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1.
Dev Cell ; 49(2): 279-292.e5, 2019 Apr 22.
Artigo em Inglês | MEDLINE | ID: mdl-31014480

RESUMO

The correct assignment of cell fate within fields of multipotent progenitors is essential for accurate tissue diversification. The first lymphatic vessels arise from pre-existing veins after venous endothelial cells become specified as lymphatic progenitors. Prox1 specifies lymphatic fate and labels these progenitors; however, the mechanisms restricting Prox1 expression and limiting the progenitor pool remain unknown. We identified a zebrafish mutant that displayed premature, expanded, and prolonged lymphatic specification. The gene responsible encodes the regulator of alternative splicing, Nova2. In zebrafish and human endothelial cells, Nova2 selectively regulates pre-mRNA splicing for components of signaling pathways and phosphoproteins. Nova2-deficient endothelial cells display increased Mapk/Erk signaling, and Prox1 expression is dynamically controlled by Erk signaling. We identify a mechanism whereby Nova2-regulated splicing constrains Erk signaling, thus limiting lymphatic progenitor cell specification. This identifies the capacity of a factor that tunes mRNA splicing to control assignment of cell fate during vascular differentiation.

2.
Dev Dyn ; 248(4): 284-295, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30801852

RESUMO

BACKGROUND: During heart morphogenesis, the cardiac chambers undergo ballooning: a process involving regionalized elongation of cardiomyocytes. Cardiomyocyte shape changes require reorganization of the actin cytoskeleton; however, the genetic regulation of this process is not well understood. RESULTS: From a forward genetic screen, we identified the zebrafish uq 23ks mutant which manifests chamber ballooning defects. Whole-genome sequencing-mapping identified a truncating mutation in the gene, myo5b. myo5b encodes an atypical myosin required for endosome recycling and, consistent with this, increased vesicles were observed in myo5b mutant cardiomyocytes. Expression of RFP-Rab11a (a recycling endosome marker) confirmed increased recycling endosomes in cardiomyocytes of myo5b mutants. To investigate potential cargo of MyoVb-associated vesicles, we examined the adherens junction protein, N-cadherin. N-cadherin appeared mispatterned at cell junctions, and an increase in the number of intracellular particles was also apparent. Co-localization with RFP-Rab11a confirmed increased N-cadherin-positive recycling endosomes, demonstrating N-cadherin trafficking is perturbed in myo5b mutants. Finally, phalloidin staining showed disorganized F-actin in myo5b cardiomyocytes, suggesting the cytoskeleton fails to remodel, obstructing chamber ballooning. CONCLUSIONS: MyoVb is required for cardiomyocyte endosomal recycling and appropriate N-cadherin localization during the onset of chamber ballooning. Cardiomyocytes lacking MyoVb are unable to reorganize their actin cytoskeleton, resulting in failed chamber ballooning. Developmental Dynamics 248:284-295, 2019. © 2019 Wiley Periodicals, Inc.

3.
RNA ; 24(4): 597-608, 2018 04.
Artigo em Inglês | MEDLINE | ID: mdl-29246928

RESUMO

MicroRNAs (miRNAs) are highly conserved ∼22 nt small noncoding RNAs that bind partially complementary sequences in target transcripts. MicroRNAs regulate both translation and transcript stability, and play important roles in development, cellular homeostasis, and disease. There are limited approaches available to agnostically identify microRNA targets transcriptome-wide, and methods using miRNA mimics, which in principle identify direct miRNA:transcript pairs, have low sensitivity and specificity. Here, we describe a novel method to identify microRNA targets using miR-29b mimics containing 3-cyanovinylcarbazole (CNVK), a photolabile nucleoside analog. We demonstrate that biotin-tagged, CNVK-containing miR-29b (CNVK-miR-29b) mimics are nontoxic in cell culture, associate with endogenous mammalian Argonaute2, are sensitive for known targets and recapitulate endogenous transcript destabilization. Partnering CNVK-miR-29b with ultra-low-input RNA sequencing, we recover ∼40% of known miR-29b targets and find conservation of the focal adhesion and apoptotic target pathways in mouse and human. We also identify hundreds of novel targets, including NRAS, HOXA10, and KLF11, with a validation rate of 71% for a subset of 73 novel target transcripts interrogated using a high-throughput luciferase assay. Consistent with previous reports, we show that both endogenous miR-29b and CNVK-miR-29b are trafficked to the nucleus, but find no evidence of nuclear-specific miR-29b transcript binding. This may indicate that miR-29b nuclear sequestration is a regulatory mechanism in itself. We suggest that CNVK-containing small RNA mimics may find applicability in other experimental models.


