncogenic transformation, in some settings their functions could also be tumor suppressive. Since numerous small molecules that affect MDM2 and MDMX have been developed for therapeutic outcomes, most if not all designed to prevent their restraint of p53, it will be essential to understand how these diverse molecules might affect the p53-independent activities of MDM2 and MDMX.

MDM2 and MDMX promote ferroptosis by PPARα-mediated lipid remodeling.

MDM2 and MDMX, negative regulators of the tumor suppressor p53, can work separately and as a heteromeric complex to restrain p53's functions. MDM2 also has pro-oncogenic roles in cells, tissues, and animals that are independent of p53. There is less information available about p53-independent roles of MDMX or the MDM2-MDMX complex. We found that MDM2 and MDMX facilitate ferroptosis in cells with or without p53. Using small molecules, RNA interference reagents, and mutant forms of MDMX, we found that MDM2 and MDMX, likely working in part as a complex, normally facilitate ferroptotic death. We observed that MDM2 and MDMX alter the lipid profile of cells to favor ferroptosis. Inhibition of MDM2 or MDMX leads to increased levels of FSP1 protein and a consequent increase in the levels of coenzyme Q10, an endogenous lipophilic antioxidant. This suggests that MDM2 and MDMX normally prevent cells from mounting an adequate defense against lipid peroxidation and thereby promote ferroptosis. Moreover, we found that PPARα activity is essential for MDM2 and MDMX to promote ferroptosis, suggesting that the MDM2-MDMX complex regulates lipids through altering PPARα activity. These findings reveal the complexity of cellular responses to MDM2 and MDMX and suggest that MDM2-MDMX inhibition might be useful for preventing degenerative diseases involving ferroptosis. Furthermore, they suggest that MDM2/MDMX amplification may predict sensitivity of some cancers to ferroptosis inducers.

Antigenic Distance between North American Swine and Human Seasonal H3N2 Influenza A Viruses as an Indication of Zoonotic Risk to Humans.

Human-to-swine transmission of influenza A virus (IAV) repeatedly occurs, leading to sustained transmission and increased diversity in swine; human seasonal H3N2 introductions occurred in the 1990s and 2010s and were maintained in North American swine. Swine H3N2 strains were subsequently associated with zoonotic infections, highlighting the need to understand the risk of endemic swine IAV to humans. We quantified antigenic distances between swine H3N2 and human seasonal vaccine strains from 1973 to 2014 using a panel of monovalent antisera raised in pigs in hemagglutination inhibition (HI) assays. Swine H3N2 lineages retained the closest antigenic similarity to human vaccine strains from the decade of incursion. Swine lineages from the 1990s were antigenically more similar to human vaccine strains of the mid-1990s but had substantial distance from recent human vaccine strains. In contrast, lineages from the 2010s were closer to human vaccine strains from 2011 and 2014 and the most antigenically distant from human vaccine strains prior to 2007. HI assays using ferret antisera demonstrated that swine lineages from the 1990s and 2010s had significant fold reductions compared to the homologous HI titer of the nearest pandemic preparedness candidate vaccine virus (CVV) or seasonal vaccine strain. The assessment of postinfection and postvaccination human serum cohorts demonstrated limited cross-reactivity to swine H3N2 from the 1990s, especially in older adults born before the 1970s. We identified swine strains to which humans are likely to lack population immunity or are not protected against by a current human seasonal vaccine or CVV to use in prioritizing future human CVV strain selection. IMPORTANCE Human H3N2 influenza A viruses spread to pigs in North America in the 1990s and more recently in the 2010s. These cross-species events led to sustained circulation and increased H3N2 diversity in pig populations. The evolution of H3N2 in swine led to a reduced similarity to human seasonal H3N2 and the vaccine strains used to protect human populations. We quantified the antigenic phenotypes and found that North American swine H3N2 lineages retained more antigenic similarity to historical human vaccine strains from the decade of incursion but had substantial differences compared to recent human vaccine strains. Additionally, pandemic preparedness vaccine strains demonstrated a loss of similarity to contemporary swine strains. Finally, human sera revealed that although these adults had antibodies against human H3N2 strains, many had limited immunity to swine H3N2, especially older adults born before 1970. Antigenic assessment of swine H3N2 provides critical information for pandemic preparedness and candidate vaccine development.

