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1.
PLoS Pathog ; 15(10): e1008093, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31600344

RESUMO

ISG20 is a broad spectrum antiviral protein thought to directly degrade viral RNA. However, this mechanism of inhibition remains controversial. Using the Vesicular Stomatitis Virus (VSV) as a model RNA virus, we show here that ISG20 interferes with viral replication by decreasing protein synthesis in the absence of RNA degradation. Importantly, we demonstrate that ISG20 exerts a translational control over a large panel of non-self RNA substrates including those originating from transfected DNA, while sparing endogenous transcripts. This activity correlates with the protein's ability to localize in cytoplasmic processing bodies. Finally, these functions are conserved in the ISG20 murine ortholog, whose genetic ablation results in mice with increased susceptibility to viral infection. Overall, our results posit ISG20 as an important defense factor able to discriminate the self/non-self origins of the RNA through translation modulation.


Assuntos
Antivirais/farmacologia , Exorribonucleases/farmacologia , Biossíntese de Proteínas , RNA Viral/metabolismo , Estomatite Vesicular/imunologia , Vesiculovirus/imunologia , Replicação Viral/efeitos dos fármacos , Animais , Exorribonucleases/fisiologia , Células HeLa , Humanos , Camundongos , Camundongos Knockout , Estabilidade de RNA , RNA Viral/genética , Estomatite Vesicular/tratamento farmacológico , Estomatite Vesicular/virologia , Vesiculovirus/efeitos dos fármacos
2.
Mol Cell ; 73(4): 803-814.e6, 2019 02 21.
Artigo em Inglês | MEDLINE | ID: mdl-30639243

RESUMO

Intron retention (IR) has emerged as an important mechanism of gene expression control, but the factors controlling IR events remain poorly understood. We observed consistent IR in one intron of the Irf7 gene and identified BUD13 as an RNA-binding protein that acts at this intron to increase the amount of successful splicing. Deficiency in BUD13 was associated with increased IR, decreased mature Irf7 transcript and protein levels, and consequently a dampened type I interferon response, which compromised the ability of BUD13-deficient macrophages to withstand vesicular stomatitis virus (VSV) infection. Global analysis of BUD13 knockdown and BUD13 cross-linking to RNA revealed a subset of introns that share many characteristics with the one found in Irf7 and are spliced in a BUD13-dependent manner. Deficiency of BUD13 led to decreased mature transcript from genes containing such introns. Thus, by acting as an antagonist to IR, BUD13 facilitates the expression of genes at which IR occurs.


Assuntos
Fator Regulador 7 de Interferon/metabolismo , Interferon Tipo I/metabolismo , Íntrons , Macrófagos/metabolismo , Proteínas de Ligação a RNA/metabolismo , Estomatite Vesicular/metabolismo , Vírus da Estomatite Vesicular Indiana/patogenicidade , Animais , Sítios de Ligação , Sequência Rica em GC , Células HEK293 , Interações Hospedeiro-Patógeno , Humanos , Fator Regulador 7 de Interferon/genética , Interferon Tipo I/imunologia , Macrófagos/imunologia , Macrófagos/virologia , Camundongos Endogâmicos C57BL , Ligação Proteica , Sítios de Splice de RNA , Processamento de RNA , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/genética , Células Vero , Estomatite Vesicular/genética , Estomatite Vesicular/imunologia , Estomatite Vesicular/virologia , Vírus da Estomatite Vesicular Indiana/imunologia
3.
J Virol Methods ; 265: 113-116, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30639413

RESUMO

This study reports the use of a site-specific recombination cloning technique for rapid development of a full-length cDNA clone that can produce infectious vesicular stomatitis New Jersey virus (VSNJV). The full-length genome of the epidemic VSNJV NJ0612NME6 strain was amplified in four overlapping cDNA fragments which were linked together and cloned into a vector plasmid by site-specific recombination. Furthermore, to derive infectious virus, three supporting plasmid vectors containing either the nucleoprotein (N), phosphoprotein (P) or polymerase (L) genes were constructed using the same cloning methodology. Recovery of recombinant VSNJV was achieved after transfecting all four vectors on into BSR-T7/5 cells, a BHK-derived cell line stably expressing T7 RNA polymerase (PMID: 9847328). In vitro characterization of recombinant and parental viruses revealed similar growth kinetics and plaque morphologies. Furthermore, experimental infection of pigs with the recombinant virus resulted in severe vesicular stomatitis with clinical signs similar to those previously reported for the parental field strain. These results validate the use of site-directed specific recombination cloning as a useful alternative method for rapid construction of stable full-length cDNA clones from vesicular stomatitis field strains. The approach reported herein contributes to the improvement of previously published methodologies for the development of full-length cDNA clones of this relevant virus.