Assuntos
Carbazóis/química , MicroRNAs/metabolismo , Nitrilos/química , RNA Antissenso/genética , Compostos de Vinila/química , Animais , Apoptose/fisiologia , Proteínas Argonauta/química , Caenorhabditis elegans/genética , Proteínas de Ciclo Celular/química , Adesões Focais/metabolismo , GTP Fosfo-Hidrolases/química , Proteínas de Homeodomínio/química , Humanos , Proteínas de Membrana/química , Camundongos , MicroRNAs/química , Proteínas Repressoras/química
4.
Biol Open ; 6(1): 125-131, 2017 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-27895053

RESUMO

The advent of genome editing has significantly altered genetic research, including research using the zebrafish model. To better understand the selectivity of the commonly used CRISPR/Cas9 system, we investigated single base pair mismatches in target sites and examined how they affect genome editing in the zebrafish model. Using two different zebrafish strains that have been deep sequenced, CRISPR/Cas9 target sites containing polymorphisms between the two strains were identified. These strains were crossed (creating heterozygotes at polymorphic sites) and CRISPR/Cas9 complexes that perfectly complement one strain injected. Sequencing of targeted sites showed biased, allele-specific editing for the perfectly complementary sequence in the majority of cases (14/19). To test utility, we examined whether phenotypes generated by F0 injection could be internally controlled with such polymorphisms. Targeting of genes bmp7a and chordin showed reduction in the frequency of phenotypes in injected 'heterozygotes' compared with injecting the strain with perfect complementarity. Next, injecting CRISPR/Cas9 complexes targeting two separate sites created deletions, but deletions were biased to selected chromosomes when one CRISPR/Cas9 target contained a polymorphism. Finally, integration of loxP sequences occurred preferentially in alleles with perfect complementarity. These experiments demonstrate that single nucleotide polymorphisms (SNPs) present throughout the genome can be utilised to increase the efficiency of in cis genome editing using CRISPR/Cas9 in the zebrafish model.

5.
Toxins (Basel) ; 8(10)2016 10 18.
Artigo em Inglês | MEDLINE | ID: mdl-27763551

RESUMO

Millions of years of evolution have fine-tuned the ability of venom peptides to rapidly incapacitate both prey and potential predators. Toxicofera reptiles are characterized by serous-secreting mandibular or maxillary glands with heightened levels of protein expression. These glands are the core anatomical components of the toxicoferan venom system, which exists in myriad points along an evolutionary continuum. Neofunctionalisation of toxins is facilitated by positive selection at functional hotspots on the ancestral protein and venom proteins have undergone dynamic diversification in helodermatid and varanid lizards as well as advanced snakes. A spectacular point on the venom system continuum is the long-glanded blue coral snake (Calliophis bivirgatus), a specialist feeder that preys on fast moving, venomous snakes which have both a high likelihood of prey escape but also represent significant danger to the predator itself. The maxillary venom glands of C. bivirgatus extend one quarter of the snake's body length and nestle within the rib cavity. Despite the snake's notoriety its venom has remained largely unstudied. Here we show that the venom uniquely produces spastic paralysis, in contrast to the flaccid paralysis typically produced by neurotoxic snake venoms. The toxin responsible, which we have called calliotoxin (δ-elapitoxin-Cb1a), is a three-finger toxin (3FTx). Calliotoxin shifts the voltage-dependence of NaV1.4 activation to more hyperpolarised potentials, inhibits inactivation, and produces large ramp currents, consistent with its profound effects on contractile force in an isolated skeletal muscle preparation. Voltage-gated sodium channels (NaV) are a particularly attractive pharmacological target as they are involved in almost all physiological processes including action potential generation and conduction. Accordingly, venom peptides that interfere with NaV function provide a key defensive and predatory advantage to a range of invertebrate venomous species including cone snails, scorpions, spiders, and anemones. Enhanced activation or delayed inactivation of sodium channels by toxins is associated with the extremely rapid onset of tetanic/excitatory paralysis in envenomed prey animals. A strong selection pressure exists for the evolution of such toxins where there is a high chance of prey escape. However, despite their prevalence in other venomous species, toxins causing delay of sodium channel inhibition have never previously been described in vertebrate venoms. Here we show that NaV modulators, convergent with those of invertebrates, have evolved in the venom of the long-glanded coral snake. Calliotoxin represents a functionally novel class of 3FTx and a structurally novel class of NaV toxins that will provide significant insights into the pharmacology and physiology of NaV. The toxin represents a remarkable case of functional convergence between invertebrate and vertebrate venom systems in response to similar selection pressures. These results underscore the dynamic evolution of the Toxicofera reptile system and reinforces the value of using evolution as a roadmap for biodiscovery.


