Search

Summary Activating mutations in the cytosolic 5′-nucleotidase II gene NT5C2 drive resistance to 6-mercaptopurine in acute lymphoblastic leukemia. Here we demonstrate that constitutively active NT5C2 mutations K359Q and L375F reconfigure the catalytic center for substrate access and catalysis in the absence of allosteric activator. In contrast, most relapse-associated mutations, which involve the arm segment and residues along the surface of the inter-monomeric cavity, disrupt a built-in switch-off mechanism responsible for turning off NT5C2. In addition, we show that the C-terminal acidic tail lost in the Q523X mutation functions to restrain NT5C2 activation. These results uncover dynamic mechanisms of enzyme regulation targeted by chemotherapy resistance-driving NT5C2 mutations, with important implications for the development of NT5C2 inhibitor therapies.

Point mutation E1099K in MMSET/NSD2 enhances its methyltranferase activity and leads to altered global chromatin methylation in lymphoid malignancies

International audience; Numerous pathological amyloid proteins spread from cell to cell during neurodegenerative disease, facilitating the propagation of cellular pathology and disease progression. Understanding the mechanism by which disease-associated amyloid protein assemblies enter target cells and induce cellular dysfunction is, therefore, key to understanding the progressive nature of such neurodegenerative diseases. In this study, we utilized an imaging-based assay to monitor the ability of disease-associated amyloid assemblies to rupture intracellular vesicles following endocytosis. We observe that the ability to induce vesicle rupture is a common feature of α-synuclein (α-syn) assemblies, as assemblies derived from WT or familial disease-associated mutant α-syn all exhibited the ability to induce vesicle rupture. Similarly, different conformational strains of WT α-syn assemblies, but not monomeric or oligomeric forms, efficiently induced vesicle rupture following endocytosis. The ability to induce vesicle rupture was not specific to α-syn, as amyloid assemblies of tau and huntingtin Exon1 with pathologic polyglutamine repeats also exhibited the ability to induce vesicle rupture. We also observe that vesicles ruptured by α-syn are positive for the autophagic marker LC3 and can accumulate and fuse into large, intracellular structures resembling Lewy bodies in vitro. Finally, we show that the same markers of vesicle rupture surround Lewy bodies in brain sections from PD patients. These data underscore the importance of this conserved endocytic vesicle rupture event as a damaging mechanism of cellular invasion by amyloid assemblies of multiple neurodegenerative disease-associated proteins, and suggest that proteinaceous inclusions such as Lewy bodies form as a consequence of continued fusion of autophagic vesicles in cells unable to degrade ruptured vesicles and their amyloid contents.

Acute lymphoblastic leukemia (ALL) is an aggressive hematological tumor resulting from the malignant transformation of lymphoid progenitors. Despite intensive chemotherapy, 20% of pediatric patients and over 50% of adult patients with ALL do not achieve a complete remission or relapse after intensified chemotherapy, making disease relapse and resistance to therapy the most substantial challenge in the treatment of this disease. Using whole-exome sequencing, we identify mutations in the cytosolic 5'-nucleotidase II gene (NT5C2), which encodes a 5'-nucleotidase enzyme that is responsible for the inactivation of nucleoside-analog chemotherapy drugs, in 20/103 (19%) relapse T cell ALLs and 1/35 (3%) relapse B-precursor ALLs. NT5C2 mutant proteins show increased nucleotidase activity in vitro and conferred resistance to chemotherapy with 6-mercaptopurine and 6-thioguanine when expressed in ALL lymphoblasts. These results support a prominent role for activating mutations in NT5C2 and increased nucleoside-analog metabolism in disease progression and chemotherapy resistance in ALL.