Assuntos
Clonagem Molecular , DNA Complementar/genética , Biologia Molecular/métodos , Recombinação Genética , Vírus da Estomatite Vesicular New Jersey/crescimento & desenvolvimento , Vírus da Estomatite Vesicular New Jersey/genética , Virologia/métodos , Animais , Linhagem Celular , Cricetinae , Suínos , Doenças dos Suínos/patologia , Doenças dos Suínos/virologia , Estomatite Vesicular/patologia , Estomatite Vesicular/virologia , Ensaio de Placa Viral
4.
Prev Vet Med ; 160: 68-75, 2018 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-30389000

RESUMO

The aim of this survey was to estimate the apparent herd-level and animal-level prevalences, as well as to identify risk factors and spatial clustering of vesicular stomatitis virus (VSV) positive herds in the state of Paraíba, semiarid of Brazil. The state was divided into three sampling strata: Sertão, Borborema and Zona da Mata/Agreste. For each sampling stratum, herd-level and animal-level prevalences were estimated by a two-stage sampling survey. First, a pre-established number of herds (primary sampling units) were randomly selected; second, within each herd, a pre-established number of cows aged ≥ 24 months were systematically selected (secondary sampling units). In total, 2279 animals were sampled from 468 herds. Serum samples were submitted to virus neutralization (VN) test for detection of antibodies to VSV using three viral strains: VSIV-3 2013SaoBento/Paraiba E, strain Indiana (VSIV-1) and VSNJV. A herd was considered positive for VSV if it included at least one positive animal in herds of up to 10 females, two positive animals in herds of 11-99 females, and three positive in herds with more than 99 females. The spatial clustering was assessed using the Cuzick-Edwards' k-nearest neighbor method and spatial scan statistics. The apparent herd-level prevalence in the state of Paraíba was 38.5% (95% CI = 35.5-41.6%), 80.6% (95% CI = 73.6-86.2%) in the region of Sertão, 7.0% (95% CI = 3.9-12.2%) in Borborema, and 2.6% (95% CI = 1.0-6.7%) in Agreste/Zona da Mata. The apparent animal-level prevalence was 26.2% (95% CI = 20.6-32.8%) in the state of Paraíba, 48.2% (95% CI = 41.5-54.9%) in Sertão, 6.3% (95% CI = 2.7-14%) in Borborema, and 3.2% 1.9% (95% CI = 0.4-8.4%) in Agreste/Zona da Mata. The risk factors identified were as follows: mixed production (milk/beef) (OR = 4.54), herd size > 23 animals (OR = 3.57), presence of cervids (OR = 15.24), rental of pastures (OR = 3.02), sharing of water sources (OR = 2.57) and presence of horses (OR = 1.69). Two significant clusters of positive herds were detected: the primary cluster covered the Sertão region and the secondary cluster covered part of the Sertão and Borborema regions. Our results suggest high VSV circulation in the bovine population of the state of Paraíba, semiarid region of Brazil, mainly in the Sertão mesoregion, and based on risk factor analysis it was possible to identify important associations that deserve more investigation on causal factors.


Assuntos
Doenças dos Bovinos/epidemiologia , Estomatite Vesicular/epidemiologia , Animais , Anticorpos Antivirais/sangue , Brasil/epidemiologia , Bovinos , Doenças dos Bovinos/virologia , Feminino , Testes de Neutralização/veterinária , Prevalência , Fatores de Risco , Análise Espacial , Estomatite Vesicular/virologia , Vírus da Estomatite Vesicular Indiana/imunologia , Vírus da Estomatite Vesicular New Jersey/imunologia
5.
J Virol ; 92(23)2018 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-30232190