Assuntos
Venenos Elapídicos/farmacologia , Elapidae , Canal de Sódio Disparado por Voltagem NAV1.4/fisiologia , Neurotoxinas/farmacologia , Agonistas do Canal de Sódio Disparado por Voltagem/farmacologia , Animais , Linhagem Celular Tumoral , Galinhas , Venenos Elapídicos/toxicidade , Células HEK293 , Humanos , Masculino , Músculo Esquelético/efeitos dos fármacos , Músculo Esquelético/inervação , Músculo Esquelético/fisiologia , Neurotoxinas/toxicidade , Agonistas do Canal de Sódio Disparado por Voltagem/toxicidade
6.
JCI Insight ; 1(8): e86914, 2016 06 02.
Artigo em Inglês | MEDLINE | ID: mdl-27699269

RESUMO

Infections are an important cause of morbidity and mortality in patients with decompensated cirrhosis and ascites. Hypothesizing that innate immune dysfunction contributes to susceptibility to infection, we assessed ascitic fluid macrophage phenotype and function. The expression of complement receptor of the immunoglobulin superfamily (CRIg) and CCR2 defined two phenotypically and functionally distinct peritoneal macrophage subpopulations. The proportion of CRIghi macrophages differed between patients and in the same patient over time, and a high proportion of CRIghi macrophages was associated with reduced disease severity (model for end-stage liver disease) score. As compared with CRIglo macrophages, CRIghi macrophages were highly phagocytic and displayed enhanced antimicrobial effector activity. Transcriptional profiling by RNA sequencing and comparison with human macrophage and murine peritoneal macrophage expression signatures highlighted similarities among CRIghi cells, human macrophages, and mouse F4/80hi resident peritoneal macrophages and among CRIglo macrophages, human monocytes, and mouse F4/80lo monocyte-derived peritoneal macrophages. These data suggest that CRIghi and CRIglo macrophages may represent a tissue-resident population and a monocyte-derived population, respectively. In conclusion, ascites fluid macrophage subset distribution and phagocytic capacity is highly variable among patients with chronic liver disease. Regulating the numbers and/or functions of these macrophage populations could provide therapeutic opportunities in cirrhotic patients.


Assuntos
Ascite/fisiopatologia , Cirrose Hepática/fisiopatologia , Macrófagos Peritoneais/citologia , Receptores de Complemento 3b/metabolismo , Idoso , Animais , Modelos Animais de Doenças , Feminino , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Pessoa de Meia-Idade , Receptores de Complemento
7.
Ann Neurol ; 79(6): 1031-1037, 2016 06.
Artigo em Inglês | MEDLINE | ID: mdl-27159321

RESUMO

Here we report whole exome sequencing (WES) on a cohort of 71 patients with persistently unresolved white matter abnormalities with a suspected diagnosis of leukodystrophy or genetic leukoencephalopathy. WES analyses were performed on trio, or greater, family groups. Diagnostic pathogenic variants were identified in 35% (25 of 71) of patients. Potentially pathogenic variants were identified in clinically relevant genes in a further 7% (5 of 71) of cases, giving a total yield of clinical diagnoses in 42% of individuals. These findings provide evidence that WES can substantially decrease the number of unresolved white matter cases. Ann Neurol 2016;79:1031-1037.


