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1.
Cell ; 181(4): 865-876.e12, 2020 05 14.
Artigo em Inglês | MEDLINE | ID: mdl-32353252

RESUMO

The coronavirus disease 2019 (COVID-19) pandemic, caused by the SARS-CoV-2 virus, has highlighted the need for antiviral approaches that can target emerging viruses with no effective vaccines or pharmaceuticals. Here, we demonstrate a CRISPR-Cas13-based strategy, PAC-MAN (prophylactic antiviral CRISPR in human cells), for viral inhibition that can effectively degrade RNA from SARS-CoV-2 sequences and live influenza A virus (IAV) in human lung epithelial cells. We designed and screened CRISPR RNAs (crRNAs) targeting conserved viral regions and identified functional crRNAs targeting SARS-CoV-2. This approach effectively reduced H1N1 IAV load in respiratory epithelial cells. Our bioinformatic analysis showed that a group of only six crRNAs can target more than 90% of all coronaviruses. With the development of a safe and effective system for respiratory tract delivery, PAC-MAN has the potential to become an important pan-coronavirus inhibition strategy.


Assuntos
Antivirais/farmacologia , Betacoronavirus/efeitos dos fármacos , Sistemas CRISPR-Cas , Vírus da Influenza A Subtipo H1N1/efeitos dos fármacos , RNA Viral/antagonistas & inibidores , Células A549 , Antibioticoprofilaxia/métodos , Sequência de Bases , Betacoronavirus/genética , Betacoronavirus/crescimento & desenvolvimento , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Simulação por Computador , Sequência Conservada , Coronavirus/efeitos dos fármacos , Coronavirus/genética , Coronavirus/crescimento & desenvolvimento , Infecções por Coronavirus/tratamento farmacológico , Células Epiteliais/virologia , Humanos , Vírus da Influenza A Subtipo H1N1/genética , Vírus da Influenza A Subtipo H1N1/crescimento & desenvolvimento , Pulmão/patologia , Pulmão/virologia , Proteínas do Nucleocapsídeo/genética , Pandemias , Filogenia , Pneumonia Viral/tratamento farmacológico , RNA Replicase/genética , Proteínas não Estruturais Virais/genética
2.
Tohoku J Exp Med ; 251(1): 27-30, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32448818

RESUMO

The number of patients infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has rapidly increased, although the WHO declared a pandemic. However, drugs that function against SARS-CoV-2 have not been established. SARS-CoV-2 has been suggested to bind angiotensin-converting enzyme 2, the receptor of the SARS coronavirus. SARS coronavirus and coronavirus 229E, the cause of the common cold, replicate through cell-surface and endosomal pathways using a protease, the type II transmembrane protease. To examine the effects of protease inhibitors on the replication of coronavirus 229E, we pretreated primary cultures of human nasal epithelial (HNE) cells with camostat or nafamostat, each of which has been used for the treatment of pancreatitis and/or disseminated intravascular coagulation. HNE cells were then infected with coronavirus 229E, and viral titers in the airway surface liquid of the cells were examined. Pretreatment with camostat (0.1-10 µg/mL) or nafamostat (0.01-1 µg/mL) reduced the titers of coronavirus 229E. Furthermore, a significant amount of type II transmembrane protease protein was detected in the airway surface liquid of HNE cells. Additionally, interferons have been reported to have antiviral effects against SARS coronavirus. The additive effects of interferons on the inhibitory effects of other candidate drugs to treat SARS-CoV-2 infection, such as lopinavir, ritonavir and favipiravir, have also been studied. These findings suggest that protease inhibitors of this type may inhibit coronavirus 229E replication in human airway epithelial cells at clinical concentrations. Protease inhibitors, interferons or the combination of these drugs may become candidate drugs to inhibit the replication of SARS-CoV-2.


Assuntos
Antivirais/farmacologia , Coronavirus Humano 229E/efeitos dos fármacos , Infecções por Coronavirus/tratamento farmacológico , Gabexato/análogos & derivados , Guanidinas/farmacologia , Pneumonia Viral/tratamento farmacológico , Inibidores de Proteases/farmacologia , Replicação Viral/efeitos dos fármacos , Betacoronavirus/efeitos dos fármacos , Células Cultivadas , Coronavirus Humano 229E/enzimologia , Coronavirus Humano 229E/fisiologia , Meios de Cultivo Condicionados , Células Epiteliais/virologia , Gabexato/farmacologia , Humanos , Mucosa Nasal/citologia , Pandemias , Cultura Primária de Células , Serina Endopeptidases/fisiologia , Glicoproteína da Espícula de Coronavírus/metabolismo , Carga Viral
4.
Arch Virol ; 165(6): 1333-1342, 2020 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-32266552