Numerous pathologic amyloid proteins spread from cell to cell during neurodegenerative disease, facilitating the propagation of cellular pathology and disease progression in a prion-like fashion. Understanding the mechanism by which amyloid assemblies enter target cells and induce dysfunction is therefore key to understanding the progressive nature of diseases like Alzheimer's, Parkinson's, Huntington's, and chronic traumatic encephalopathy. In this study, we utilized an imaging-based assay to monitor the ability of amyloid assemblies to induce the rupture of intracellular vesicles following endocytosis, as well as to elucidate the cellular consequences of this damaging mechanism of invasion. We observe that induction of vesicle rupture is a conserved ability of fibrillar amyloid assemblies of alpha-synuclein, tau, and polyglutamine-rich huntingtin. Endocytic vesicle rupture potency is strongly influenced by strain conformation and is increased by assembly phosphorylation. Vesicles ruptured by alpha-synuclein can accumulate and fuse into large, intracellular structures resembling Lewy bodies in vitro, and the same markers of vesicle rupture surround Lewy bodies in brain sections from Parkinson's patients. Finally, ruptured vesicles containing alpha-synuclein can be observed in the extracellular environment and can be seen trafficking from cell to cell. These data underscore the importance of endocytic vesicle rupture as a conserved mechanism of cellular invasion by multiple disease-associated amyloid protein assemblies, implicate this process in the formation of proteinaceous inclusions such as Lewy bodies, and suggest that this form of cellular damage can serve as both a driving force and a vector for amyloid protein release and subsequent transmission to neighboring cells.

Drosophila Morgue is a unique ubiquitination protein that facilitates programmed cell death and associates with DIAP1, a critical cell death inhibitor with E3 ubiquitin ligase activity. Morgue possesses a unique combination of functional domains typically associated with distinct types of ubiquitination enzymes. This includes an F box characteristic of the substrate-binding subunit in Skp, Cullin, and F box (SCF)-type ubiquitin E3 ligase complexes and a variant ubiquitin E2 conjugase domain where the active site cysteine is replaced by a glycine. Morgue also contains a single C4-type zinc finger motif. This architecture suggests potentially novel ubiquitination activities for Morgue. In this study, we address the evolutionary origins of this distinctive protein utilizing a combination of bioinformatics and molecular biology approaches. We find that Morgue exhibits widespread but restricted phylogenetic distribution among metazoans. Morgue proteins were identified in a wide range of Protostome phyla, including Arthropoda, Annelida, Mollusca, Nematoda, and Platyhelminthes. However, with one potential exception, Morgue was not detected in Deuterostomes, including Chordates, Hemichordates, or Echinoderms. Morgue was also not found in Ctenophora, Cnidaria, Placozoa, or Porifera. Characterization of Morgue sequences within specific animal lineages suggests that gene deletion or acquisition has occurred during divergence of nematodes and that at least one arachnid expresses an atypical form of Morgue consisting only of the variant E2 conjugase domain. Analysis of the organization of several morgue genes suggests that exon-shuffling events have contributed to the evolution of the Morgue protein. These results suggest that Morgue mediates conserved and distinctive ubiquitination functions in specific cell death pathways.

Activating mutations in cytosolic 5'-nucleotidase II (NT5C2) drive resistance to 6-mercaptopurine (6-MP) in relapsed acute lymphoblastic leukemia (ALL). These gain-of function alleles encode proteins with increased nucleotidase activity, yet their specific molecular mechanisms are poorly understood. Allosteric activation of NT5C2 is primarily mediated by a marked conformational change in the helix A region, which reconfigures the catalytic center for substrate access and catalysis. Here we demonstrate that constitutively active NT5C2 mutant proteins (K359Q and L375F) adopt active helix A region structures in the absence of allosteric activator. Moreover, crystallographic, structural modeling, genetic, and functional antibody analyses identify a switch-off mechanism mediated by the dynamic mobilization of Asp407 in the tip region of the arm segment which, guided by positively charged residues located in the surface of a basic inter-monomeric cavity exposed upon allosteric activation, turns off NT5C2 by interacting with and disrupting the helical conformation of helix A. Loss of this regulatory mechanism in NT5C2 mutants involving the tip region of the arm segment and the inter-monomeric cavity drives increased nucleotidase activity and allosteric response. Finally, increased allosteric activation by the Q523X mutation results from loss of the NT5C2 C-terminal acidic tail segment, which promotes a closed inactive state in basal conditions. These results uncover dynamic mechanisms responsible for NT5C2 regulation targeted by chemotherapy resistance-driving NT5C2 mutations, with important implications for the development of NT5C2 inhibitor therapies. This abstract is also being presented as Poster 05. Citation Format: Chelsea L. Dieck, Gannie Tzoneva, Farhad Forouhar, Zachary Carpenter, Marta Sanchez-Martin, Alberto Ambesi-Impiombato, Renate Kirschner-Schwabe, Scott Lew, Jayaraman Seetharaman, Liang Tong, Adolfo Ferrando. Mechanisms of NT5C2 activating mutations driving thiopurine resistance in relapsed lymphoblastic leukemia [abstract]. In: Proceedings of the Second AACR Conference on Hematologic Malignancies: Translating Discoveries to Novel Therapies; May 6-9, 2017; Boston, MA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(24_Suppl):Abstract nr PR02.