Assuntos
Análise Mutacional de DNA , Exoma/genética , Leucoencefalopatias/diagnóstico , Leucoencefalopatias/genética , Substância Branca/patologia , Adolescente , Adulto , Criança , Pré-Escolar , Feminino , Humanos , Leucoencefalopatias/patologia , Masculino , Mutação , Adulto Jovem
8.
Mol Pain ; 122016.
Artigo em Inglês | MEDLINE | ID: mdl-27130590

RESUMO

BACKGROUND: Dorsal root ganglion neuron-derived immortal cell lines including ND7/23 and F-11 cells have been used extensively as in vitro model systems of native peripheral sensory neurons. However, while it is clear that some sensory neuron-specific receptors and ion channels are present in these cell lines, a systematic comparison of the molecular targets expressed by these cell lines with those expressed in intact peripheral neurons is lacking. RESULTS: In this study, we examined the expression of RNA transcripts in the human neuroblastoma-derived cell line, SH-SY5Y, and two dorsal root ganglion hybridoma cell lines, F-11 and ND7/23, using Illumina next-generation sequencing, and compared the results with native whole murine dorsal root ganglions. The gene expression profiles of these three cell lines did not resemble any specific defined dorsal root ganglion subclass. The cell lines lacked many markers for nociceptive sensory neurons, such as the Transient receptor potential V1 gene, but expressed markers for both myelinated and unmyelinated neurons. Global gene ontology analysis on whole dorsal root ganglions and cell lines showed similar enrichment of biological process terms across all samples. CONCLUSIONS: This paper provides insights into the receptor repertoire expressed in common dorsal root ganglion neuron-derived cell lines compared with whole murine dorsal root ganglions, and illustrates the limits and potentials of these cell lines as tools for neuropharmacological exploration.


Assuntos
Gânglios Espinais/metabolismo , Modelos Biológicos , Neurônios/metabolismo , Transcriptoma/genética , Animais , Biomarcadores/metabolismo , Cálcio/metabolismo , Linhagem Celular , Gânglios Espinais/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Ontologia Genética , Células HEK293 , Humanos , Canais Iônicos/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Neurônios/efeitos dos fármacos , Ocitocina/farmacologia , Receptores Acoplados a Proteínas-G/genética , Receptores Acoplados a Proteínas-G/metabolismo , Transcriptoma/efeitos dos fármacos , Canais de Receptores Transientes de Potencial/metabolismo , Vasopressinas/farmacologia
10.
Nature ; 526(7574): 564-8, 2015 Oct 22.
Artigo em Inglês | MEDLINE | ID: mdl-26444236

RESUMO

The human kidney contains up to 2 million epithelial nephrons responsible for blood filtration. Regenerating the kidney requires the induction of the more than 20 distinct cell types required for excretion and the regulation of pH, and electrolyte and fluid balance. We have previously described the simultaneous induction of progenitors for both collecting duct and nephrons via the directed differentiation of human pluripotent stem cells. Paradoxically, although both are of intermediate mesoderm in origin, collecting duct and nephrons have distinct temporospatial origins. Here we identify the developmental mechanism regulating the preferential induction of collecting duct versus kidney mesenchyme progenitors. Using this knowledge, we have generated kidney organoids that contain nephrons associated with a collecting duct network surrounded by renal interstitium and endothelial cells. Within these organoids, individual nephrons segment into distal and proximal tubules, early loops of Henle, and glomeruli containing podocytes elaborating foot processes and undergoing vascularization. When transcription profiles of kidney organoids were compared to human fetal tissues, they showed highest congruence with first trimester human kidney. Furthermore, the proximal tubules endocytose dextran and differentially apoptose in response to cisplatin, a nephrotoxicant. Such kidney organoids represent powerful models of the human organ for future applications, including nephrotoxicity screening, disease modelling and as a source of cells for therapy.


Assuntos
Linhagem da Célula , Células-Tronco Pluripotentes Induzidas/citologia , Modelos Biológicos , Néfrons/citologia , Néfrons/embriologia , Organogênese , Organoides/citologia , Animais , Técnicas de Cocultura , Células Alimentadoras , Feto/anatomia & histologia , Feto/citologia , Feto/embriologia , Fibroblastos/citologia , Humanos , Túbulos Renais Coletores/citologia , Túbulos Renais Proximais/citologia , Túbulos Renais Proximais/embriologia , Túbulos Renais Proximais/fisiologia , Mesoderma/citologia , Camundongos , Néfrons/anatomia & histologia , Néfrons/fisiologia , Organoides/embriologia , Técnicas de Cultura de Tecidos
11.
Genes Dev ; 29(15): 1618-30, 2015 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-26253536