RESUMO

Equine infectious anemia (EIA), a disease caused by equine infectious anemia virus (EIAV), is considered an obstacle to the development of the horse industry. There is no treatment or vaccine available for EIA, and its pathogenesis, as well as the immune response against the virus, is not fully understood. Therefore, an immunohistochemistry assay was developed for the detection of viral antigens in tissues of equids naturally infected with EIAV. Sections of organs of six equids from Apodi-RN, Brazil, that tested positive for EIA by serological tests (ELISA and AGID) were fixed in 10% formalin solution and embedded in paraffin. Immunohistochemistry was performed using a polyclonal anti-EIAV antibody. EIAV antigens were observed in red spleen pulp cells and hepatic sinusoids, as well as bronchiolar and alveolar epithelial cells of the lungs and proximal and distal tubules of the kidneys. The presence of EIAV in the spleen and liver was expected due to viral tropism by macrophages, which are abundantly present in these organs. However, EIAV was also found in lung and kidney epithelial cells, indicating that the virus infects cell types other than macrophages. In conclusion, the immunohistochemical assay standardized in this study was able to detect EIAV antigens in spleen, liver, kidney and lung cells from naturally infected EIAV equids. Immunostaining observed in the spleen confirms viral tropism by mononuclear phagocytes; however, the presence of EIAV in lung and kidney epithelial cells indicates that virus may be eliminated in urine and/or oronasal secretions, suggesting new routes for viral excretion.


Assuntos
Anemia Infecciosa Equina/virologia , Vírus da Anemia Infecciosa Equina/isolamento & purificação , Animais , Antígenos Virais/análise , Brasil , DNA Viral/genética , Células Epiteliais/patologia , Células Epiteliais/virologia , Anemia Infecciosa Equina/imunologia , Anemia Infecciosa Equina/patologia , Cavalos/virologia , Vírus da Anemia Infecciosa Equina/classificação , Rim/patologia , Rim/virologia , Leucócitos Mononucleares/virologia , Fígado/patologia , Fígado/virologia , Pulmão/patologia , Pulmão/virologia , Reação em Cadeia da Polimerase , Testes Sorológicos , Baço/patologia , Baço/virologia
6.
Sichuan Da Xue Xue Bao Yi Xue Ban ; 51(2): 171-177, 2020 Mar.
Artigo em Chinês | MEDLINE | ID: mdl-32220184

RESUMO

Objective: To investigate the effects of dihydroartemis (DHA) on influenza A virus (IAV) A/PR/8/34 (H1N1) induces the pro-inflammatory factor and protein of extracellular signal regulated kinase (ERK) signaling pathway expression in bronchial epithelial cells. Methods: The BEAS-2B cells were treated with different concentrations of DHA (i.e.,0, 12.5, 25,50 and 100 µmol/L) for 24 h and the effect of DHA on the viability of BEAS-2B cells were measure by CCK8 method. The BEAS-2B cells were absorbed with IAV for 1 h, and then were treated with different concentrations of DHA (i.e., 12.5, 25 and 50 µmol/L) for 24 h, meanwhile, the normal control group and IAV group were established. The mRNA and protein expression levels of tumor necrosis factor-α (TNF-α) and interleukin (IL-6) were measured by real time quantitative PCR (RT-qPCR) and enzyme linked immunosorbent assay (ELISA), the expression levels of phospho-ERK (p-ERK) proteins were tested by Western blot (WB). Then, an ERK agonist (20 ng/mL) was used to treat BEAS-2B cells (the groups were divided into normal control group, DHA group, DHA+IAV group, ERK agonist group and DHA+IAV+ERK agonist group) for 24 h, and to observe the effect of DHA on inhibiting IAV induce the TNF-α, IL-6 and p-ERK expression in the BEAS-2B cells. Results: The BEAS-2B cells viability was not significantly different from that of the normal control group after treatment with DHA (i.e., 12.5, 25, and 50 µmol/L). The expression levels of TNF-α, IL-6 mRNA and TNF-α, IL-6, p-ERK protein in IAV group were significantly up-regulated compared with that in the normal control group ( P<0.05), meanwhile, compared with the IAV group, the expression levels of TNF-α, IL-6 mRNA and TNF-α, IL-6, p-ERK protein showed dose-dependent decrease in IAV+DHA group ( P<0.05). However, ERK agonists attenuated the DHA inhibit IAV induced the proinflammatory factors TNF-α, IL-6 secretion and the p-ERK protein expression of ERK signaling pathway in BEAS-2B cells. Conclusion: These data suggest that DHA can inhibit IAV induces the TNF-α and IL-6 expression in BEAS-2B cells through ERK signaling pathway.


