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1.
J Infect Dis ; 219(11): 1688-1696, 2019 05 05.
Artigo em Inglês | MEDLINE | ID: mdl-30395249

RESUMO

BACKGROUND: In 2016, an influenza A(H7N2) virus outbreak occurred in cats in New York City's municipal animal shelters. One human infection was initially detected. METHODS: We conducted a serological survey using a novel approach to rule out cross-reactive antibodies to other seasonal influenza viruses to determine whether additional A(H7N2) human infections had occurred and to assess exposure risk. RESULTS: Of 121 shelter workers, one had serological evidence of A(H7N2) infection, corresponding to a seroprevalence of 0.8% (95% confidence interval, .02%-4.5%). Five persons exhibited low positive titers to A(H7N2) virus, indicating possible infection; however, we could not exclude cross-reactive antibody responses to seasonal influenza viruses. The remaining 115 persons were seronegative. The seropositive person reported multiple direct cat exposures without using personal protective equipment and mild illness with subjective fever, runny nose, and sore throat. CONCLUSIONS: We identified a second case of A(H7N2) infection from this outbreak, providing further evidence of cat-to-human transmission of A(H7N2) virus.


Assuntos
Anticorpos Antivirais/sangue , Surtos de Doenças/veterinária , Vírus da Influenza A Subtipo H7N2/imunologia , Influenza Aviária/virologia , Influenza Humana/virologia , Infecções por Orthomyxoviridae/veterinária , Adulto , Idoso , Animais , Aves , Gatos , Reações Cruzadas , Feminino , Humanos , Vírus da Influenza A Subtipo H7N2/isolamento & purificação , Influenza Humana/transmissão , Masculino , Pessoa de Meia-Idade , Cidade de Nova Iorque/epidemiologia , Infecções por Orthomyxoviridae/epidemiologia , Infecções por Orthomyxoviridae/virologia , Estudos Soroepidemiológicos , Zoonoses
2.
PLoS Pathog ; 4(11): e1000219, 2008 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-19043551

RESUMO

Small interfering RNAs regulate gene expression in diverse biological processes, including heterochromatin formation and DNA elimination, developmental regulation, and cell differentiation. In the single-celled eukaryote Entamoeba histolytica, we have identified a population of small RNAs of 27 nt size that (i) have 5'-polyphosphate termini, (ii) map antisense to genes, and (iii) associate with an E. histolytica Piwi-related protein. Whole genome microarray expression analysis revealed that essentially all genes to which antisense small RNAs map were not expressed under trophozoite conditions, the parasite stage from which the small RNAs were cloned. However, a number of these genes were expressed in other E. histolytica strains with an inverse correlation between small RNA and gene expression level, suggesting that these small RNAs mediate silencing of the cognate gene. Overall, our results demonstrate that E. histolytica has an abundant 27 nt small RNA population, with features similar to secondary siRNAs from C. elegans, and which appear to regulate gene expression. These data indicate that a silencing pathway mediated by 5'-polyphosphate siRNAs extends to single-celled eukaryotic organisms.


Assuntos
Entamoeba histolytica/genética , Regulação da Expressão Gênica , Polifosfatos , RNA Interferente Pequeno/fisiologia , Proteínas de Ligação a RNA/metabolismo , Animais , Inativação Gênica , Genômica , RNA Interferente Pequeno/isolamento & purificação
3.
PLoS One ; 10(7): e0133740, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26230096

RESUMO

Dicer enzymes process double-stranded RNA (dsRNA) into small RNAs that target gene silencing through the RNA interference (RNAi) pathway. Dicer enzymes are complex, multi-domain RNaseIII proteins, however structural minimalism of this protein has recently emerged in parasitic and fungal systems. The most minimal Dicer, Saccharomyces castellii Dicer1, has a single RNaseIII domain and two double stranded RNA binding domains. In the protozoan parasite Entamoeba histolytica 27nt small RNAs are abundant and mediate silencing, yet no canonical Dicer enzyme has been identified. Although EhRNaseIII does not exhibit robust dsRNA cleavage in vitro, it can process dsRNA in the RNAi-negative background of Saccharomyces cerevisiae, and in conjunction with S. castellii Argonaute1 can partially reconstitute the RNAi pathway. Thus, although EhRNaseIII lacks the domain architecture of canonical or minimal Dicer enzymes, it has dsRNA processing activity that contributes to gene silencing via RNAi. Our data advance the understanding of small RNA biogenesis in Entamoeba as well as broaden the spectrum of non-canonical Dicer enzymes that contribute to the RNAi pathway.