RESUMO

The lymphatic vasculature plays roles in tissue fluid balance, immune cell trafficking, fatty acid absorption, cancer metastasis, and cardiovascular disease. Lymphatic vessels form by lymphangiogenesis, the sprouting of new lymphatics from pre-existing vessels, in both development and disease contexts. The apical signaling pathway in lymphangiogenesis is the VEGFC/VEGFR3 pathway, yet how signaling controls cellular transcriptional output remains unknown. We used a forward genetic screen in zebrafish to identify the transcription factor mafba as essential for lymphatic vessel development. We found that mafba is required for the migration of lymphatic precursors after their initial sprouting from the posterior cardinal vein. mafba expression is enriched in sprouts emerging from veins, and we show that mafba functions cell-autonomously during lymphatic vessel development. Mechanistically, Vegfc signaling increases mafba expression to control downstream transcription, and this regulatory relationship is dependent on the activity of SoxF transcription factors, which are essential for mafba expression in venous endothelium. Here we identify an indispensable Vegfc-SoxF-Mafba pathway in lymphatic development.


Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Linfangiogênese/genética , Vasos Linfáticos/embriologia , Fator de Transcrição MafB/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Transdução de Sinais , Fator C de Crescimento do Endotélio Vascular/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Animais , Movimento Celular/genética , Embrião não Mamífero , Fator de Transcrição MafB/genética , Mutação , Proteínas do Tecido Nervoso/genética , Receptor 3 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Peixe-Zebra/embriologia , Proteínas de Peixe-Zebra/genética
13.
Nat Genet ; 47(1): 73-7, 2015 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-25420144

RESUMO

Temple-Baraitser syndrome (TBS) is a multisystem developmental disorder characterized by intellectual disability, epilepsy, and hypoplasia or aplasia of the nails of the thumb and great toe. Here we report damaging de novo mutations in KCNH1 (encoding a protein called ether à go-go, EAG1 or KV10.1), a voltage-gated potassium channel that is predominantly expressed in the central nervous system (CNS), in six individuals with TBS. Characterization of the mutant channels in both Xenopus laevis oocytes and human HEK293T cells showed a decreased threshold of activation and delayed deactivation, demonstrating that TBS-associated KCNH1 mutations lead to deleterious gain of function. Consistent with this result, we find that two mothers of children with TBS, who have epilepsy but are otherwise healthy, are low-level (10% and 27%) mosaic carriers of pathogenic KCNH1 mutations. Consistent with recent reports, this finding demonstrates that the etiology of many unresolved CNS disorders, including epilepsies, might be explained by pathogenic mosaic mutations.


Assuntos
Epilepsia/genética , Canais de Potássio Éter-A-Go-Go/genética , Hallux/anormalidades , Deficiência Intelectual/genética , Mutação de Sentido Incorreto , Unhas Malformadas/genética , Polegar/anormalidades , Sequência de Aminoácidos , Animais , Criança , Pré-Escolar , Sequência Conservada , Canais de Potássio Éter-A-Go-Go/química , Canais de Potássio Éter-A-Go-Go/fisiologia , Éxons/genética , Feminino , Células HEK293 , Humanos , Lactente , Masculino , Dados de Sequência Molecular , Mosaicismo , Oócitos , Conformação Proteica , Proteínas Recombinantes de Fusão/metabolismo , Homologia de Sequência de Aminoácidos , Xenopus laevis
14.
J Virol ; 87(8): 4768-71, 2013 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-23388708

RESUMO

Influenza A viruses are characterized by their ability to evade host immunity, even in vaccinated individuals. To determine how prior immunity shapes viral diversity in vivo, we studied the intra- and interhost evolution of equine influenza virus in vaccinated horses. Although the level and structure of genetic diversity were similar to those in naïve horses, intrahost bottlenecks may be more stringent in vaccinated animals, and mutations shared among horses often fall close to putative antigenic sites.