Assuntos
Antivirais/farmacologia , Artemisininas/farmacologia , Vírus da Influenza A Subtipo H1N1/patogenicidade , Vírus da Influenza A , Interleucina-6/metabolismo , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Fator de Necrose Tumoral alfa/metabolismo , Brônquios , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/virologia , Humanos , Fator de Transcrição STAT1
7.
Int J Oral Sci ; 12(1): 8, 2020 02 24.
Artigo em Inglês | MEDLINE | ID: mdl-32094336

RESUMO

It has been reported that ACE2 is the main host cell receptor of 2019-nCoV and plays a crucial role in the entry of virus into the cell to cause the final infection. To investigate the potential route of 2019-nCov infection on the mucosa of oral cavity, bulk RNA-seq profiles from two public databases including The Cancer Genome Atlas (TCGA) and Functional Annotation of The Mammalian Genome Cap Analysis of Gene Expression (FANTOM5 CAGE) dataset were collected. RNA-seq profiling data of 13 organ types with para-carcinoma normal tissues from TCGA and 14 organ types with normal tissues from FANTOM5 CAGE were analyzed in order to explore and validate the expression of ACE2 on the mucosa of oral cavity. Further, single-cell transcriptomes from an independent data generated in-house were used to identify and confirm the ACE2-expressing cell composition and proportion in oral cavity. The results demonstrated that the ACE2 expressed on the mucosa of oral cavity. Interestingly, this receptor was highly enriched in epithelial cells of tongue. Preliminarily, those findings have explained the basic mechanism that the oral cavity is a potentially high risk for 2019-nCoV infectious susceptibility and provided a piece of evidence for the future prevention strategy in dental clinical practice as well as daily life.


Assuntos
Betacoronavirus , Infecções por Coronavirus/transmissão , Mucosa Bucal , Peptidil Dipeptidase A/metabolismo , Pneumonia Viral/transmissão , Língua , Betacoronavirus/patogenicidade , Infecções por Coronavirus/genética , Bases de Dados Genéticas , Células Epiteliais/metabolismo , Células Epiteliais/virologia , Perfilação da Expressão Gênica , Humanos , Mucosa Bucal/enzimologia , Mucosa Bucal/virologia , Pneumonia Viral/genética , Língua/metabolismo , Língua/virologia
8.
N Engl J Med ; 382(8): 727-733, 2020 02 20.
Artigo em Inglês | MEDLINE | ID: mdl-31978945

RESUMO

In December 2019, a cluster of patients with pneumonia of unknown cause was linked to a seafood wholesale market in Wuhan, China. A previously unknown betacoronavirus was discovered through the use of unbiased sequencing in samples from patients with pneumonia. Human airway epithelial cells were used to isolate a novel coronavirus, named 2019-nCoV, which formed a clade within the subgenus sarbecovirus, Orthocoronavirinae subfamily. Different from both MERS-CoV and SARS-CoV, 2019-nCoV is the seventh member of the family of coronaviruses that infect humans. Enhanced surveillance and further investigation are ongoing. (Funded by the National Key Research and Development Program of China and the National Major Project for Control and Prevention of Infectious Disease in China.).


Assuntos
Betacoronavirus/isolamento & purificação , Infecções por Coronavirus/virologia , Pulmão/diagnóstico por imagem , Pneumonia Viral/virologia , Adulto , Betacoronavirus/genética , Betacoronavirus/ultraestrutura , Líquido da Lavagem Broncoalveolar/virologia , Células Cultivadas , China , Infecções por Coronavirus/diagnóstico por imagem , Infecções por Coronavirus/patologia , Células Epiteliais/patologia , Células Epiteliais/virologia , Feminino , Genoma Viral , Humanos , Pulmão/patologia , Pulmão/virologia , Masculino , Microscopia Eletrônica de Transmissão , Pessoa de Meia-Idade , Filogenia , Pneumonia Viral/diagnóstico por imagem , Pneumonia Viral/patologia , Radiografia Torácica , Sistema Respiratório/patologia , Sistema Respiratório/virologia
9.
Vet Res ; 50(1): 110, 2019 Dec 19.
Artigo em Inglês | MEDLINE | ID: mdl-31856906

RESUMO

Intestinal epithelium functions as a barrier to protect multicellular organisms from the outside world. It consists of epithelial cells closely connected by intercellular junctions, selective gates which control paracellular diffusion of solutes, ions and macromolecules across the epithelium and keep out pathogens. Rotavirus is one of the major enteric viruses causing severe diarrhea in humans and animals. It specifically infects the enterocytes on villi of small intestines. The polarity of rotavirus replication in their target enterocytes and the role of intestinal epithelial integrity were examined in the present study. Treatment with EGTA, a drug that chelates calcium and disrupts the intercellular junctions, (i) significantly enhanced the infection of rotavirus in primary enterocytes, (ii) increased the binding of rotavirus to enterocytes, but (iii) considerably blocked internalization of rotavirus. After internalization, rotavirus was resistant to EGTA treatment. To investigate the polarity of rotavirus infection, the primary enterocytes were cultured in a transwell system and infected with rotavirus at either the apical or the basolateral surface. Rotavirus preferentially infected enterocytes at the basolateral surface. Restriction of infection through apical inoculation was overcome by EGTA treatment. Overall, our findings demonstrate that integrity of the intestinal epithelium is crucial in the host's innate defense against rotavirus infection. In addition, the intercellular receptor is located basolaterally and disruption of intercellular junctions facilitates the binding of rotavirus to their receptor at the basolateral surface.