Assuntos
Entamoeba histolytica/genética , Inativação Gênica/fisiologia , Clivagem do RNA/genética , Interferência de RNA/fisiologia , RNA de Cadeia Dupla/metabolismo , Ribonuclease III/metabolismo , Entamoeba histolytica/enzimologia , Genes de Protozoários/genética , Genes de Protozoários/fisiologia , Organismos Geneticamente Modificados/genética , Organismos Geneticamente Modificados/fisiologia , Clivagem do RNA/fisiologia , Ribonuclease III/fisiologia , Saccharomyces/genética , Saccharomyces/fisiologia , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/fisiologia
4.
PLoS One ; 9(9): e106477, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25198343

RESUMO

The RNA interference pathway in the protist Entamoeba histolytica plays important roles in permanent gene silencing as well as in the regulation of virulence determinants. Recently, a novel RNA interference (RNAi)-based silencing technique was developed in this parasite that uses a gene endogenously silenced by small RNAs as a "trigger" to induce silencing of other genes that are fused to it. Fusion to a trigger gene induces the production of gene-specific antisense small RNAs, resulting in robust and permanent silencing of the cognate gene. This approach has silenced multiple genes including those involved in virulence and transcriptional regulation. We now demonstrate that all tested genes of the amebic RNAi pathway are unable to be silenced using the trigger approach, including Argonaute genes (Ago2-1, Ago2-2, and Ago2-3), RNaseIII, and RNA-dependent RNA polymerase (RdRP). In all situations (except for RdRP), fusion to a trigger successfully induces production of gene-specific antisense small RNAs to the cognate gene. These small RNAs are capable of silencing a target gene in trans, indicating that they are functional; despite this, however, they cannot silence the RNAi pathway genes. Interestingly, when a trigger is fused to RdRP, small RNA induction to RdRP does not occur, a unique phenotype hinting that either RdRP is highly resistant to being a target of small RNAs or that small RNA generation may be controlled by RdRP. The inability of the small RNA pathway to silence RNAi genes in E. histolytica, despite the generation of functional small RNAs to these loci suggest that epigenetic factors may protect certain genomic loci and thus determine susceptibility to small RNA mediated silencing.


Assuntos
Entamoeba histolytica/genética , Inativação Gênica , Genes de Protozoários , Interferência de RNA , Entamoeba histolytica/patogenicidade , Virulência/genética
5.
Future Microbiol ; 6(1): 103-17, 2011 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-21162639

RESUMO

Entamoeba histolytica is a major health threat to people in developing countries, where it causes invasive diarrhea and liver abscesses. The study of this important human pathogen has been hindered by a lack of tools for genetic manipulation. Recently, a number of genetic approaches based on variations of the RNAi method have been successfully developed and cloning of endogenous small-interfering RNAs from E. histolytica revealed an abundant population of small RNAs with an unusual 5´-polyphosphate structure. However, little is known about the implications of these findings to amebic biology or the mechanisms of gene silencing in this organism. In this article we review the literature relevant to RNAi in E. histolytica, discuss its implications for advances in gene silencing in this organism and outline potential future directions towards understanding the repertoire of RNAi and its impact on the biology of this deep-branching eukaryotic parasite.