Evolution of protein trafficking in kinetoplastid parasites: Complexity and pathogenesis.

The kinetoplastida and their close relatives are unicellular organisms prevalent within the biosphere and important for significant impacts on global health, economy and ecosystems. They are, under most models, an early branching lineage. Individual species adapted to highly diverse environments by adopting complex life styles; parasitic species can infect a wide range of eukaryotic hosts, while many relatives are free-living and some autotrophic from acquiring a plastid for photosynthesis. Adaptation is especially evident in the evolution of kinetoplastid cell surface architecture and is supported by endomembrane trafficking and serves as a platform for interaction with its environment. Here we summarize and discuss recent genomic and experimental studies of the protein trafficking system in kinetoplastids, with focus on the composition and function of the surface as well as mechanisms for constructing, maintaining and regulating the cell surface proteome. We hope this provides a broad view of how protein trafficking contributes to the intricate and dynamic host-parasite interfaces that are critical for successful environmental adaptation of this highly important lineage.

Evolution of the endomembrane systems of trypanosomatids - conservation and specialisation.

Parasite surfaces support multiple functions required for survival within their hosts, and maintenance and functionality of the surface depends on membrane trafficking. To understand the evolutionary history of trypanosomatid trafficking, where multiple lifestyles and mechanisms of host interactions are known, we examined protein families central to defining intracellular compartments and mediating transport, namely Rabs, SNAREs and RabGAPs, across all available Euglenozoa genomes. Bodonids possess a large trafficking repertoire, which is mainly retained by the Trypanosoma cruzi group, with extensive losses in other lineages, particularly African trypanosomes and phytomonads. There are no large-scale expansions or contractions from an inferred ancestor, excluding direct associations between parasitism or host range. However, we observe stepwise secondary losses within Rab and SNARE cohorts (but not RabGAPs). Major changes are associated with endosomal and late exocytic pathways, consistent with the diversity in surface proteomes between trypanosomatids and mechanisms of interaction with the host. Along with the conserved core family proteins, several lineage-specific members of the Rab (but not SNARE) family were found. Significantly, testing predictions of SNARE complex composition by proteomics confirms generalised retention of function across eukaryotes.

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.

Comparison of 2016-17 and Previous Epizootics of Highly Pathogenic Avian Influenza H5 Guangdong Lineage in Europe.

We analyzed the highly pathogenic avian influenza (HPAI) H5 epizootic of 2016-17 in Europe by epidemiologic and genetic characteristics and compared it with 2 previous epizootics caused by the same H5 Guangdong lineage. The 2016-17 epizootic was the largest in Europe by number of countries and farms affected and greatest diversity of wild birds infected. We observed significant differences among the 3 epizootics regarding region affected, epidemic curve, seasonality, and outbreak duration, making it difficult to predict future HPAI epizootics. However, we know that in 2005-06 and 2016-17 the initial peak of wild bird detections preceded the peak of poultry outbreaks within Europe. Phylogenetic analysis of 2016-17 viruses indicates 2 main pathways into Europe. Our findings highlight the need for global surveillance of viral changes to inform disease preparedness, detection, and control.

Unconventional T-cell recognition of an arthritogenic epitope of proteoglycan aggrecan released from degrading cartilage.

It has been proposed that peptide epitopes bind to MHC class II molecules to form distinct structural conformers of the same MHC II-peptide complex termed type A and type B, and that the two conformers of the same peptide-MHC II complex are recognized by distinct CD4 T cells, termed type A and type B T cells. Both types recognize short synthetic peptides but only type A recognize endosomally processed intact antigen. Type B T cells that recognize self peptides from exogenously degraded proteins have been shown to escape negative selection during thymic development and so have the potential to contribute to the pathogenesis of autoimmunity. We generated and characterized mouse CD4 T cells specific for an arthritogenic epitope of the candidate joint autoantigen proteoglycan aggrecan. Cloned T-cell hybridomas specific for a synthetic peptide containing the aggrecan epitope showed two distinct response patterns based on whether they could recognize processed intact aggrecan. Fine mapping demonstrated that both types of T-cell recognized the same core epitope. The results are consistent with the generation of aggrecan-specific type A and type B T cells. Type B T cells were activated by supernatants released from degrading cartilage, indicating the presence of antigenic extracellular peptides or fragments of aggrecan. Type B T cells could play a role in the pathogenesis of proteoglycan-induced arthritis in mice, a model for rheumatoid arthritis, by recognizing extracellular peptides or protein fragments of joint autoantigens released by inflamed cartilage.