Assuntos
Enterócitos/virologia , Células Epiteliais/virologia , Mucosa Intestinal/citologia , Rotavirus/classificação , Rotavirus/fisiologia , Animais , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Técnicas de Cocultura/veterinária , Ácido Egtázico/farmacologia , Enterócitos/efeitos dos fármacos , Miofibroblastos/fisiologia , Suínos , Internalização do Vírus , Replicação Viral
10.
Vet Microbiol ; 239: 108462, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31767100

RESUMO

In contrast to human influenza viruses that replicate in the respiratory tract and are airborne transmitted, avian viruses also replicate in gut epithelial cells and are transmitted via the fecal-oral route. On this route, the virus is exposed to destructive fluids of the digestive tract, which are acidic and contain the proteases pepsin (gizzard) or chymotrypsin and trypsin (intestine). Only the latter enzyme activates virus by cleaving hemagglutinin (HA) into HA1 and HA2 subunits. We mimicked the passage of viruses through the gastrointestinal tract by treating them with digestive fluids from chicken and determined titers and integrity of HA by western-blot. Gizzard fluid completely inactivated virions and degrades HA even at a high dilution, but only if the pH was kept acidic. If the fluid is diluted with neutral buffer (mimicking virus uptake with seawater) particles were more resistant. Virions containing an uncleaved HA were even activated suggesting that gastric juice contains a trypsin-like protease. Undiluted intestinal fluid inactivated particles and destroyed HA, but diluted fluid activated virions. A virus isolated from the duck´s intestine is more tolerant against intestinal fluid compared to fowl plague virus suggesting that the former is better adapted to grow in the intestine. We also demonstrate that influenza viruses replicate to high titers in a novel chicken epithelial gut cell line. While viruses with a monobasic HA cleavage site require addition of trypsin, these cells effectively process HA with a polybasic cleavage site, which could be blocked with an inhibitor of the cellular furin protease.


Assuntos
Trato Gastrointestinal/virologia , Hemaglutininas/metabolismo , Influenza Aviária/virologia , Animais , Galinhas , Células Epiteliais/citologia , Células Epiteliais/virologia , Suco Gástrico/química , Suco Gástrico/enzimologia , Concentração de Íons de Hidrogênio , Técnicas In Vitro , Secreções Intestinais/química , Secreções Intestinais/enzimologia , Inativação de Vírus , Replicação Viral/fisiologia
11.
Vet Microbiol ; 237: 108400, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31585640

RESUMO

The entry mechanism of porcine epidemic diarrhea virus (PEDV) remains unclear, especially the virus receptor. Our previous study revealed a potential correlation between integrin αvß3 and PEDV infection. In the current study, the effect of overexpression, silencing, antibody inhibition, and co-expression with porcine aminopeptidase N (pAPN) of integrin αvß3 on PEDV infection was investigated and analyzed in African green monkey Vero E6 cells and porcine intestinal epithelial cells (IECs) using the classical strain CV777 and variant strain HM2017 of PEDV. Integrin αvß3 significantly enhanced the replication of the classical and variant strains of PEDV in Vero E6 cells and IECs. The integrin αv and ß3 subunits were both involved in the enhancement of PEDV infection, the Arg-Gly-Asp peptides targeting integrin αvß3 significantly inhibited replication of PEDV in Vero E6 cells, and co-expression of integrin αvß3 with pAPN significantly enhanced replication of PEDV in Vero E6 and BHK-21 cells. These results demonstrate that integrin αvß3 enhances PEDV replication in Vero E6 cells and IECs. These data provide novel insights into the entry mechanism of PEDV.


Assuntos
Células Epiteliais/virologia , Integrina alfaVbeta3/metabolismo , Vírus da Diarreia Epidêmica Suína/fisiologia , Cultura de Vírus/veterinária , Replicação Viral/fisiologia , Animais , Regulação da Expressão Gênica , Inativação Gênica , Mucosa Intestinal/citologia , Vírus da Diarreia Epidêmica Suína/classificação , Suínos , Células Vero
12.
BMC Res Notes ; 12(1): 639, 2019 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-31570108