Relapse acute lymphoblastic leukemia (ALL) is associated with chemotherapy resistance and poor prognosis. Persistent leukemia initiating cells with increased self-renewal capacity, clonal heterogeneity and selection of resistance-driving genetic alterations have been proposed as drivers of leukemia relapse. Gain of function mutations in the 5'-Nucleotidase, Cytosolic II (NT5C2) gene are selectively present in relapsed ALL and confer resistance to chemotherapy with 6-mercaptopurine (6-MP). Yet, the specific mechanisms mediating constitutive activation of NT5C2 remain unknown. Here we performed detailed genetic, enzymatic, structural and functional analyses of relapsed leukemia-associated NT5C2 mutant alleles uncovering previously unrecognized functional heterogeneity in their mechanisms of action. Specifically, detailed nucleotidase assays in the presence of increased concentration of allosteric activators across 10 relapse-associated NT5C2 mutant proteins and analyses of the crystal structures of 7 distinct mutant proteins and wild type NT5C2 in the presence and absence of allosteric factors defined three classes of NT5C2 mutants with distinct mechanisms of action. Class I NT5C2 mutations force an active configuration of the NT5C2 catalytic center in the absence of allosteric activator. In contrast, class II NT5C2 mutations disrupt a novel switch-off mechanism mediated by the dynamic mobilization of the tip region of the arm segment responsible for returning the enzyme to its basal inactive configuration following allosteric activation. Finally, increased allosteric response in the class III p.Gln523* truncating mutation results from loss of the C-terminal acidic tail of NT5C2, which functions as a lock stabilizing the enzyme in its inactive basal configuration. The proposed role of the arm domain region and the C-terminal segment were verified in enzymatic assays incubating wild type NT5C2 with antibodies recognizing these regulatory elements, which phenocopied the increased enzymatic activity and response to allosteric effector of NT5C2 mutations involving these domains. In all, these results identify distinct molecular mechanisms mediating NT5C2 regulation targeted by activating NT5C2 mutant alleles and serve as a framework for the functional annotation of clinically-relevant NT5C2 mutations in relapsed ALL. Finally, the prominent role of the allosteric binding site as critical mediator of the mechanisms mediating thhe increased nucleotidase activity of NT5C2 mutant proteins point to this region as a preferred target for the design of NT5C2 inhibitors for the reversal of chemotherapy resistance in relapsed ALL. Figure 1 Relapse associated NT5C2 mutations and crystal structure of basal and activated wild type human NT5C2. (a) Graphical representation of relapse associated mutations in B-ALL (black) and T-ALL (red).(b-d) In vitro nucleotidase assays assessing the enzymatic activity of (b)class I, (c) class II, and (d)class IIINT5C2 mutations in the presence of increased concentrations of ATP. (e) A Ribbon diagram of the basal structure of wild type NT5C2-X537, displaying the HAD domain (cyan) and its three extensions: N-terminal segment (orange), arm (pink), and C-terminal segment (marine). The mutation sites are depicted as red solid spheres for Cα of each mutated amino acid. The phosphate (yellow for phosphorus) and Mg ions (dark green) are depicted as stick models and solid spheres respectively. (f) A Ribbon diagram of the activated structure of the full-length wild type NT5C2, in which the allosteric helix A (αA) is shown in dark purple. (g,h) Ribbon and surface (for subunit B) depictions of basal and activated dimers of (g) NT5C2-X537 and the (h) full-length, respectively. (i,j) Ribbon diagrams of the basal (i) NT5C2-X537 and activated (j) full-length wild type tetramers, respectively. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