RESUMO

Vesicular stomatitis virus Indiana strain G protein (VSVind.G) is the most commonly used envelope glycoprotein to pseudotype lentiviral vectors (LV) for experimental and clinical applications. Recently, G proteins derived from other vesiculoviruses (VesG), for example, Cocal virus, have been proposed as alternative LV envelopes with possible advantages over VSVind.G. Well-characterized antibodies that recognize VesG will be useful for vesiculovirus research, development of G protein-containing advanced therapy medicinal products (ATMPs), and deployment of VSVind-based vaccine vectors. Here, we show that one commercially available monoclonal antibody, 8G5F11, binds to and neutralizes G proteins from three strains of VSV, as well as Cocal and Maraba viruses, whereas the other commercially available monoclonal anti-VSVind.G antibody, IE9F9, binds to and neutralizes only VSVind.G. Using a combination of G protein chimeras and site-directed mutations, we mapped the binding epitopes of IE9F9 and 8G5F11 on VSVind.G. IE9F9 binds close to the receptor binding site and competes with soluble low-density lipoprotein receptor (LDLR) for binding to VSVind.G, explaining its mechanism of neutralization. In contrast, 8G5F11 binds close to a region known to undergo conformational changes when the G protein moves to its postfusion structure, and we propose that 8G5F11 cross-neutralizes VesGs by inhibiting this.IMPORTANCE VSVind.G is currently regarded as the gold-standard envelope glycoprotein to pseudotype lentiviral vectors. However, recently other G proteins derived from vesiculoviruses have been proposed as alternative envelopes. Here, we investigated two commercially available anti-VSVind.G monoclonal antibodies for their ability to cross-react with other vesiculovirus G proteins, identified the epitopes they recognize, and explored their neutralization activity. We have identified 8G5F11, for the first time, as a cross-neutralizing antibody against several vesiculovirus G proteins. Furthermore, we elucidated the two different neutralization mechanisms employed by these two monoclonal antibodies. Understanding how cross-neutralizing antibodies interact with other G proteins may be of interest in the context of host-pathogen interaction and coevolution, as well as providing the opportunity to modify the G proteins and improve G protein-containing medicinal products and vaccine vectors.


Assuntos
Anticorpos Monoclonais/imunologia , Anticorpos Neutralizantes/imunologia , Antígenos Virais/imunologia , Epitopos/imunologia , Glicoproteínas de Membrana/imunologia , Estomatite Vesicular/imunologia , Vírus da Estomatite Vesicular Indiana/imunologia , Proteínas do Envelope Viral/imunologia , Sequência de Aminoácidos , Anticorpos Monoclonais/metabolismo , Anticorpos Neutralizantes/metabolismo , Antígenos Virais/genética , Antígenos Virais/metabolismo , Reações Cruzadas , Epitopos/metabolismo , Células HEK293 , Humanos , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Testes de Neutralização , Filogenia , Homologia de Sequência , Estomatite Vesicular/metabolismo , Estomatite Vesicular/virologia , Proteínas do Envelope Viral/genética , Proteínas do Envelope Viral/metabolismo
6.
Vaccine ; 36(41): 6061-6069, 2018 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-30219365

RESUMO

The ability to rapidly and accurately determine viral infectivity can help improve the speed of vaccine product development and manufacturing. Current methods to determine infectious viral titers, such as the end-point dilution (50% tissue culture infective dose, TCID50) and plaque assays are slow, labor intensive, and often subjective. In order to accelerate virus quantification, Laser Force Cytology (LFC) was used to monitor vesicular stomatitis virus (VSV) infection in Vero (African green monkey kidney) cells. LFC uses a combination of optical and fluidic forces to interrogate single cells without the use of labels or antibodies. Using a combination of variables measured by the Radiance™ LFC instrument (LumaCyte), an infection metric was developed that correlates well with the viral titer as measured by TCID50 and shortens the timeframe from infection to titer determination from 3 days to 16 h (a 4.5 fold reduction). A correlation was also developed between in-process cellular measurements and the viral titer of collected supernatant, demonstrating the potential for real-time infectivity measurements. Overall, these results demonstrate the utility of LFC as a tool for rapid infectivity measurements throughout the vaccine development process.


Assuntos
Estomatite Vesicular/virologia , Vesiculovirus/isolamento & purificação , Vesiculovirus/patogenicidade , Animais , Anticorpos Antivirais/imunologia , Técnicas Citológicas , Células Vero , Vírus da Estomatite Vesicular Indiana/imunologia , Vírus da Estomatite Vesicular Indiana/isolamento & purificação , Vírus da Estomatite Vesicular Indiana/patogenicidade , Vesiculovirus/imunologia
7.
Sci Rep ; 8(1): 10669, 2018 Jul 13.
Artigo em Inglês | MEDLINE | ID: mdl-30006542