Assuntos
Evolução Molecular , Doenças dos Cavalos/prevenção & controle , Vírus da Influenza A Subtipo H3N8/genética , Vírus da Influenza A Subtipo H3N8/imunologia , Infecções por Orthomyxoviridae/veterinária , RNA Viral/genética , Animais , Variação Genética , Doenças dos Cavalos/imunologia , Doenças dos Cavalos/virologia , Cavalos , Vírus da Influenza A Subtipo H3N8/isolamento & purificação , Dados de Sequência Molecular , Infecções por Orthomyxoviridae/imunologia , Infecções por Orthomyxoviridae/prevenção & controle , Infecções por Orthomyxoviridae/virologia , Seleção Genética , Análise de Sequência de DNA
15.
Retrovirology ; 10: 8, 2013 Jan 18.
Artigo em Inglês | MEDLINE | ID: mdl-23331949

RESUMO

BACKGROUND: Dynamic changes in Human Immunodeficiency Virus 1 (HIV-1) sequence diversity and divergence are associated with immune control during primary infection and progression to AIDS. Consensus sequencing or single genome amplification sequencing of the HIV-1 envelope (env) gene, in particular the variable (V) regions, is used as a marker for HIV-1 genome diversity, but population diversity is only minimally, or semi-quantitatively sampled using these methods. RESULTS: Here we use second generation deep sequencing to determine inter-and intra-patient sequence heterogeneity and to quantify minor variants in a cohort of individuals either receiving or not receiving antiretroviral treatment following seroconversion; the SPARTAC trial. We show, through a cross-sectional study of sequence diversity of the env V3 in 30 antiretroviral-naive patients during primary infection that considerable population structure diversity exists, with some individuals exhibiting highly constrained plasma virus diversity. Diversity was independent of clinical markers (viral load, time from seroconversion, CD4 cell count) of infection. Serial sampling over 60 weeks of non-treated individuals that define three initially different diversity profiles showed that complex patterns of continuing HIV-1 sequence diversification and divergence could be readily detected. Evidence for minor sequence turnover, emergence of new variants and re-emergence of archived variants could be inferred from this analysis. Analysis of viral divergence over the same time period in patients who received short (12 weeks, ART12) or long course antiretroviral therapy (48 weeks, ART48) and a non-treated control group revealed that ART48 successfully suppressed viral divergence while ART12 did not have a significant effect. CONCLUSIONS: Deep sequencing is a sensitive and reliable method for investigating the diversity of the env V3 as an important component of HIV-1 genome diversity. Detailed insights into the complex early intra-patient dynamics of env V3 diversity and divergence were explored in antiretroviral-naïve recent seroconverters. Long course antiretroviral therapy, initiated soon after seroconversion and administered for 48 weeks, restricts HIV-1 divergence significantly. The effect of ART12 and ART48 on clinical markers of HIV infection and progression is currently investigated in the SPARTAC trial.


Assuntos
Terapia Antirretroviral de Alta Atividade , Genes env , Variação Genética , Anticorpos Anti-HIV/sangue , Proteína gp120 do Envelope de HIV/genética , Infecções por HIV/tratamento farmacológico , HIV-1/genética , Fragmentos de Peptídeos/genética , Estudos de Coortes , Estudos Transversais , Infecções por HIV/virologia , HIV-1/efeitos dos fármacos , HIV-1/imunologia , Humanos , Mutação , Análise de Sequência de DNA/métodos , Fatores de Tempo , Resultado do Tratamento
16.
PLoS Pathog ; 8(5): e1002730, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22693449

RESUMO

Influenza viruses are characterized by an ability to cross species boundaries and evade host immunity, sometimes with devastating consequences. The 2009 pandemic of H1N1 influenza A virus highlights the importance of pigs in influenza emergence, particularly as intermediate hosts by which avian viruses adapt to mammals before emerging in humans. Although segment reassortment has commonly been associated with influenza emergence, an expanded host-range is also likely to be associated with the accumulation of specific beneficial point mutations. To better understand the mechanisms that shape the genetic diversity of avian-like viruses in pigs, we studied the evolutionary dynamics of an Eurasian Avian-like swine influenza virus (EA-SIV) in naïve and vaccinated pigs linked by natural transmission. We analyzed multiple clones of the hemagglutinin 1 (HA1) gene derived from consecutive daily viral populations. Strikingly, we observed both transient and fixed changes in the consensus sequence along the transmission chain. Hence, the mutational spectrum of intra-host EA-SIV populations is highly dynamic and allele fixation can occur with extreme rapidity. In addition, mutations that could potentially alter host-range and antigenicity were transmitted between animals and mixed infections were commonplace, even in vaccinated pigs. Finally, we repeatedly detected distinct stop codons in virus samples from co-housed pigs, suggesting that they persisted within hosts and were transmitted among them. This implies that mutations that reduce viral fitness in one host, but which could lead to fitness benefits in a novel host, can circulate at low frequencies.