Peripheral T cell lymphomas (PTCLs) are a heterogeneous and poorly understood group of non-Hodgkin lymphomas. Here we combined whole-exome sequencing of 12 tumor-normal DNA pairs, RNA sequencing analysis and targeted deep sequencing to identify new genetic alterations in PTCL transformation. These analyses identified highly recurrent epigenetic factor mutations in TET2, DNMT3A and IDH2 as well as a new highly prevalent RHOA mutation encoding a p.Gly17Val alteration present in 22 of 35 (67%) angioimmunoblastic T cell lymphoma (AITL) samples and in 8 of 44 (18%) PTCL, not otherwise specified (PTCL-NOS) samples. Mechanistically, the RHOA Gly17Val protein interferes with RHOA signaling in biochemical and cellular assays, an effect potentially mediated by the sequestration of activated guanine-exchange factor (GEF) proteins. In addition, we describe new and recurrent, albeit less frequent, genetic defects including mutations in FYN, ATM, B2M and CD58 implicating SRC signaling, impaired DNA damage response and escape from immune surveillance mechanisms in the pathogenesis of PTCL.

Acute Lymphoblastic Leukemia (ALL) is the most common malignancy in children. Altogether 90% of pediatric ALL patients achieve a complete hematologic remission with current high dose combination chemotherapy and 80% of them remain leukemia free. However, the outcome for patients showing refractory disease or those whose leukemia relapses after an initial transient response remains disappointingly poor with cure rates of less than 40%. To investigate genetic drivers of relapse and resistance and explore the specific roles of clonal evolution in disease progression and relapse here we performed whole-exome sequence analysis of matched diagnosis, germline (remission) and relapse DNA samples in a panel of 55 pediatric ALL patients including 33 T-cell ALLs and 22 B-cell precursor ALLs. These analyses identified an average of 9 mutations present in diagnostic samples and 17 mutations in relapsed leukemia DNAs. Phylogenetic tree analysis for each of the 48 cases with optimal variant call parameters analyzing their clonal evolution dynamics during disease progression, combined with whole genome sequencing of targeted samples with low exonic mutation input, showed that branched evolution in which relapse clones contain some, but not all genetic lesions present in the major clone at diagnosis as the primary mechanism driving tumor progression and relapse present in 45/48 (94%) cases. In addition, and consistent with previous reports we identified the presence of chemotherapy associated mutations in NT5C2 (10/55), TP53 (3/55), CREBBP (4/55) and the NR3C1 glucocorticoid receptor gene (2/55). However, and most strikingly, 23/27 (85%) recurrently mutated genes in this series with mutations preferentially selected or retained at the time of relapse (mutation never lost in the relapse clone) were not implicated in relapse ALL before (HTR3A, MED12, USP9X, CACNA1H, ODZ3, AACS, SAMD4A, ANO5, PAPPA, NAALADL2, HIST3H2A, FZD7, TBX15, NEB, GREB1L, PLXNA4, SGK223, TSC1, PTPRG, FGF10, SYCP2, TRPM3 and EYS). A branched pattern of genetic evolution and the presence of recurrent mutations selected at relapse support that chemotherapy imposes a strong Darwinian genetic selection in leukemic cell populations. In this context it is worth noting that RAS-MAPK pathway activating mutations in NRAS, KRAS and PTPN11 were present in 24/55 (44%) cases in our series. Interestingly, some leukemias showed retention or emergence of RAS mutant clones at relapse, while in others, RAS mutant clones present at diagnosis were replaced by RAS wild type populations, supporting a role for both positive and negative selection evolutionary pressures in clonal evolution of RAS-mutant leukemia. Most notably, and in agreement with this hypothesis, inducible expression of mutant KRAS in human ALL lines demonstrate that oncogenic KRAS G12D induces methotrexate resistance, but also improves leukemia response to vincristine; a phenotype perfectly recapitulated in a isogenic ALL leukemia model generated from a conditional inducible Kras G12D knockin mice. Mechanistically, KRAS G12 expression induces MAPK dependent abrogation of methotrexate induced apoptosis. Moreover, Kras mutant tumors show enhanced G2/M cell cycle arrest and apoptosis upon spindle poisoning with vincristine, a phenotype linked with increased PLK phosphorylation and transcriptional down-regulation of mitotic genes. Finally clonal competition assays demonstrate that the differential response to methotrexate and vincristine in isogenic Kras wild type and Kras mutant ALL cells results in clonal dominance of Kras G12D populations in cultures treated with methotrexate, while Kras wild type cells are selected the context of vincristine treatment. In all these results show novel insight on the genetics and mechanisms of clonal selection, disease progression and relapse in ALL and demonstrate a previously unrecognized dual role of RAS mutations in chemotherapy response. Disclosures Loh: Abbvie: Research Funding; Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees.