RESUMO

Viral fusion proteins are essential for enveloped virus infection. These proteins mediate fusion between the virus envelope and host cellular membrane to release the viral genome into cells. Vesicular stomatitis virus G (VSV G) protein is a typical type III viral fusion protein. To study the mechanism of VSV G protein mediated membrane fusion, we set up a cell-cell fusion system in which cells are marked by different fluorescent proteins. Taking advantage of this system, we performed real-time recording and quantitative analysis of the cell fusion mediated by VSV G. We found that the time scale required for VSV G mediated cell-cell fusion was approximately 1-2 minutes. Next, we specifically examined the function of the transmembrane (TM) region of VSV G protein in membrane fusion by replacing the TM region with those of other fusion proteins. The TM region replacements dramatically impaired VSV G protein function in the cell-cell fusion assay and diminished VSV G mediated lentivirus and recombinant VSV infection efficiency. Further experiments implied that the TM region played a role in the transition from hemi-fusion to full fusion. Several residues within the TM region were identified as important for membrane fusion. Overall, our findings unraveled the important function of the TM region in VSV G mediated viral fusion.


Assuntos
Fusão de Membrana , Glicoproteínas de Membrana/metabolismo , Estomatite Vesicular/virologia , Vírus da Estomatite Vesicular Indiana/patogenicidade , Proteínas do Envelope Viral/metabolismo , Proteínas Virais de Fusão/metabolismo , Animais , Células CHO , Membrana Celular/metabolismo , Membrana Celular/virologia , Cricetulus , Células HEK293 , Células HeLa , Humanos , Microscopia Intravital , Glicoproteínas de Membrana/genética , Microscopia Confocal , Mutação , Domínios Proteicos/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Células Vero , Vírus da Estomatite Vesicular Indiana/genética , Proteínas do Envelope Viral/genética , Proteínas Virais de Fusão/genética
8.
Vet Microbiol ; 219: 30-39, 2018 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-29778202

RESUMO

Vesicular stomatitis virus (VSV) can cause serious vesicular lesions in pigs, and the matrix (M) protein is its predominant virulence factor. Dendritic cells (DCs) act as the bridge between innate and adaptive immune responses. However, the susceptibility of porcine DCs to VSV infection and the role of M protein in modulating the function of infected DCs are still poorly defined. Thus, this study aimed to determine the ability of virulent wild-type VSV(wtVSV) and two attenuated M protein variants (VSVΔM51 and VSVMT) to induce maturation of porcine monocyte-derived DCs (MoDCs) in vitro. It was found that both wtVSV and the M protein mutant VSVs could productively replicate in porcine MoDCs. Infection with wtVSV resulted in weak proinflammatory cytokine responses and interfered with DC maturation via downregulation of the costimulatory molecule complex CD80/86. Whilst VSVΔM51 could activate porcine MoDCs, VSVMT, a highly attenuated recombinant VSV with triple mutations in the M protein, induced a potent maturation of MoDCs, as evidenced by efficient cytokine induction, and upregulation of CD80/86 and MHC class II. Overall, our findings reveal that porcine MoDCs are differentially activated by VSV, dependent on the presence of a functional M protein. M protein plays a crucial role in modulating porcine DC-VSV interactions. The data further support the potential use of VSVMT as a vaccine vector for pigs.


Assuntos
Células Dendríticas/virologia , Monócitos/virologia , Vírus da Estomatite Vesicular Indiana/genética , Proteínas da Matriz Viral/farmacologia , Animais , Moléculas de Adesão Celular/imunologia , Células Dendríticas/efeitos dos fármacos , Células Dendríticas/imunologia , Interleucina-1beta/biossíntese , Interleucina-1beta/imunologia , Monócitos/imunologia , Monócitos/fisiologia , Proteínas Mutantes/genética , Proteínas Mutantes/imunologia , Proteínas Mutantes/farmacologia , Suínos , Estomatite Vesicular/virologia , Vírus da Estomatite Vesicular Indiana/efeitos dos fármacos , Vírus da Estomatite Vesicular Indiana/imunologia , Vírus da Estomatite Vesicular Indiana/patogenicidade , Proteínas da Matriz Viral/genética
9.
J Orthop Res ; 36(9): 2562-2569, 2018 09.
Artigo em Inglês | MEDLINE | ID: mdl-29637599

