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1.
iScience ; 26(8): 107323, 2023 Aug 18.
Artigo em Inglês | MEDLINE | ID: mdl-37529105

RESUMO

Lymphocyte depletion is a distinctive feature of Ebola virus (EBOV) disease. The ectodomain of EBOV glycoprotein (GP) is cleaved off the surface of infected cells into circulation as shed GP. To test the hypothesis that shed GP induces lymphocyte death, we cultured primary human B, NK, or T cells with shed GP in vitro. We found that shed GP dependably decreased B, NK, and T cell viability across donors. B and NK cells exhibited higher susceptibility than T cells. Continuous monitoring revealed shed GP began to kill B and NK cells by 4 h and T cells by 5 h. We also demonstrated that shed GP-induced lymphocyte death can be both caspase dependent and caspase independent. Our data are evidence that the cytotoxic effect of shed GP on lymphocytes may contribute to EBOV disease and highlight the need for further research to clarify mechanisms of shed GP-induced death.

2.
Cardiol Rev ; 2022 Oct 25.
Artigo em Inglês | MEDLINE | ID: mdl-36730913

RESUMO

Viral infections have been linked to a variety of cardiac pathology, which may include acute myocarditis, dilated cardiomyopathy, heart failure, cardiogenic shock, pericarditis, acute coronary syndromes, and arrhythmias. We performed a systematic review of literature focusing on the cardiovascular effects of various viral infections, as well as providing an update on the current understanding of the pathophysiology of Coronavirus disease-2019 (COVID-19). Cardiac manifestations of viral illnesses are usually self-limiting, have variable clinical presentations, and require sufficient clinical suspicion for diagnosis and optimal management.

4.
Cell Host Microbe ; 29(5): 834-848.e13, 2021 05 12.
Artigo em Inglês | MEDLINE | ID: mdl-33794184

RESUMO

Argonaute (AGO) proteins bind small RNAs to silence complementary RNA transcripts, and they are central to RNA interference (RNAi). RNAi is critical for regulation of gene expression and antiviral defense in Aedes aegypti mosquitoes, which transmit Zika, chikungunya, dengue, and yellow fever viruses. In mosquitoes, AGO1 mediates miRNA interactions, while AGO2 mediates siRNA interactions. We applied AGO-crosslinking immunoprecipitation (AGO-CLIP) for both AGO1 and AGO2, and we developed a universal software package for CLIP analysis (CLIPflexR), identifying 230 small RNAs and 5,447 small RNA targets that comprise a comprehensive RNAi network map in mosquitoes. RNAi network maps predicted expression levels of small RNA targets in specific tissues. Additionally, this resource identified unexpected, context-dependent AGO2 target preferences, including endogenous viral elements and 3'UTRs. Finally, contrary to current thinking, mosquito AGO2 repressed imperfect targets. These findings expand our understanding of small RNA networks and have broad implications for the study of antiviral RNAi.


Assuntos
Aedes/enzimologia , Aedes/genética , Proteínas Argonautas/metabolismo , Proteínas de Insetos/metabolismo , Interferência de RNA , RNA Viral/metabolismo , Vírus/metabolismo , Aedes/virologia , Animais , Proteínas Argonautas/genética , Imunoprecipitação , Proteínas de Insetos/genética , RNA Viral/genética , Vírus/genética
5.
Sci Rep ; 11(1): 736, 2021 01 12.
Artigo em Inglês | MEDLINE | ID: mdl-33436886

RESUMO

Aedes (Ae.) aegypti and Ae. albopictus mosquitoes transmit arthropod-borne diseases around the globe, causing ~ 700,000 deaths each year. Genetic mutants are valuable tools to interrogate both fundamental vector biology and mosquito host factors important for viral infection. However, very few genetic mutants have been described in mosquitoes in comparison to model organisms. The relative ease of applying CRISPR/Cas9-based gene editing has transformed genome engineering and has rapidly increased the number of available gene mutants in mosquitoes. Yet, in vivo studies may not be practical for screening large sets of mutants or possible for laboratories that lack insectaries. Thus, it would be useful to adapt CRISPR/Cas9 systems to common mosquito cell lines. In this study, we generated and characterized a mosquito optimized, plasmid-based CRISPR/Cas9 system for use in U4.4 (Ae. albopictus) and Aag2 (Ae. aegypti) cell lines. We demonstrated highly efficient editing of the AGO1 locus and isolated U4.4 and Aag2 cell lines with reduced AGO1 expression. Further, we used homology-directed repair to establish knock-in Aag2 cell lines with a 3xFLAG-tag at the N-terminus of endogenous AGO1. These experimentally verified plasmids are versatile, cost-effective, and efficiently edit immune competent mosquito cell lines that are widely used in arbovirus studies.