RESUMO

OBJECTIVE: Survivors of Ebola virus disease (EVD) are at risk of developing blinding intraocular inflammation-or uveitis-which is associated with retinal pigment epithelial (RPE) scarring and persistence of live Zaire ebolavirus (EBOV) within the eye. As part of a large research project aimed at defining the human RPE cell response to being infected with EBOV, this work focused on the microRNAs (miRNAs) associated with the infection. RESULTS: Using RNA-sequencing, we detected 13 highly induced and 2 highly repressed human miRNAs in human ARPE-19 RPE cells infected with EBOV, including hsa-miR-1307-5p, hsa-miR-29b-3p and hsa-miR-33a-5p (up-regulated), and hsa-miR-3074-3p and hsa-miR-27b-5p (down-regulated). EBOV-miR-1-5p was also found in infected RPE cells. Through computational identification of putative miRNA targets, we predicted a broad range of regulatory activities, including effects on innate and adaptive immune responses, cellular metabolism, cell cycle progression, apoptosis and autophagy. The most highly-connected molecule in the miR-target network was leucine-rich repeat kinase 2, which is involved in neuroinflammation and lysosomal processing. Our findings should stimulate new studies on the impact of miRNA changes in EBOV-infected RPE cells to further understanding of intraocular viral persistence and the pathogenesis of uveitis in EVD survivors.


Assuntos
Ebolavirus/genética , Células Epiteliais/metabolismo , Regulação da Expressão Gênica , Redes Reguladoras de Genes , Interações Hospedeiro-Patógeno/genética , MicroRNAs/genética , Imunidade Adaptativa/genética , Apoptose/genética , Autofagia/genética , Ciclo Celular/genética , Linhagem Celular , Ebolavirus/crescimento & desenvolvimento , Ebolavirus/patogenicidade , Células Epiteliais/imunologia , Células Epiteliais/virologia , Perfilação da Expressão Gênica , Interações Hospedeiro-Patógeno/imunologia , Humanos , Imunidade Inata/genética , MicroRNAs/classificação , MicroRNAs/imunologia , Pigmentos da Retina , Transdução de Sinais
13.
PLoS Pathog ; 15(10): e1008071, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31584998

RESUMO

The Epstein Barr virus (EBV) is linked to the development of two major epithelial malignancies, gastric carcinoma and nasopharyngeal carcinoma. This study evaluates the effects of EBV on cellular expression in a gastric epithelial cell line infected with or without EBV and a nasopharyngeal carcinoma cell line containing EBV. The cells were grown in vitro and as tumors in vivo. The effects on cellular expression were determined using both 2D DIGE proteomics and high throughput RNA sequencing. The data identify multiple pathways that were uniquely activated in vitro. RNA sequences mapping to the mouse genome were identified in both the EBV positive and negative tumor samples in vivo, although, differences between the EBV positive and negative cells were not apparent. However, the tumors appeared to be grossly distinct. The majority of the identified canonical pathways based on two fold changes in expression had decreased activity within the tumors in vivo. Identification of the predicted upstream regulating factors revealed that in vitro the regulating factors were primarily protein transcriptional regulators. In contrast, in vivo the predicted regulators were frequently noncoding RNAs. Hierarchical clustering distinguished the cell lines and tumors, the EBV positive tumors from the EBV negative tumors, and the NPC tumors from the gastric tumors and cell lines. The delineating genes were changed greater than 4 fold and were frequently regulated by protein transcription factors. These data suggest that EBV distinctly affects cellular expression in gastric tumors and NPC and that growth in vivo requires activation of fewer cellular signaling pathways. It is likely that the broad changes in cellular expression that occur at low levels are controlled by regulatory viral and cellular RNAs while major changes are affected by induced protein regulators.


Assuntos
Biomarcadores/análise , Células Epiteliais/metabolismo , Infecções por Vírus Epstein-Barr/metabolismo , Regulação Viral da Expressão Gênica , Herpesvirus Humano 4/isolamento & purificação , Neoplasias Nasofaríngeas/metabolismo , Neoplasias Gástricas/metabolismo , Animais , Apoptose , Proliferação de Células , Células Epiteliais/virologia , Infecções por Vírus Epstein-Barr/genética , Infecções por Vírus Epstein-Barr/virologia , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Neoplasias Nasofaríngeas/genética , Neoplasias Nasofaríngeas/virologia , Neoplasias Gástricas/genética , Neoplasias Gástricas/virologia , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de Xenoenxerto
14.
Virus Genes ; 55(6): 779-785, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31552622

RESUMO

Epstein-Barr virus (EBV) is a widely prevalent pathogen currently infecting over 90% of the human population and is associated with various lymphomas and carcinomas. Lytic replication of EBV is regulated by the expression of the immediate-early genes BZLF1 and BRLF1. In B lymphocytes, BZLF1 transcripts have been shown to be processed to a fully spliced form, as well as zDelta, a spliced variant containing only the first and third exons. While splice variants have been reported in nasopharyngeal carcinoma biopsies, alternative splicing of BZLF1 in EBV-positive epithelial cell lines has not yet been characterized. In this study, we identified the consistent expression of three distinct BZLF1 transcripts in the EBV-positive epithelial cell lines D98/HR1, AGS-BDneo, and AGS-BX1. These BZLF1 transcripts consisted of not only the normally spliced variant but also a completely unspliced and a spliced variant containing exons one and three only. In contrast, we detected only the normally spliced version of the BZLF1 transcript in B-cell lines (B95-8, IM-9, Raji and Daudi). Previous work has also demonstrated that inhibition of the mTOR pathway, via rapamycin, altered total levels of BZLF1 transcripts. We examined the production of specific transcript variants under rapamycin treatment and found that rapamycin alters the production of transcripts in a cell-type, as well as transcripts in variant-type, manners. The expression of these transcript variants may play a role in modulating the replication cycle of EBV within epithelial cells.