Intermittent MEK inhibition with GITR co-stimulation rescues T-cell function for increased efficacy with CTLA-4 blockade in solid tumor models.

MEK inhibitors (MEKis) have shown limited success as a treatment for MAPK/ERK pathway-dependent cancers due to various resistance mechanisms tumor cells can employ. CH5126766 (CKI27) is an inhibitor that binds to MEK and prevents release of RAF, reducing the relief of negative feedback commonly observed with other MEKis. We observed that CKI27 increased MHC expression on tumor cells and improved T cell-mediated killing. Yet, CKI27 also decreased T-cell proliferation, activation, and cytolytic activity by inhibiting the MAPK/ERK pathway that is activated downstream of T cell-receptor signaling. Therefore, we aimed to balance the positive and negative immunomodulatory effects of MEKis for optimal combination with immunotherapy. Intermittent administration of CKI27 allowed T cells to partially recover and co-stimulation via GITR and OX-40 agonist antibodies completely alleviated inhibition of function. In Kras mutant lung and colon tumor mouse models, intermittent CKI27 and anti-GITR significantly decreased tumor growth and prolonged survival when further combined with CTLA-4 immune checkpoint blockade. Moreover, this triple combination increased CD8+ and CD4+ T-cell proliferation, activation, and effector/memory subsets in the tumor draining lymph nodes and tumors and led to intratumoral regulatory T cell (Treg) destabilization. These data, collectively, will allow for more informed decisions when optimizing combination regimens by overcoming resistance, reducing toxicity, and generating long-term immune responses.

APR-246 increases tumor antigenicity independent of p53.

We previously reported that activation of p53 by APR-246 reprograms tumor-associated macrophages to overcome immune checkpoint blockade resistance. Here, we demonstrate that APR-246 and its active moiety, methylene quinuclidinone (MQ) can enhance the immunogenicity of tumor cells directly. MQ treatment of murine B16F10 melanoma cells promoted activation of melanoma-specific CD8+ T cells and increased the efficacy of a tumor cell vaccine using MQ-treated cells even when the B16F10 cells lacked p53. We then designed a novel combination of APR-246 with the TLR-4 agonist, monophosphoryl lipid A, and a CD40 agonist to further enhance these immunogenic effects and demonstrated a significant antitumor response. We propose that the immunogenic effect of MQ can be linked to its thiol-reactive alkylating ability as we observed similar immunogenic effects with the broad-spectrum cysteine-reactive compound, iodoacetamide. Our results thus indicate that combination of APR-246 with immunomodulatory agents may elicit effective antitumor immune response irrespective of the tumor's p53 mutation status.

GLS2 Is a Tumor Suppressor and a Regulator of Ferroptosis in Hepatocellular Carcinoma.

Glutamine synthase 2 (GLS2) is a key regulator of glutaminolysis and has been previously implicated in activities consistent with tumor suppression. Here we generated Gls2 knockout (KO) mice that develop late-occurring B-cell lymphomas and hepatocellular carcinomas (HCC). Further, Gls2 KO mice subjected to the hepatocarcinogenic Stelic Animal Model (STAM) protocol produce larger HCC tumors than seen in wild-type (WT) mice. GLS2 has been shown to promote ferroptosis, a form of cell death characterized by iron-dependent accumulation of lipid peroxides. In line with this, GLS2 deficiency, either in cells derived from Gls2 KO mice or in human cancer cells depleted of GLS2, conferred significant resistance to ferroptosis. Mechanistically, GLS2, but not GLS1, increased lipid reactive oxygen species (ROS) production by facilitating the conversion of glutamate to α-ketoglutarate (αKG), thereby promoting ferroptosis. Ectopic expression of WT GLS2 in a human hepatic adenocarcinoma xenograft model significantly reduced tumor size; this effect was nullified by either expressing a catalytically inactive form of GLS2 or by blocking ferroptosis. Furthermore, analysis of cancer patient datasets supported a role for GLS2-mediated regulation of ferroptosis in human tumor suppression. These data suggest that GLS2 is a bona fide tumor suppressor and that its ability to favor ferroptosis by regulating glutaminolysis contributes to its tumor suppressive function. SIGNIFICANCE: This study demonstrates that the key regulator of glutaminolysis, GLS2, can limit HCC in vivo by promoting ferroptosis through αKG-dependent lipid ROS, which in turn might lay the foundation for a novel therapeutic approach.