Assuntos
Aviadenovirus/genética , Evolução Molecular , Vírus da Influenza A Subtipo H1N1/genética , Infecções por Orthomyxoviridae/virologia , Doenças dos Suínos/virologia , Animais , Anticorpos Antivirais/imunologia , Aviadenovirus/imunologia , Clonagem Molecular , Regulação Viral da Expressão Gênica , Interações Hospedeiro-Patógeno , Vírus da Influenza A Subtipo H1N1/imunologia , Vacinas contra Influenza , Infecções por Orthomyxoviridae/imunologia , Suínos , Doenças dos Suínos/imunologia , Vacinação , Eliminação de Partículas Virais
17.
PLoS Pathog ; 8(12): e1003081, 2012 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-23308065

RESUMO

The ability of influenza A viruses (IAVs) to cross species barriers and evade host immunity is a major public health concern. Studies on the phylodynamics of IAVs across different scales - from the individual to the population - are essential for devising effective measures to predict, prevent or contain influenza emergence. Understanding how IAVs spread and evolve during outbreaks is critical for the management of epidemics. Reconstructing the transmission network during a single outbreak by sampling viral genetic data in time and space can generate insights about these processes. Here, we obtained intra-host viral sequence data from horses infected with equine influenza virus (EIV) to reconstruct the spread of EIV during a large outbreak. To this end, we analyzed within-host viral populations from sequences covering 90% of the infected yards. By combining gene sequence analyses with epidemiological data, we inferred a plausible transmission network, in turn enabling the comparison of transmission patterns during the course of the outbreak and revealing important epidemiological features that were not apparent using either approach alone. The EIV populations displayed high levels of genetic diversity, and in many cases we observed distinct viral populations containing a dominant variant and a number of related minor variants that were transmitted between infectious horses. In addition, we found evidence of frequent mixed infections and loose transmission bottlenecks in these naturally occurring populations. These frequent mixed infections likely influence the size of epidemics.


Assuntos
Coinfecção/transmissão , Surtos de Doenças , Doenças dos Cavalos/transmissão , Vírus da Influenza A Subtipo H3N8/genética , Infecções por Orthomyxoviridae/transmissão , Animais , Coinfecção/epidemiologia , Coinfecção/genética , Variação Genética , Doenças dos Cavalos/epidemiologia , Doenças dos Cavalos/genética , Cavalos , Vírus da Influenza A Subtipo H3N8/imunologia , Mucosa Nasal/virologia , Infecções por Orthomyxoviridae/epidemiologia , Infecções por Orthomyxoviridae/genética , RNA Viral/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa
18.
J Virol ; 86(1): 11-8, 2012 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-22013031

RESUMO

Virus gene sequencing and phylogenetics can be used to study the epidemiological dynamics of rapidly evolving viruses. With complete genome data, it becomes possible to identify and trace individual transmission chains of viruses such as influenza virus during the course of an epidemic. Here we sequenced 153 pandemic influenza H1N1/09 virus genomes from United Kingdom isolates from the first (127 isolates) and second (26 isolates) waves of the 2009 pandemic and used their sequences, dates of isolation, and geographical locations to infer the genetic epidemiology of the epidemic in the United Kingdom. We demonstrate that the epidemic in the United Kingdom was composed of many cocirculating lineages, among which at least 13 were exclusively or predominantly United Kingdom clusters. The estimated divergence times of two of the clusters predate the detection of pandemic H1N1/09 virus in the United Kingdom, suggesting that the pandemic H1N1/09 virus was already circulating in the United Kingdom before the first clinical case. Crucially, three clusters contain isolates from the second wave of infections in the United Kingdom, two of which represent chains of transmission that appear to have persisted within the United Kingdom between the first and second waves. This demonstrates that whole-genome analysis can track in fine detail the behavior of individual influenza virus lineages during the course of a single epidemic or pandemic.