RESUMO

Sarcomas are associated with a high incidence of lung metastasis, which leads to a high-risk of cancer death. This study was performed to explore the pre-clinical theranostic potential of a novel fully functional recombinant vesicular stomatitis virus carrying imaging gene Katushka (rVSV-K), as virotherapy and circulating tumor cells (CTCs) detection in the syngeneic mouse model of osteosarcoma with spontaneous pulmonary metastases. Recombinant VSV-K was generated and evaluated in vitro on human and murine osteosarcoma cells. Spontaneous osteosarcoma metastases were established in immune-competent mice by implanting subcutaneously syngeneic osteosarcoma LM8 cells. The vector was injected into the tumor-bearing mice via jugular vein either once or repeatedly. To assess effectiveness, primary tumor growth and development of lung metastasis as well as survival were evaluated. We found that rVSV-K efficiently replicated in and killed all osteosarcoma cell lines in time-dependent manners. Both single or repeated systemic injections of the virus did not inhibit the growth of the primary tumor, but the repeated administration could effectively suppress the development of lung metastases and was likely responsible for the observed increase in survival. Furthermore, we demonstrated, for the first time, that CTCs in blood samples from syngeneic osteosarcoma-bearing mice were successfully detected by utilizing rVSV-K ex vivo. Our results show that repeated systemic injections of rVSV-K are an effective anti-metastatic agent against osteosarcoma in immune-competent mice and this virus to be a useful tool for detection of osteosarcoma CTCs, suggesting that further development of future viral-based theranostic approach in patients with osteosarcoma is warranted. © 2018 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:2562-2569, 2018.


Assuntos
Metástase Neoplásica , Terapia Viral Oncolítica , Osteossarcoma/terapia , Estomatite Vesicular/virologia , Replicação Viral , Animais , Neoplasias Ósseas , Linhagem Celular Tumoral , Modelos Animais de Doenças , Humanos , Neoplasias Pulmonares/secundário , Masculino , Camundongos , Pessoa de Meia-Idade , Transplante de Neoplasias , Vírus da Estomatite Vesicular Indiana/fisiologia
10.
Nat Commun ; 9(1): 1029, 2018 03 12.
Artigo em Inglês | MEDLINE | ID: mdl-29531262

RESUMO

Vesicular stomatitis virus (VSV) is an oncolytic rhabdovirus and its glycoprotein G is widely used to pseudotype other viruses for gene therapy. Low-density lipoprotein receptor (LDL-R) serves as a major entry receptor for VSV. Here we report two crystal structures of VSV G in complex with two distinct cysteine-rich domains (CR2 and CR3) of LDL-R, showing that their binding sites on G are identical. We identify two basic residues on G, which are essential for its interaction with CR2 and CR3. Mutating these residues abolishes VSV infectivity even though VSV can use alternative receptors, indicating that all VSV receptors are members of the LDL-R family. Collectively, our data suggest that VSV G has specifically evolved to interact with receptor CR domains. These structural insights into the interaction between VSV G and host cell receptors provide a basis for the design of recombinant viruses with an altered tropism.


Assuntos
Glicoproteínas de Membrana/metabolismo , Receptores de LDL/química , Receptores de LDL/metabolismo , Receptores Virais/química , Receptores Virais/metabolismo , Estomatite Vesicular/metabolismo , Vírus da Estomatite Vesicular Indiana/metabolismo , Proteínas do Envelope Viral/metabolismo , Humanos , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/genética , Família Multigênica , Ligação Proteica , Domínios Proteicos , Receptores de LDL/genética , Receptores Virais/genética , Estomatite Vesicular/genética , Estomatite Vesicular/virologia , Vírus da Estomatite Vesicular Indiana/química , Vírus da Estomatite Vesicular Indiana/genética , Proteínas do Envelope Viral/química , Proteínas do Envelope Viral/genética
11.
J Virol Methods ; 257: 7-11, 2018 07.
Artigo em Inglês | MEDLINE | ID: mdl-29601843

RESUMO

Vesicular stomatitis is an infectious disease that occurs mainly in countries of the Western Hemisphere and affects cattle, swine and horses. The clinical symptoms in cattle and swine are similar to foot-and-mouth disease and include vesicular ulceration of the tongue and mouth. The disease requires a rapid and accurate differential diagnosis, aiming for immediate implementation of control measures. The objective of the present study was to develop and perform validation tests of multiplex RT-qPCR(s) for the detection of RNA from Alagoas vesiculovirus, considering the parameters of sensitivity and analytical specificity, analytical performance (repeatability and reproducibility criteria) and the uncertainty of the measurement. The threshold cycle values obtained in triplicate from each sample were evaluated by considering the variations between days, analysts and equipment in an analysis of variance aimed at determining the variances of repeatability and reproducibility. The results showed that RT-qPCRs had excellent sensitivity and specificity in the detection of RNA of the Alagoas vesiculovirus. The validation parameters showed low coefficients of variation and were equivalent to those found in other validation studies, indicating that the tests presented excellent repeatability and reproducibility.