Assuntos
Aedes/genética , Sistemas CRISPR-Cas , Fatores de Iniciação em Eucariotos/genética , Edição de Genes/métodos , Mosquitos Vetores/genética , Plasmídeos/genética , Animais , Linhagem Celular , Fatores de Iniciação em Eucariotos/antagonistas & inibidores , Genoma de Inseto
6.
Cell ; 184(1): 133-148.e20, 2021 01 07.
Artigo em Inglês | MEDLINE | ID: mdl-33338421

RESUMO

Flaviviruses pose a constant threat to human health. These RNA viruses are transmitted by the bite of infected mosquitoes and ticks and regularly cause outbreaks. To identify host factors required for flavivirus infection, we performed full-genome loss of function CRISPR-Cas9 screens. Based on these results, we focused our efforts on characterizing the roles that TMEM41B and VMP1 play in the virus replication cycle. Our mechanistic studies on TMEM41B revealed that all members of the Flaviviridae family that we tested require TMEM41B. We tested 12 additional virus families and found that SARS-CoV-2 of the Coronaviridae also required TMEM41B for infection. Remarkably, single nucleotide polymorphisms present at nearly 20% in East Asian populations reduce flavivirus infection. Based on our mechanistic studies, we propose that TMEM41B is recruited to flavivirus RNA replication complexes to facilitate membrane curvature, which creates a protected environment for viral genome replication.


Assuntos
Infecções por Flavivirus/genética , Flavivirus/fisiologia , Proteínas de Membrana/metabolismo , Animais , Povo Asiático/genética , Autofagia , COVID-19/genética , COVID-19/metabolismo , COVID-19/virologia , Sistemas CRISPR-Cas , Linhagem Celular , Infecções por Flavivirus/imunologia , Infecções por Flavivirus/metabolismo , Infecções por Flavivirus/virologia , Técnicas de Inativação de Genes , Estudo de Associação Genômica Ampla , Interações Hospedeiro-Patógeno , Humanos , Imunidade Inata , Proteínas de Membrana/genética , Polimorfismo de Nucleotídeo Único , SARS-CoV-2/fisiologia , Replicação Viral , Vírus da Febre Amarela/fisiologia , Zika virus/fisiologia
7.
Diagn Microbiol Infect Dis ; 99(1): 115226, 2021 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-33070027

RESUMO

We describe 3 adult patients who did not have COVID-19 but instead had a treatable tick-borne infection. In each case, however, the duration of time until diagnosis was delayed due to issues that have arisen because of the COVID-19 pandemic. These issues need to be addressed to preserve patient well-being.


Assuntos
COVID-19/epidemiologia , Pandemias , Doenças Transmitidas por Carrapatos/diagnóstico , Adulto , Idoso , Diagnóstico Tardio , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , SARS-CoV-2 , Doenças Transmitidas por Carrapatos/etiologia , Doenças Transmitidas por Carrapatos/terapia
8.
bioRxiv ; 2020 Oct 11.
Artigo em Inglês | MEDLINE | ID: mdl-33052348

RESUMO

Flaviviruses pose a constant threat to human health. These RNA viruses are transmitted by the bite of infected mosquitoes and ticks and regularly cause outbreaks. To identify host factors required for flavivirus infection we performed full-genome loss of function CRISPR-Cas9 screens. Based on these results we focused our efforts on characterizing the roles that TMEM41B and VMP1 play in the virus replication cycle. Our mechanistic studies on TMEM41B revealed that all members of the Flaviviridae family that we tested require TMEM41B. We tested 12 additional virus families and found that SARS-CoV-2 of the Coronaviridae also required TMEM41B for infection. Remarkably, single nucleotide polymorphisms (SNPs) present at nearly twenty percent in East Asian populations reduce flavivirus infection. Based on our mechanistic studies we hypothesize that TMEM41B is recruited to flavivirus RNA replication complexes to facilitate membrane curvature, which creates a protected environment for viral genome replication. HIGHLIGHTS: TMEM41B and VMP1 are required for both autophagy and flavivirus infection, however, autophagy is not required for flavivirus infection.TMEM41B associates with viral proteins and likely facilitates membrane remodeling to establish viral RNA replication complexes.TMEM41B single nucleotide polymorphisms (SNPs) present at nearly twenty percent in East Asian populations reduce flavivirus infection.TMEM41B-deficient cells display an exaggerated innate immune response upon high multiplicity flavivirus infection.