Assuntos
Células Epiteliais/virologia , Infecções por Vírus Epstein-Barr/genética , Herpesvirus Humano 4/genética , Transativadores/genética , Linfócitos B/virologia , Linhagem Celular , Células Epiteliais/efeitos dos fármacos , Infecções por Vírus Epstein-Barr/virologia , Regulação Viral da Expressão Gênica/genética , Herpesvirus Humano 4/efeitos dos fármacos , Herpesvirus Humano 4/patogenicidade , Humanos , Regiões Promotoras Genéticas/genética , Transdução de Sinais/efeitos dos fármacos , Sirolimo/farmacologia , Serina-Treonina Quinases TOR/genética , Ativação Viral/genética , Latência Viral/genética
15.
Nat Commun ; 10(1): 4066, 2019 09 06.
Artigo em Inglês | MEDLINE | ID: mdl-31492846

RESUMO

Human enteroviruses (HEVs) of the family Picornaviridae, which comprises non-enveloped RNA viruses, are ubiquitous worldwide. The majority of EV proteins are derived from viral polyproteins encoded by a single open reading frame (ORF). Here, we characterize a second ORF in HEVs that is crucial for viral intestinal infection. Disruption of ORF2p expression decreases the replication capacity of EV-A71 in human intestinal epithelial cells (IECs). Ectopic expression of ORF2p proteins derived from diverse enteric enteroviruses sensitizes intestinal cells to the replication of ORF2p-defective EV-A71 and respiratory enterovirus EV-D68. We show that the highly conserved WIGHPV domain of ORF2p is important for ORF2p-dependent viral intestinal infection. ORF2p expression is required for EV-A71 particle release from IECs and can support productive EV-D68 infection in IECs by facilitating virus release. Our results indicate that ORF2p is a determining factor for enteric enterovirus replication in IECs.


Assuntos
Enterovirus/genética , Fases de Leitura Aberta/genética , Vírus de RNA/genética , Replicação Viral/genética , Sequência de Aminoácidos , Sequência de Bases , Enterovirus/fisiologia , Infecções por Enterovirus/transmissão , Infecções por Enterovirus/virologia , Células Epiteliais/virologia , Fezes/virologia , Células HT29 , Interações Hospedeiro-Patógeno/genética , Humanos , Intestinos/citologia , Intestinos/virologia , Vírus de RNA/fisiologia , Proteínas Virais/genética , Proteínas Virais/metabolismo
16.
Immunopharmacol Immunotoxicol ; 41(5): 527-537, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31505962

RESUMO

Background: Pattern recognition receptors form an essential part of the host defenses against pathogens, in particular in the intestinal epithelium. However, despite their importance relatively little is understood about the regulation of their expression. Increasing evidence suggesting that epigenetic mechanisms such as DNA methylation and histone acetylation have substantial effects on gene expression and regulation. Epigenetic modifying drugs are now used to treat certain cancers but not a lot is known about their effects on the innate immune system. Thus, we set out to examine the role of such drugs in the expression and function of Toll-like receptors. Methods: Using the HCT116 epithelial cell line, we determined the effects of genetic knockout of the DNA methyltransferases enzymes (DNMTs), as well as pharmacological inhibition of the DNMTs and histone deacetylase complexes (HDACs) on TLR responses to their ligands. Results: Our initial results showed that anti-viral responses were affected by changes in the epigenome, with TLR3 responses showing the most dramatic differences. We determined that inhibition of methylation and acetylation inhibited poly I:C induced increases in signaling protein phosphorylation, as well as increases in cytokine mRNA expression and release. We also observed that treatment with epigenetic modifying drugs were leading to large increases in IRF8 expression, a protein that is a known negative regulator of TLR3. When we overexpressed IRF8 in our WT cells we noticed inhibition of poly I:C responses. Conclusion: This research highlighted the potential immunoregulatory role of epigenetic modifying drugs specifically in response to viral stimulation.