The TLX1 and TLX3 transcription factor oncogenes have a key role in the pathogenesis of T cell acute lymphoblastic leukemia (T-ALL)(1,2). Here we used reverse engineering of global transcriptional networks to decipher the oncogenic regulatory circuit controlled by TLX1 and TLX3. This systems biology analysis defined T cell leukemia homeobox 1 (TLX1) and TLX3 as master regulators of an oncogenic transcriptional circuit governing T-ALL. Notably, a network structure analysis of this hierarchical network identified RUNX1 as a key mediator of the T-ALL induced by TLX1 and TLX3 and predicted a tumor-suppressor role for RUNX1 in T cell transformation. Consistent with these results, we identified recurrent somatic loss-of-function mutations in RUNX1 in human T-ALL. Overall, these results place TLX1 and TLX3 at the top of an oncogenic transcriptional network controlling leukemia development, show the power of network analyses to identify key elements in the regulatory circuits governing human cancer and identify RUNX1 as a tumor-suppressor gene in T-ALL.

Peripheral T-cell lymphomas (PTCLs) are a heterogeneous and poorly understood group of aggressive non Hodgkin lymphomas with poor prognosis. To gain further insight on the genetics and pathogenic mechanisms of aggressive PTCLs we performed whole exome sequencing of matched tumor and normal DNA samples from 12 PTCL patients including 6 PTCL not otherwise specified (PTCL-NOS) tumors, 3 angioimunoablastic (AITL) T-cell lymphomas, 2 nasal type NK-/T-cell lymphomas and one enteropathy-associated T-cell lymphoma (EATL). This analysis identified 288 candidate coding somatic mutations in 268 genes and a mean mutation load of 24 non synonymous mutations per sample (range 4 – 57). Among these we noted the presence of a recurrent heterozygous mutation in the RHOA small GTPase gene (RHOA G17V) present in two independent AITL samples and one PTCL NOS biopsy. Analysis of a broad and diverse panel of 126 PTCL samples identified the presence of the RHOA G17V allele in 32 samples with a high prevalence in AITL (24/36, 67%, P < 0.001) and PTCL NOS cases (8/44, 18%, P < 0.002). The RHOA protein belongs to the Rho family of small GTPases, a group of Ras-like proteins responsible for linking a variety of cell-surface receptors to different intracellular signaling proteins. As is the case for RAS and most other small GTPases, RHOA activation is mediated by guanine exchange factors (GEFs), which catalyze the switch of RHOA from an inactive GDP-bound to an active GTP-bound state. Thus, and to test the functional significance of the RHOA G17V mutation we analyzed the capacity of this mutant to load GTP. This analysis revealed that RHOA G17V fails to incorporate GTP in response to an activated GEF in vitro. Moreover, and consistent with its inability to bind GTP, RHOA G17V failed to interact with rhotekin, a RHOA effector protein that selectively interacts with the GTP-bound active form of RHOA. However and most notably, the lack of RHOA G17V activation is not the result of a defect in RHOA-GEF interaction as RHOA G17V pull down assays demonstrated effective binding of this mutant protein to activated GEF proteins in T-cells. Based on these results we proposed an inhibitory role for RHOA G17V via sequestration of active GEF proteins. Consistently, while forced activation of RHOA signaling by GFP-RHOA overexpression induced loss of adhesion and round cell morphology in HEK293T cells, transfection of GFP-RHOA-G17V induced increased elongation and cellular protrusions as result of RHOA inactivation. In addition, immunoflourescence analysis of actin stress fiber formation by RHOA demonstrated effective abrogation of RHOA mediated cytoskeleton remodeling in cells expressing RHOA G17V. Overall these results show novel insight on the genetic basis of PTCLs and demonstrate a prominent role for RHOA G17V in the pathogenesis of AITL via disruption of RHOA signaling. Disclosures: No relevant conflicts of interest to declare.