Antigenic Characterization and Pandemic Risk Assessment of North American H1 Influenza A Viruses Circulating in Swine.

The first pandemic of the 21st century was caused by an H1N1 influenza A virus (IAV) introduced from pigs into humans, highlighting the importance of swine as reservoirs for pandemic viruses. Two major lineages of swine H1 circulate in North America: the 1A classical swine lineage (including that of the 2009 H1N1 pandemic) and the 1B human seasonal-like lineage. Here, we investigated the evolution of these H1 IAV lineages in North American swine and their potential pandemic risk. We assessed the antigenic distance between the HA of representative swine H1 and human seasonal vaccine strains (1978 to 2015) in hemagglutination inhibition (HI) assays using a panel of monovalent antisera raised in pigs. Antigenic cross-reactivity varied by strain but was associated with genetic distance. Generally, the swine 1A lineage viruses that seeded the 2009 H1 pandemic were antigenically most similar to the H1 pandemic vaccine strains, with the exception of viruses in the genetic clade 1A.1.1.3, which had a two-amino acid deletion mutation near the receptor-binding site, which dramatically reduced antibody recognition. The swine 1B lineage strains, which arose from previously circulating (pre-2009 pandemic) human seasonal viruses, were more antigenically similar to pre-2009 human seasonal H1 vaccine viruses than post-2009 strains. Human population immunity was measured by cross-reactivity in HI assays to representative swine H1 strains. There was a broad range of titers against each swine strain that was not associated with age, sex, or location. However, there was almost no cross-reactivity in human sera to the 1A.1.1.3 and 1B.2.1 genetic clades of swine viruses, and the 1A.1.1.3 and 1B.2.1 clades were also the most antigenically distant to the human vaccine strains. Our data demonstrate that the antigenic distances of representative swine strains from human vaccine strains represent an important part of the rational assessment of swine IAV for zoonotic risk research and pandemic preparedness prioritization. IMPORTANCE Human H1 influenza A viruses (IAV) spread to pigs in North America, resulting in a sustained circulation of two major groups of H1 viruses in swine. We quantified the genetic diversity of H1 in swine and measured antigenic phenotypes. We demonstrated that the swine H1 lineages were significantly different from the human vaccine strains and that this antigenic dissimilarity increased over time as the viruses evolved in swine. Pandemic preparedness vaccine strains for human vaccines also demonstrated a loss in similarity with contemporary swine strains. Human sera revealed a range of responses to swine IAV, including two groups of viruses with little to no immunity. The surveillance and risk assessment of IAV diversity in pig populations are essential to detect strains with reduced immunity in humans and provide critical information for pandemic preparedness.

Increased p53 expression induced by APR-246 reprograms tumor-associated macrophages to augment immune checkpoint blockade.

In addition to playing a major role in tumor cell biology, p53 generates a microenvironment that promotes antitumor immune surveillance via tumor-associated macrophages. We examined whether increasing p53 signaling in the tumor microenvironment influences antitumor T cell immunity. Our findings indicate that increased p53 signaling induced either pharmacologically with APR-246 (eprenetapopt) or in p53-overexpressing transgenic mice can disinhibit antitumor T cell immunity and augment the efficacy of immune checkpoint blockade. We demonstrated that increased p53 expression in tumor-associated macrophages induces canonical p53-associated functions such as senescence and activation of a p53-dependent senescence-associated secretory phenotype. This was linked with decreased expression of proteins associated with M2 polarization by tumor-associated macrophages. Our preclinical data led to the development of a clinical trial in patients with solid tumors combining APR-246 with pembrolizumab. Biospecimens from select patients participating in this ongoing trial showed that there was a suppression of M2-polarized myeloid cells and increase in T cell proliferation with therapy in those who responded to the therapy. Our findings, based on both genetic and a small molecule-based pharmacological approach, suggest that increasing p53 expression in tumor-associated macrophages reprograms the tumor microenvironment to augment the response to immune checkpoint blockade.