Assuntos
Doenças dos Bovinos/diagnóstico , Doenças dos Cavalos/diagnóstico , Reação em Cadeia da Polimerase em Tempo Real/métodos , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Doenças dos Suínos/diagnóstico , Estomatite Vesicular/diagnóstico , Vesiculovirus/isolamento & purificação , Animais , Bovinos , Doenças dos Bovinos/virologia , Doenças dos Cavalos/virologia , Cavalos , Reação em Cadeia da Polimerase Multiplex/métodos , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Suínos , Doenças dos Suínos/virologia , Estomatite Vesicular/virologia , Vesiculovirus/genética
12.
Zebrafish ; 15(2): 124-132, 2018 04.
Artigo em Inglês | MEDLINE | ID: mdl-29304309

RESUMO

The zebrafish, Danio rerio, has become recognized as a valuable model for infectious diseases. Here we evaluated the susceptibility of zebrafish to be infected with the mammalian vesicular stomatitis virus (VSV). Both zebrafish cells and embryos were highly susceptible to VSV infection. Mortalities exceeded 80% in infected embryos and were preceded by the invasion of the central nervous system by VSV. Live imaging of the infection with GFP-VSV as well as virus titration from infected fish confirmed the viral replication. Immunohistochemical analysis of embryonic fish provided evidence of viral antigens as well as of the apoptosis marker caspase-3 in the brain, eye, liver, pronephros, and skeletal muscle. So far, this is the first report describing the susceptibility of zebrafish to the mammalian virus VSV.


Assuntos
Doenças dos Peixes/virologia , Estomatite Vesicular/virologia , Vírus da Estomatite Vesicular Indiana/fisiologia , Peixe-Zebra , Animais , Apoptose , Caspase 3/metabolismo , Células Cultivadas , Embrião não Mamífero/patologia , Embrião não Mamífero/virologia , Doenças dos Peixes/patologia , Regulação da Expressão Gênica , Proteínas de Fluorescência Verde/metabolismo , Estomatite Vesicular/patologia , Replicação Viral , Peixe-Zebra/embriologia
13.
Hum Vaccin Immunother ; 14(4): 994-1002, 2018 04 03.
Artigo em Inglês | MEDLINE | ID: mdl-29206076

RESUMO

V920, rVSVΔG-ZEBOV-GP, is a recombinant vesicular stomatitis-Zaire ebolavirus vaccine which has shown an acceptable safety profile and provides a protective immune response against Ebola virus disease (EVD) induced by Zaire ebolavirus in humans. The purpose of this study was to determine whether the V920 vaccine is capable of replicating in arthropod cell cultures of relevant vector species and of replicating in live mosquitoes. While the V920 vaccine replicated well in Vero cells, no replication was observed in Anopheles or Aedes mosquito, Culicoides biting midge, or Lutzomyia sand fly cells, nor in live Culex or Aedes mosquitoes following exposure through intrathoracic inoculation or feeding on a high-titer infectious blood meal. The insect taxa selected for use in this study represent actual and potential epidemic vectors of VSV. V920 vaccine inoculated into Cx. quinquefasciatus and Ae. aegypti mosquitoes demonstrated persistence of replication-competent virus following inoculation, consistent with the recognized biological stability of the vaccine, but no evidence for active virus replication in live mosquitoes was observed. Following administration of an infectious blood meal to Ae. aegypti and Cx. quinquefasciatus mosquitoes at a titer several log10 PFU more concentrated than would be observed in vaccinated individuals, no infection or dissemination of V920 was observed in either mosquito species. In vitro and in vivo data gathered during this study support minimal risk of the vector-borne potential of the V920 vaccine.


Assuntos
Artrópodes/imunologia , Vacinas contra Ebola/imunologia , Ebolavirus/imunologia , Doença pelo Vírus Ebola/imunologia , Mosquitos Vetores/imunologia , Vacinas Sintéticas/imunologia , Proteínas do Envelope Viral/imunologia , Aedes/imunologia , Aedes/virologia , Animais , Artrópodes/virologia , Culex/imunologia , Culex/virologia , Doença pelo Vírus Ebola/prevenção & controle , Humanos , Células Vero , Estomatite Vesicular/imunologia , Estomatite Vesicular/prevenção & controle , Estomatite Vesicular/virologia
14.
Nat Immunol ; 19(1): 41-52, 2018 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-29242538