Peripheral T-cell lymphomas (PTCLs) are a heterogeneous group of aggressive non Hodgkin lymphomas with poor prognosis. Molecular and cytogenetic studies have shown a prominent role for kinase fusion oncogenes, mostly NPM-ALK, in ALK+ anaplastic large cell lymphomas (ALCLs) and ITK-SYK kinase in unspecified PTCLs. To gain further insight on the genetics and pathogenic mechanisms of aggressive PTCLs we performed an integrated mutation analysis using whole exome sequencing (n=12) and RNAseq (n=35) data. This analysis identified 288 candidate coding somatic mutations in 268 genes including known recurrent mutations in the TET2, DNMT3A and IDH2 epigenetic factor genes and pointed to the FYN kinase gene as a new recurrently mutated oncogene in T-cell transformation. The FYN tyrosine kinase is, with LCK, the predominant SRC family kinase found in T lymphocytes and plays an important role in T-cell activation upon T-cell receptor (TCR) stimulation. FYN mutations in PTCL included a FYN L174R mutation detected in one AITL patient sample, a FYN R176C allele recurrently found in two PTCL NOS cases and a FYN Y531H mutation present in a PTCL NOS sample. Notably, each of these alleles are predicted to specifically disrupt the inhibition of FYN kinase activity by the C terminal SRC kinase (CSK). Thus, structure analysis of FYN and FYN mutant proteins predicted that FYN L174R and, most prominently, FYN R176C and FYN Y531H can disrupt the inhibitory interaction of the FYN SH2 domain with the CSK-phosphorylated Y531. Consistently, pull down assays using GST-FYN-SH2 recombinant proteins and biotinylated C-terminal FYN peptides encompassing Y531 showed abrogation of the interaction between FYN-SH2 and P-Y531 in each of these mutants. In agreement with these results, expression of FYN L174R, FYN R176C and FYN Y531H resulted in increased levels of FYN activation. Moreover, CSK expression effectively inhibited wild type FYN, but failed to abrogate FYN L174R, FYN R176C or FYN Y531H activation. In contrast, pharmacologic kinase inhibition with dasatinib, a multikinase inhibitor which blocks ABL1 and SRC kinases, effectively abrogated the activity of FYN L174R, FYN R176C and FYN Y531H mutant proteins and suppressed the growth of cells transformed via expression of activated FYN mutant alleles. Overall these results support an oncogenic role for FYN activating mutations in the pathogenesis of PTCL and support a role for SRC kinase inhibitors for the treatment of this disease. Disclosures: No relevant conflicts of interest to declare.