Assuntos
Antivirais/farmacologia , Epigênese Genética/efeitos dos fármacos , Células Epiteliais/efeitos dos fármacos , Inibidores de Histona Desacetilases/farmacologia , Poli I-C/farmacologia , Receptor 3 Toll-Like/metabolismo , DNA (Citosina-5-)-Metiltransferases/antagonistas & inibidores , DNA (Citosina-5-)-Metiltransferases/genética , Metilação de DNA/efeitos dos fármacos , Decitabina/farmacologia , Células Epiteliais/enzimologia , Células Epiteliais/imunologia , Células Epiteliais/virologia , Técnicas de Silenciamento de Genes , Células HCT116 , Histona Desacetilases/metabolismo , Humanos , Ácidos Hidroxâmicos/farmacologia , Imunidade Inata/efeitos dos fármacos , Imunidade Inata/genética , Mucosa Intestinal/efeitos dos fármacos , Mucosa Intestinal/enzimologia , Mucosa Intestinal/imunologia , Mucosa Intestinal/virologia , Proteínas Repressoras/antagonistas & inibidores , Proteínas Repressoras/genética , Receptor 3 Toll-Like/genética
17.
Biomed Pharmacother ; 118: 109359, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31545243

RESUMO

As one of the highly contagious forms, herpes simplex virus type 2 (HSV-2) commonly caused severe genital diseases and closely referred to the HIV infection. The lack of effective vaccines and drug-resistance proclaimed the preoccupation for alternative antiviral agents against HSV-2. Molecules bearing indole nucleus presented diverse biological properties involving antiviral and anti-inflammatory activities. In this study, one of the indole molecules, arbidol derivative (ARD) was designed and synthesized prior to the evaluation of its anti-HSV-2 activity. Our data showed that the ARD effectively suppressed HSV-2-induced cytopathic effects and the generation of progeny virus, with 50% effective concentrations of 3.386 and 1.717 µg/mL, respectively. The results of the time-course assay suggested that the ARD operated in a dual antiviral way by interfering virus entry and impairing the earlier period of viral cycle during viral DNA synthesis. The ARD-mediated HSV-2 inhibition was partially attained by blocking NF-κB pathways and down-regulating the expressions of several inflammatory cytokines. Furthermore, in vivo studies showed that oral administration of ARD protected BALB/c mice from intravaginal HSV-2 challenge by alleviating serious vulval lesions and histopathological changes in the target organs. Besides, the treatment with ARD also made the levels of viral protein, NF-κB protein and inflammatory cytokines lower, in consistent with the in-vitro studies. Collectively, ARD unveiled therapeutic potential for the prevention and treatment of HSV-2 infections.


Assuntos
Colo do Útero/patologia , Colo do Útero/virologia , Células Epiteliais/virologia , Herpesvirus Humano 2/efeitos dos fármacos , Indóis/farmacologia , Animais , Antivirais/química , Antivirais/farmacologia , Citocinas/metabolismo , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/patologia , Feminino , Humanos , Indóis/química , Indóis/toxicidade , Camundongos Endogâmicos BALB C , NF-kappa B/metabolismo , Receptores Toll-Like/metabolismo , Vagina/efeitos dos fármacos , Vagina/patologia , Vagina/virologia , Replicação Viral/efeitos dos fármacos
18.
PLoS Pathog ; 15(8): e1007963, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31381610

RESUMO

Human respiratory syncytial virus (RSV) is the leading viral cause of acute pediatric lower respiratory tract infections worldwide, with no available vaccine or effective antiviral drug. To gain insight into virus-host interactions, we performed a genome-wide siRNA screen. The expression of over 20,000 cellular genes was individually knocked down in human airway epithelial A549 cells, followed by infection with RSV expressing green fluorescent protein (GFP). Knockdown of expression of the cellular ATP1A1 protein, which is the major subunit of the Na+,K+-ATPase of the plasma membrane, had one of the strongest inhibitory effects on GFP expression and viral titer. Inhibition was not observed for vesicular stomatitis virus, indicating that it was RSV-specific rather than a general effect. ATP1A1 formed clusters in the plasma membrane very early following RSV infection, which was independent of replication but dependent on the attachment glycoprotein G. RSV also triggered activation of ATP1A1, resulting in signaling by c-Src-kinase activity that transactivated epidermal growth factor receptor (EGFR) by Tyr845 phosphorylation. ATP1A1 signaling and activation of both c-Src and EGFR were found to be required for efficient RSV uptake. Signaling events downstream of EGFR culminated in the formation of macropinosomes. There was extensive uptake of RSV virions into macropinosomes at the beginning of infection, suggesting that this is a major route of RSV uptake, with fusion presumably occurring in the macropinosomes rather than at the plasma membrane. Important findings were validated in primary human small airway epithelial cells (HSAEC). In A549 cells and HSAEC, RSV uptake could be inhibited by the cardiotonic steroid ouabain and the digitoxigenin derivative PST2238 (rostafuroxin) that bind specifically to the ATP1A1 extracellular domain and block RSV-triggered EGFR Tyr845 phosphorylation. In conclusion, we identified ATP1A1 as a host protein essential for macropinocytic entry of RSV into respiratory epithelial cells, and identified PST2238 as a potential anti-RSV drug.