Assuntos
Entamoeba histolytica/genética , Interferência de RNA , RNA de Protozoário/genética , RNA Interferente Pequeno/genética , Sequência de Bases , Entamoeba histolytica/citologia , Entamoeba histolytica/metabolismo , Humanos , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , RNA de Cadeia Dupla/genética
6.
EMBO J ; 26(4): 1094-104, 2007 Feb 21.
Artigo em Inglês | MEDLINE | ID: mdl-17290222

RESUMO

In most eukaryotes, sphingolipids (SLs) are critical membrane components and signaling molecules. However, mutants of the trypanosomatid protozoan Leishmania lacking serine palmitoyltransferase (spt2-) and SLs grow well, although they are defective in stationary phase differentiation and virulence. Similar phenotypes were observed in sphingolipid (SL) mutant lacking the degradatory enzyme sphingosine 1-phosphate lyase (spl-). This epistatic interaction suggested that a metabolite downstream of SLs was responsible. Here we show that unlike other organisms, the Leishmania SL pathway has evolved to be the major route for ethanolamine (EtN) synthesis, as EtN supplementation completely reversed the viability and differentiation defects of both mutants. Thus Leishmania has undergone two major metabolic shifts: first in de-emphasizing the metabolic roles of SLs themselves in growth, signaling, and maintenance of membrane microdomains, which may arise from the unique combination of abundant parasite lipids; Second, freed of typical SL functional constraints and a lack of alternative routes to produce EtN, Leishmania redirected SL metabolism toward bulk EtN synthesis. Our results thus reveal a striking example of remodeling of the SL metabolic pathway in Leishmania.


Assuntos
Evolução Biológica , Vias Biossintéticas/genética , Etanolamina/metabolismo , Leishmania major/genética , Leishmania major/metabolismo , Redes e Vias Metabólicas/genética , Esfingolipídeos/metabolismo , Aldeído Liases/deficiência , Aldeído Liases/genética , Animais , Sequência de Bases , Clonagem Molecular , Feminino , Pé/microbiologia , Pé/patologia , Leishmania major/patogenicidade , Leishmania major/ultraestrutura , Espectrometria de Massas , Camundongos , Camundongos Endogâmicos BALB C , Microscopia Eletrônica de Transmissão , Dados de Sequência Molecular , Mutação/genética , Análise de Sequência de DNA , Serina C-Palmitoiltransferase/deficiência , Serina C-Palmitoiltransferase/genética
7.
J Virol ; 77(9): 5305-12, 2003 May.
Artigo em Inglês | MEDLINE | ID: mdl-12692232

RESUMO

Virus-induced apoptosis of infected cells can limit both the time and the cellular machinery available for virus replication. Hence, many viruses have evolved strategies to specifically inhibit apoptosis. However, Aleutian mink disease parvovirus (ADV) is the first example of a DNA virus that not only induces apoptosis but also utilizes caspase activity to facilitate virus replication. To determine the function of caspase activity during ADV replication, virus-infected cell lysates or purified ADV proteins were incubated with various purified caspases. Caspases cleaved the major nonstructural protein of ADV (NS1) at two caspase recognition sequences, whereas ADV structural proteins could not be cleaved. Importantly, the NS1 products could be identified in ADV-infected cells but were not present in infected cells pretreated with caspase inhibitors. By mutating putative caspase cleavage sites (D to E), we mapped the two cleavage sites to amino acid residues NS1:227 (INTD downward arrow S) and NS1:285 (DQTD downward arrow S). Replication of ADV containing either of these mutations was reduced 10(3)- to 10(4)-fold compared to that of wild-type virus, and a construct containing both mutations was replication defective. Immunofluorescent studies revealed that cleavage was required for nuclear localization of NS1. The requirement for caspase activity during permissive replication suggests that limitation of caspase activation and apoptosis in vivo may be a novel approach to restricting virus replication.


Assuntos
Vírus da Doença Aleutiana do Vison/fisiologia , Vírus da Doença Aleutiana do Vison/patogenicidade , Caspases/metabolismo , Proteínas não Estruturais Virais/metabolismo , Replicação Viral , Animais , Apoptose , Gatos , Células Cultivadas , Imunofluorescência , Proteínas Recombinantes de Fusão/metabolismo , Proteínas não Estruturais Virais/química , Proteínas não Estruturais Virais/genética
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