Acute lymphoblastic leukemia (ALL) is an aggressive hematological tumor resulting from the malignant transformation of lymphoid progenitors. Despite intensive chemotherapy, 20% of pediatric and over 50% of adult ALL patients fail to achieve a complete remission or relapse after intensified chemotherapy, making disease relapse and resistance to therapy the most significant challenge in the treatment of this disease. Using whole exome sequencing, here we identify mutations in NT5C2, a 5’-nucleotidase enzyme responsible for inactivation of nucleoside analog chemotherapy drugs, in 20/103 (19%) relapse T-ALLs and in 1/35 (3%) relapse B-precursor ALLs analyzed. NT5C2 mutant proteins show increased nucleotidase activity in vitro and conferred resistance to chemotherapy with 6-mercaptopurine and 6-thioguanine when expressed in ALL lymphoblasts. These results support a prominent role for activating mutations in NT5C2 and increased nucleoside analog metabolism in disease progression and chemotherapy resistance in ALL. Citation Format: Gannie Tzoneva, Arianne Perez-Garcia, Zachary Carpenter, Hossein Khiabanian, Valeria Tosello, Maddalena Allegretta, Elisabeth Paietta, Janis Racevkis, Jakob M. Rowe, Martin S. Tallman, Maddalena Paganin, Jana Hoff, Renate Kirschner-Schwabe, Teresa Palomero, Raul Rabadan, Adolfo Ferrando. Activating mutations in the NT5C2 nucleotidase gene drive chemotherapy resistance in relapsed ALL. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4595. doi:10.1158/1538-7445.AM2013-4595

From September to December 2011, 162 New England harbor seals died in an outbreak of pneumonia. Sequence analysis of postmortem samples revealed the presence of an avian H3N8 influenza A virus, similar to a virus circulating in North American waterfowl since at least 2002 but with mutations that indicate recent adaption to mammalian hosts. These include a D701N mutation in the viral PB2 protein, previously reported in highly pathogenic H5N1 avian influenza viruses infecting people. Lectin staining and agglutination assays indicated the presence of the avian-preferred SAα-2,3 and mammalian SAα-2,6 receptors in seal respiratory tract, and the ability of the virus to agglutinate erythrocytes bearing either the SAα-2,3 or the SAα-2,6 receptor. The emergence of this A/harbor seal/Massachusetts/1/2011 virus may herald the appearance of an H3N8 influenza clade with potential for persistence and cross-species transmission., IMPORTANCE The emergence of new strains of influenza virus is always of great public concern, especially when the infection of a new mammalian host has the potential to result in a widespread outbreak of disease. Here we report the emergence of an avian influenza virus (H3N8) in New England harbor seals which caused an outbreak of pneumonia and contributed to a U.S. federally recognized unusual mortality event (UME). This outbreak is particularly significant, not only because of the disease it caused in seals but also because the virus has naturally acquired mutations that are known to increase transmissibility and virulence in mammals. Monitoring the spillover and adaptation of avian viruses in mammalian species is critically important if we are to understand the factors that lead to both epizootic and zoonotic emergence.

Background: Clonal expansion is a process in which a single organism reproduces asexually, giving rise to a diversifying population. It is pervasive in nature, from within-host pathogen evolution to emergent infectious disease outbreaks. Standard phylogenetic tools rely on full-length genomes of individual pathogens or population consensus sequences (phased genotypes). Although high-throughput sequencing technologies are able to sample population diversity, the short sequence reads inherent to them preclude assessing whether two reads originate from the same clone (unphased genotypes). This obstacle severely limits the application of phylogenetic methods and investigation of within-host dynamics of acute infections using this rich data source. Methods: We introduce two measures of diversity to study the evolution of clonal populations using unphased genomic data, which eliminate the need to construct full-length genomes. Our method follows a maximum likelihood approach to estimate evolutionary rates and times to the most recent common ancestor, based on a relaxed molecular clock model; independent of a growth model. Deviations from neutral evolution indicate the presence of selection and bottleneck events. Results: We evaluated our methods in silico and then compared it against existing approaches with the well-characterized 2009 H1N1 influenza pandemic. We then applied our method to high-throughput genomic data from marburgvirus-infected non-human primates and inferred the time of infection and the intra-host evolutionary rate, and identified purifying selection in viral populations. Conclusions: Our method has the power to make use of minor variants present in less than 1% of the population and capture genomic diversification within days of infection, making it an ideal tool for the study of acute RNA viral infection dynamics.