RESUMO

Prolonged activation of interferon-STAT1 signaling is closely related to inflammatory autoimmune disorders, and therefore the identification of negative regulators of these pathways is important. Through high-content screening of 115 mouse RING-domain E3 ligases, we identified the E3 ubiquitin ligase RNF2 as a potent inhibitor of interferon-dependent antiviral responses. RNF2 deficiency substantially enhanced interferon-stimulated gene (ISG) expression and antiviral responses. Mechanistically, nuclear RNF2 directly bound to STAT1 after interferon stimulation and increased K33-linked polyubiquitination of the DNA-binding domain of STAT1 at position K379, in addition to promoting the disassociation of STAT1/STAT2 from DNA and consequently suppressing ISG transcription. Our study provides insight into the regulation of interferon-dependent responses via a previously unrecognized post-translational modification of STAT1 in the nucleus.


Assuntos
DNA/metabolismo , Interferon Tipo I/farmacologia , Lisina/metabolismo , Complexo Repressor Polycomb 1/metabolismo , Fator de Transcrição STAT1/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Animais , Antivirais/farmacologia , Linhagem Celular , Expressão Gênica/efeitos dos fármacos , Lisina/genética , Macrófagos/metabolismo , Macrófagos/virologia , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Complexo Repressor Polycomb 1/genética , Ligação Proteica/efeitos dos fármacos , Fator de Transcrição STAT1/genética , Fator de Transcrição STAT2/genética , Fator de Transcrição STAT2/metabolismo , Ubiquitina-Proteína Ligases/genética , Ubiquitinação/efeitos dos fármacos , Estomatite Vesicular/genética , Estomatite Vesicular/prevenção & controle , Estomatite Vesicular/virologia , Vírus da Estomatite Vesicular Indiana/fisiologia
15.
Vet Microbiol ; 212: 59-66, 2017 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-29173589

RESUMO

The matrix protein of vesicular stomatitis virus (VSV) performs multiple functions during viral genome replication and virion production and is involved in modulating multiple host signaling pathways that favor virus replication. To perform numerous functions within infected cells, the M protein needs to recruit cellular partners. To better understand the role of M during VSV replication, we looked for interacting partners by using the two-hybrid system. The eukaryotic translation initiation factor 3, subunit i (eIF3i) was identified to be an M-binding partner, and this interaction was validated by GST pull-down and laser confocal assays. Through a mutagenesis analysis, we found that some mutants of M between amino acids 122 and 181 impaired but did not completely abolish the M-eIF3i interaction. Furthermore, the knockdown of eIF3i by RNA interference decreased viral replication and transcription in the early stages but led to increase in later stages. VSV transcription was increased at 4h post-infection but was not changed at 8 and 12h post-infection after the over-expression of eIF3i. Finally, we also demonstrated that VSV could inhibit the activity of Akt1 and that the knockdown of eIF3i inhibited the expression of the ISGs regulated by phospho-Akt1. These results indicated that eIF3i may affect VSV growth by regulating the host antiviral response in HeLa cells.


Assuntos
Fator de Iniciação 3 em Eucariotos/metabolismo , Estomatite Vesicular/prevenção & controle , Vírus da Estomatite Vesicular Indiana/crescimento & desenvolvimento , Proteínas da Matriz Viral/metabolismo , Animais , Linhagem Celular , Fator de Iniciação 3 em Eucariotos/genética , Humanos , Mesocricetus , Estomatite Vesicular/virologia , Vírus da Estomatite Vesicular Indiana/genética , Proteínas da Matriz Viral/genética , Replicação Viral
16.
Infect Genet Evol ; 55: 112-116, 2017 11.
Artigo em Inglês | MEDLINE | ID: mdl-28882516

RESUMO

Chandipura virus (CHPV) is found to be associated with sporadic encephalitis outbreaks in humans in India since 1965. We report here, the investigation of CHPV activity during the period of June-August 2015 in the state of Gujarat, which revealed 24.44% positivity among 45 referred encephalitis cases. Phylogenetic study of the G gene sequences of strains from Gujarat 2015 along with available sequences of additional strains from different geographical locations and isolation years (1965-2015), indicated the relatedness of the 2015 strain to a group of the CHPV prototype strain of 1965 and the earliest outbreak strains of 2003. Analyses of selection pressure in the G gene revealed positively selected sites within the signal peptide region and a putative CHPV epitope. These results indicate a probable role of G protein-based immune selection and underline the need for continued surveillance to monitor genetic and antigenic variations in the CHPV.