Calreticulin mutant myeloproliferative neoplasms induce MHC-I skewing, which can be overcome by an optimized peptide cancer vaccine.

The majority of JAK2V617F-negative myeloproliferative neoplasms (MPNs) have disease-initiating frameshift mutations in calreticulin (CALR), resulting in a common carboxyl-terminal mutant fragment (CALRMUT), representing an attractive source of neoantigens for cancer vaccines. However, studies have shown that CALRMUT-specific T cells are rare in patients with CALRMUT MPN for unknown reasons. We examined class I major histocompatibility complex (MHC-I) allele frequencies in patients with CALRMUT MPN from two independent cohorts. We observed that MHC-I alleles that present CALRMUT neoepitopes with high affinity are underrepresented in patients with CALRMUT MPN. We speculated that this was due to an increased chance of immune-mediated tumor rejection by individuals expressing one of these MHC-I alleles such that the disease never clinically manifested. As a consequence of this MHC-I allele restriction, we reasoned that patients with CALRMUT MPN would not efficiently respond to a CALRMUT fragment cancer vaccine but would when immunized with a modified CALRMUT heteroclitic peptide vaccine approach. We found that heteroclitic CALRMUT peptides specifically designed for the MHC-I alleles of patients with CALRMUT MPN efficiently elicited a CALRMUT cross-reactive CD8+ T cell response in human peripheral blood samples but not to the matched weakly immunogenic CALRMUT native peptides. We corroborated this effect in vivo in mice and observed that C57BL/6J mice can mount a CD8+ T cell response to the CALRMUT fragment upon immunization with a CALRMUT heteroclitic, but not native, peptide. Together, our data emphasize the therapeutic potential of heteroclitic peptide-based cancer vaccines in patients with CALRMUT MPN.

p53 Frameshift Mutations Couple Loss-of-Function with Unique Neomorphic Activities.

p53 mutations that result in loss of transcriptional activity are commonly found in numerous types of cancer. While the majority of these are missense mutations that map within the central DNA-binding domain, truncations and/or frameshift mutations can also occur due to various nucleotide substitutions, insertions, or deletions. These changes result in mRNAs containing premature stop codons that are translated into a diverse group of C-terminally truncated proteins. Here we characterized three p53 frameshift mutant proteins expressed from the endogenous TP53 locus in U2OS osteosarcoma and HCT116 colorectal cancer cell lines. These mutants retain intact DNA-binding domains but display altered oligomerization properties. Despite their abnormally high expression levels, they are mostly transcriptionally inactive and unable to initiate a stimuli-induced transcriptional program characteristic of wild-type p53. However, one of these variant p53 proteins, I332fs*14, which resembles naturally expressed TAp53 isoforms ß and γ, retains some residual antiproliferative activity and can induce cellular senescence in HCT116 cells. Cells expressing this mutant also display decreased motility in migration assays. Hence, this p53 variant exhibits a combination of loss-of-gain and gain-of-function characteristics, distinguishing it from both wild type p53 and p53 loss. IMPLICATIONS: p53 frameshift mutants display a mixture of residual antiproliferative and neomorphic functions that may be differentially exploited for targeted therapy.

Swine Influenza A Viruses and the Tangled Relationship with Humans.

Influenza A viruses (IAVs) are the causative agents of one of the most important viral respiratory diseases in pigs and humans. Human and swine IAV are prone to interspecies transmission, leading to regular incursions from human to pig and vice versa. This bidirectional transmission of IAV has heavily influenced the evolutionary history of IAV in both species. Transmission of distinct human seasonal lineages to pigs, followed by sustained within-host transmission and rapid adaptation and evolution, represent a considerable challenge for pig health and production. Consequently, although only subtypes of H1N1, H1N2, and H3N2 are endemic in swine around the world, extensive diversity can be found in the hemagglutinin (HA) and neuraminidase (NA) genes, as well as the remaining six genes. We review the complicated global epidemiology of IAV in swine and the inextricably entangled implications for public health and influenza pandemic planning.

p21 can be a barrier to ferroptosis independent of p53.