Assuntos
Pinocitose , Infecções por Vírus Respiratório Sincicial/complicações , Vírus Sincicial Respiratório Humano/patogenicidade , Infecções Respiratórias/prevenção & controle , ATPase Trocadora de Sódio-Potássio/antagonistas & inibidores , Proteínas Virais/metabolismo , Internalização do Vírus , Células A549 , Cardiotônicos/farmacologia , Digitoxigenina/química , Digitoxigenina/farmacologia , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/enzimologia , Células Epiteliais/virologia , Receptores ErbB/metabolismo , Ensaios de Triagem em Larga Escala , Humanos , Ouabaína/farmacologia , RNA Interferente Pequeno/genética , Infecções por Vírus Respiratório Sincicial/virologia , Sistema Respiratório/efeitos dos fármacos , Sistema Respiratório/enzimologia , Sistema Respiratório/virologia , Infecções Respiratórias/epidemiologia , Infecções Respiratórias/virologia , Transdução de Sinais , ATPase Trocadora de Sódio-Potássio/genética , Proteínas Virais/genética
19.
Virol J ; 16(1): 99, 2019 08 08.
Artigo em Inglês | MEDLINE | ID: mdl-31395061

RESUMO

BACKGROUND: Both vector borne and sexual transmission of Zika virus (ZIKV) involve infection of epithelial cells in the initial stages of infection. Epithelial cells are unique in their ability to form polarized monolayers and their barrier function. Cell polarity induces an asymmetry in the epithelial monolayer, which is maintained by tight junctions and specialized sorting machinery. This differential localization can have a potential impact of virus infection. Asymmetrical distribution of a viral receptor can restrict virus entry to a particular membrane while polarized sorting can lead to a directional release of virions. The present study examined the impact of cell polarity on ZIKV infection and release. METHODS: A polarized Caco-2 cell model we described previously was used to assess ZIKV infection. Transepithelial resistance (TEER) was used to assess epithelial cell polarity, and virus infection was measured by immunofluorescence microscopy and qRT-PCR. Cell permeability was measured using a fluorescein leakage assay. Statistical significance was calculated using one-way ANOVA and significance was set at p < 0.05. RESULTS: Using the Caco-2 cell model for polarized epithelial cells, we report that Zika virus preferentially infects polarized cells from the apical route and is released vectorially through the basolateral route. Our data also indicates that release occurs without disruption of cell permeability. CONCLUSIONS: Our results show that ZIKV has directional infection and egress in a polarized cell system. This mechanism of directional infection may be one of the mechanisms that enables the cross the epithelial barrier effectively without a disruption in cell monolayer integrity. Elucidation of entry and release characteristics of Zika virus in polarized epithelial cells can lead to better understanding of virus dissemination in the host, and can help in developing effective therapeutic interventions.


Assuntos
Polaridade Celular , Células Epiteliais/virologia , Internalização do Vírus , Zika virus/fisiologia , Células CACO-2 , Humanos , Microscopia de Fluorescência , Receptores Virais/fisiologia
20.
Vet Microbiol ; 235: 220-228, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31383305

RESUMO

The highly virulent porcine epidemic diarrhea virus (PEDV) variants cause the death of mainly neonatal piglets, but how the viruses spread within the gastro-intestinal tract in a temporal and spatial manner has remained poorly characterized but is critical to understand the viral pathogenesis. In this study, we used the Chinese PEDV epidemic strain BJ2011C as a model organism and took advantage of the newly developed RNAscope in situ hybridization technology to investigate the tempo-spatial infection dynamics in neonatal piglets. We found that the PEDV strain BJ2011C could quickly colonize the small intestine, which occurred in just 6 h post infection, with virus shedding starting at 6 hpi and peaking at 24 hpi. Jejunum was the first target tissue for infection and then ileum, followed by infrequent infection of duodenum. In these tissues, the virus nucleic acids were mainly present in the villous epithelial cells but not in crypt cells. Interestingly, the viral RNAs were not detectable by RNAscope in large intestines although tissue damages could be discerned by H & E staining. Overall, our results provide useful information about spread dynamics and tissue preference of PEDV epidemic strain BJ2011C.


Assuntos
Infecções por Coronavirus/patologia , Intestino Delgado/virologia , Vírus da Diarreia Epidêmica Suína/patogenicidade , RNA Viral/isolamento & purificação , Doenças dos Suínos/patologia , Animais , Animais Recém-Nascidos , Diarreia/veterinária , Diarreia/virologia , Células Epiteliais/virologia , Hibridização in Situ Fluorescente , Jejuno/citologia , Jejuno/virologia , Suínos , Doenças dos Suínos/virologia , Eliminação de Partículas Virais
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