9.
Artigo em Inglês | MEDLINE | ID: mdl-33106261

RESUMO

Erythema migrans is the most common clinical manifestation of Lyme disease, with concomitant subjective symptoms occurring in ∼65% of cases in the United States. We evaluated the impact of having been started on antibiotic treatment before study enrollment on 12 particular symptoms for 38 subjects with erythema migrans versus 52 untreated subjects. There were no significant differences in the frequency of having at least one symptom or in the symptom severity score on study entry. However, the frequency of having at least one symptom was significantly greater for those who had received <7 days of antibiotic treatment than for those who had been treated for ≥7 days (23/24 [95.8%] versus 8/14 [57.1%], P = 0.006). In addition, the percentage of subjects who were males was significantly lower among the group on treatment than among the untreated study subjects (13/38 [34.2%] versus 34/52 [65.4%], P = 0.005). In conclusion, based on these findings, combining untreated and treated groups of patients with erythema migrans for research study analyses may have limitations and, depending on the study objectives, might not be preferred. Additional studies are warranted to better understand the day-to-day impact of antibiotic treatment on the presence, type, and severity of symptoms in patients with early Lyme disease.


Assuntos
Eritema Migrans Crônico , Doença de Lyme , Antibacterianos/uso terapêutico , Eritema/tratamento farmacológico , Eritema Migrans Crônico/tratamento farmacológico , Feminino , Humanos , Doença de Lyme/tratamento farmacológico , Masculino
10.
PLoS One ; 14(10): e0224440, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31648237

RESUMO

Even when successfully surviving an infection, a host often fails to eliminate a pathogen completely and may sustain substantial pathogen burden for the remainder of its life. Using systemic bacterial infection in Drosophila melanogaster, we characterize chronic infection by three bacterial species from different genera - Providencia rettgeri, Serratia marcescens, and Enterococcus faecalis-following inoculation with a range of doses. To assess the consequences of these chronic infections, we determined the expression of antimicrobial peptide genes, survival of secondary infection, and starvation resistance after one week of infection. While higher infectious doses unsurprisingly lead to higher risk of death, they also result in higher chronic bacterial loads among the survivors for all three infections. All three chronic infections caused significantly elevated expression of antimicrobial peptide genes at one week post-infection and provided generalized protection again secondary bacterial infection. Only P. rettgeri infection significantly influenced resistance to starvation, with persistently infected flies dying more quickly under starvation conditions relative to controls. These results suggest that there is potentially a generalized mechanism of protection against secondary infection, but that other impacts on host physiology may depend on the specific pathogen. We propose that chronic infections in D. melanogaster could be a valuable tool for studying tolerance of infection, including impacts on host physiology and behavior.


Assuntos
Drosophila melanogaster/microbiologia , Interações Hospedeiro-Patógeno , Animais , Peptídeos Catiônicos Antimicrobianos/genética , Carga Bacteriana , Doença Crônica , Drosophila melanogaster/citologia , Drosophila melanogaster/genética , Drosophila melanogaster/fisiologia , Espaço Extracelular/microbiologia , Regulação da Expressão Gênica , Inanição/microbiologia
11.
J Vis Exp ; (99): e52613, 2015 May 13.
Artigo em Inglês | MEDLINE | ID: mdl-25992475

RESUMO

The fruit fly Drosophila melanogaster is one of the premier model organisms for studying the function and evolution of immune defense. Many aspects of innate immunity are conserved between insects and mammals, and since Drosophila can readily be genetically and experimentally manipulated, they are powerful for studying immune system function and the physiological consequences of disease. The procedure demonstrated here allows infection of flies by introduction of bacteria directly into the body cavity, bypassing epithelial barriers and more passive forms of defense and allowing focus on systemic infection. The procedure includes protocols for the measuring rates of host mortality, systemic pathogen load, and degree of induction of the host immune system. This infection procedure is inexpensive, robust and quantitatively repeatable, and can be used in studies of functional genetics, evolutionary life history, and physiology.


Assuntos
Infecções Bacterianas/imunologia , Drosophila melanogaster/imunologia , Animais , Modelos Animais de Doenças , Proteínas de Drosophila/imunologia , Imunidade Inata/imunologia , Masculino , Fenótipo
12.
Infect Immun ; 82(10): 4380-9, 2014 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-25092914

RESUMO

The route of infection can profoundly affect both the progression and outcome of disease. We investigated differences in Drosophila melanogaster defense against infection after bacterial inoculation into two sites--the abdomen and the thorax. Thorax inoculation results in increased bacterial proliferation and causes high mortality within the first few days of infection. In contrast, abdomen inoculation results in minimal mortality and lower bacterial loads than thorax inoculation. Inoculation into either site causes systemic infection. Differences in mortality and bacterial load are due to injury of the thorax and can be recapitulated by abdominal inoculation coupled with aseptic wounding of the thorax. This altered resistance appears to be independent of classical immune pathways and opens new avenues of research on the role of injury during defense against infection.


Assuntos
Drosophila melanogaster/fisiologia , Traumatismos Abdominais/imunologia , Animais , Infecções Bacterianas/imunologia , Infecções Bacterianas/microbiologia , Carga Bacteriana , Modelos Animais de Doenças , Resistência à Doença , Drosophila melanogaster/imunologia , Feminino , Masculino , Análise de Sobrevida , Traumatismos Torácicos/imunologia
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