Traditionally, the p21 protein has been viewed as limiting cancer progression and promoting aging. In contrast, there are reports that p21 can enhance cancer survival and limit tissue damage, depending on the tissue of origin and type of stressor involved. Here, we provide evidence to support these latter two roles of p21 by exploring its ability to regulate ferroptosis. Ferroptosis is a form of cell death that is associated with certain degenerative diseases, some of which are aging-related. Our results reveal a correlation between p21 protein levels in cell lines that are resistant to ferroptosis (p21 high) versus cell lines that are sensitive and easily undergo ferroptosis (p21 low). We also show that p21 levels themselves are differentially regulated in response to ferroptosis in a p53-independent manner. Further, experimentally altering the abundance of p21 protein inverts the ferroptosis-sensitivity of both resistant and sensitive human cancer cell lines. Our data also indicate that the interaction of p21 with CDKs is crucial for its ability to restrict the progression of ferroptosis. While this study was performed in cancer cell lines, our results support the potential of p21 to aid in maintenance of healthy tissues by blocking the damage incurred due to ferroptosis.

Regional Transmission and Reassortment of 2.3.4.4b Highly Pathogenic Avian Influenza (HPAI) Viruses in Bulgarian Poultry 2017/18.

Between 2017 and 2018, several farms across Bulgaria reported outbreaks of H5 highly-pathogenic avian influenza (HPAI) viruses. In this study we used genomic and traditional epidemiological analyses to trace the origin and subsequent spread of these outbreaks within Bulgaria. Both methods indicate two separate incursions, one restricted to the northeastern region of Dobrich, and another largely restricted to Central and Eastern Bulgaria including places such as Plovdiv, Sliven and Stara Zagora, as well as one virus from the Western region of Vidin. Both outbreaks likely originate from different European 2.3.4.4b virus ancestors circulating in 2017. The viruses were likely introduced by wild birds or poultry trade links in 2017 and have continued to circulate, but due to lack of contemporaneous sampling and sequences from wild bird viruses in Bulgaria, the precise route and timing of introduction cannot be determined. Analysis of whole genomes indicates a complete lack of reassortment in all segments but the matrix protein gene (MP), which presents as multiple smaller clusters associated with different European 2.3.4.4b viruses. Ancestral reconstruction of host states of the hemagglutinin (HA) gene of viruses involved in the outbreaks suggests that transmission is driven by domestic ducks into galliform poultry. Thus, according to present evidence, we suggest the surveillance of domestic ducks as they are an epidemiologically relevant species for subclinical infection. Monitoring the spread due to movement between farms within regions and links to poultry production systems in European countries can help to predict and prevent future outbreaks. The 2.3.4.4b lineage which caused the largest recorded poultry epidemic in Europe continues to circulate, and the risk of further transmission by wild birds during migration remains.

Detection of H3N8 influenza A virus with multiple mammalian-adaptive mutations in a rescued Grey seal (Halichoerus grypus) pup.

Avian influenza A viruses (IAVs) in different species of seals display a spectrum of pathogenicity, from sub-clinical infection to mass mortality events. Here we present an investigation of avian IAV infection in a 3- to 4-month-old Grey seal (Halichoerus grypus) pup, rescued from St Michael's Mount, Cornwall in 2017. The pup underwent medical treatment but died after two weeks; post-mortem examination and histology indicated sepsis as the cause of death. IAV NP antigen was detected by immunohistochemistry in the nasal mucosa, and sensitive real-time reverse transcription polymerase chain reaction assays detected trace amounts of viral RNA within the lower respiratory tract, suggesting that the infection may have been cleared naturally. IAV prevalence among Grey seals may therefore be underestimated. Moreover, contact with humans during the rescue raised concerns about potential zoonotic risk. Nucleotide sequencing revealed the virus to be of subtype H3N8. Combining a GISAID database BLAST search and time-scaled phylogenetic analyses, we inferred that the seal virus originated from an unsampled, locally circulating (in Northern Europe) viruses, likely from wild Anseriformes. From examining the protein alignments, we found several residue changes in the seal virus that did not occur in the bird viruses, including D701N in the PB2 segment, a rare mutation, and a hallmark of mammalian adaptation of bird viruses. IAVs of H3N8 subtype have been noted for their particular ability to cross the species barrier and cause productive infections, including historical records suggesting that they may have caused the 1889 pandemic. Therefore, infections such as the one we report here may be of interest to pandemic surveillance and risk and help us better understand the determinants and drivers of mammalian adaptation in influenza.