Assuntos
Evolução Molecular , Genoma Viral , Vírus da Influenza A Subtipo H1N1/classificação , Vírus da Influenza A Subtipo H1N1/genética , Influenza Humana/virologia , Adolescente , Adulto , Criança , Humanos , Vírus da Influenza A Subtipo H1N1/isolamento & purificação , Influenza Humana/epidemiologia , Dados de Sequência Molecular , Pandemias , Filogenia , Reino Unido , Adulto Jovem
19.
J Virol ; 84(14): 6943-54, 2010 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-20444896

RESUMO

Determining the evolutionary basis of cross-species transmission and immune evasion is key to understanding the mechanisms that control the emergence of either new viruses or novel antigenic variants with pandemic potential. The hemagglutinin glycoprotein of influenza A viruses is a critical host range determinant and a major target of neutralizing antibodies. Equine influenza virus (EIV) is a significant pathogen of the horse that causes periodical outbreaks of disease even in populations with high vaccination coverage. EIV has also jumped the species barrier and emerged as a novel respiratory pathogen in dogs, canine influenza virus. We studied the dynamics of equine influenza virus evolution in horses at the intrahost level and how this evolutionary process is affected by interhost transmission in a natural setting. To this end, we performed clonal sequencing of the hemagglutinin 1 gene derived from individual animals at different times postinfection. Our results show that despite the population consensus sequence remaining invariant, genetically distinct subpopulations persist during the course of infection and are also transmitted, with some variants likely to change antigenicity. We also detected a natural case of mixed infection in an animal infected during an outbreak of equine influenza, raising the possibility of reassortment between different strains of virus. In sum, our data suggest that transmission bottlenecks may not be as narrow as originally perceived and that the genetic diversity required to adapt to new host species may be partially present in the donor host and potentially transmitted to the recipient host.


Assuntos
Evolução Molecular , Doenças dos Cavalos/transmissão , Doenças dos Cavalos/virologia , Vírus da Influenza A Subtipo H3N8 , Infecções por Orthomyxoviridae/veterinária , Animais , Surtos de Doenças/veterinária , Cães , Glicoproteínas de Hemaglutininação de Vírus da Influenza/classificação , Glicoproteínas de Hemaglutininação de Vírus da Influenza/genética , Glicoproteínas de Hemaglutininação de Vírus da Influenza/imunologia , Doenças dos Cavalos/epidemiologia , Doenças dos Cavalos/genética , Cavalos , Humanos , Evasão da Resposta Imune , Vírus da Influenza A Subtipo H3N8/genética , Vírus da Influenza A Subtipo H3N8/imunologia , Vírus da Influenza A Subtipo H3N8/patogenicidade , Influenza Humana/epidemiologia , Influenza Humana/genética , Influenza Humana/transmissão , Influenza Humana/virologia , Funções Verossimilhança , Mutação , Infecções por Orthomyxoviridae/epidemiologia , Infecções por Orthomyxoviridae/genética , Infecções por Orthomyxoviridae/transmissão , Infecções por Orthomyxoviridae/virologia , Filogenia
20.
J Virol ; 84(10): 5329-35, 2010 May.
Artigo em Inglês | MEDLINE | ID: mdl-20219907

RESUMO

The patterns and dynamics of evolution in acutely infecting viruses within individual hosts are largely unknown. To this end, we investigated the intrahost variation of canine influenza virus (CIV) during the course of experimental infections in naïve and partially immune dogs and in naturally infected dogs. Tracing sequence diversity in the gene encoding domain 1 of the hemagglutinin (HA1) protein over the time course of infection provided information on the patterns and processes of intrahost viral evolution and revealed some of the effects of partial host immunity. Viral populations sampled on any given day were generally characterized by mean pairwise genetic diversities between 0.1 and 0.2% and by mutational spectra that changed considerably on different days. Some observed mutations may have affected antigenicity or host range, including reversions of CIV host-associated mutations. Patterns of sequence diversity differed between naïve and vaccinated dogs, with some presumably antigenic mutations transiently reaching high frequency in the latter. CIV populations are therefore characterized by the rapid generation and clearance of genetic diversity. Potentially advantageous mutations arise readily during the course of single infections and may give rise to antigenic escape or host range variants.


Assuntos
Doenças do Cão/virologia , Evolução Molecular , Vírus da Influenza A/classificação , Vírus da Influenza A/isolamento & purificação , Infecções por Orthomyxoviridae/veterinária , Polimorfismo Genético , Animais , Análise por Conglomerados , Doenças do Cão/imunologia , Cães , Hemaglutininas Virais/genética , Vírus da Influenza A/genética , Vírus da Influenza A/imunologia , Dados de Sequência Molecular , Mutação de Sentido Incorreto , Infecções por Orthomyxoviridae/imunologia , Infecções por Orthomyxoviridae/virologia , RNA Viral/genética , Análise de Sequência de DNA
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