Assuntos
Surtos de Doenças , Estomatite Vesicular/epidemiologia , Estomatite Vesicular/virologia , Vesiculovirus/genética , Proteínas Virais de Fusão/genética , Sequência de Aminoácidos , Variação Genética , Humanos , Índia/epidemiologia , Filogenia , Análise de Sequência de DNA , Vesiculovirus/classificação
17.
J Immunol ; 199(4): 1372-1381, 2017 08 15.
Artigo em Inglês | MEDLINE | ID: mdl-28687662

RESUMO

Accumulating evidence shows that innate immune responses are associated with extracellular nucleotides, particularly ATP. In this article, we demonstrate extensive protection of ATP/P2X7 signaling in a host against viral infection. Interestingly, we observed a significant increase in ATP as a danger signal in vesicular stomatitis virus (VSV)-infected cell supernatant and VSV-infected mice in an exocytosis- and pannexin channel-dependent manner. Furthermore, extracellular ATP reduces the replication of VSV, Newcastle disease virus, murine leukemia virus, and HSV in vivo and in vitro through the P2X7 receptor. Meanwhile, ATP significantly increases IFN-ß expression in a concentration- and time-dependent manner. Mechanistically, ATP facilitates IFN-ß secretion through P38/JNK/ATF-2 signaling pathways, which are crucial in promoting antiviral immunity. Taken together, these results demonstrate the protective role of extracellular ATP and P2X7 in viral infection and suggest a potential therapeutic role for ATP/P2X7 in viral diseases.


Assuntos
Trifosfato de Adenosina/metabolismo , Interferon beta/biossíntese , Receptores Purinérgicos P2X7/metabolismo , Estomatite Vesicular/imunologia , Vírus da Estomatite Vesicular Indiana/fisiologia , Trifosfato de Adenosina/farmacologia , Animais , Imunidade Inata , Interferon beta/genética , Interferon beta/imunologia , Vírus da Leucemia Murina/efeitos dos fármacos , Vírus da Leucemia Murina/imunologia , Medições Luminescentes , Camundongos , Vírus da Doença de Newcastle/efeitos dos fármacos , Vírus da Doença de Newcastle/imunologia , Células RAW 264.7 , Receptores Purinérgicos P2X7/imunologia , Transdução de Sinais , Simplexvirus/efeitos dos fármacos , Simplexvirus/imunologia , Estomatite Vesicular/virologia , Vírus da Estomatite Vesicular Indiana/efeitos dos fármacos , Vírus da Estomatite Vesicular Indiana/imunologia , Replicação Viral/efeitos dos fármacos
18.
Methods Mol Biol ; 1628: 53-63, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28573610

RESUMO

Members of the family Filoviridae are filamentous, enveloped, and nonsegmented negative-stranded RNA viruses that can cause severe hemorrhagic disease in humans and nonhuman primates with high mortality rates. Current efforts to analyze the structure and biology of these viruses as well as the development of antivirals have been hindered by the necessity of biosafety level 4 containment (BSL4). Here, we outline how to produce and work with Ebola virus glycoprotein bearing vesicular stomatitis virus (VSV) pseudovirions. These pseudovirions can be safely used to evaluate early steps of the filovirus life cycle without need for BSL4 containment. Virus gene expression in the transduced cells is easy to assess since the pseudovirions encode a reporter gene in place of the VSV G glycoprotein gene. Adoption of VSV for use as a pseudovirion system for filovirus GP has significantly expanded access for researchers to study specific aspects of the viral life cycle outside of BSL4 containment and has allowed substantial growth of filovirus research.


Assuntos
Ebolavirus/patogenicidade , Glicoproteínas de Membrana/genética , Estomatite Vesicular/virologia , Vesiculovirus/patogenicidade , Proteínas do Envelope Viral/genética , Contenção de Riscos Biológicos , Ebolavirus/genética , Genes Reporter/genética , Humanos , Vírus de RNA/genética , Vírus de RNA/patogenicidade , Estomatite Vesicular/patologia , Vesiculovirus/genética , Vírion/genética , Internalização do Vírus
19.
Sci Signal ; 10(482)2017 Jun 06.
Artigo em Inglês | MEDLINE | ID: mdl-28588082

RESUMO

The unfolded protein response (UPR) is an ancient cellular pathway that detects and alleviates protein-folding stresses. The UPR components X-box binding protein 1 (XBP1) and inositol-requiring enzyme 1α (IRE1α) promote type I interferon (IFN) responses. We found that Xbp1-deficient mouse embryonic fibroblasts and macrophages had impaired antiviral resistance. However, this was not because of a defect in type I IFN responses but rather an inability of Xbp1-deficient cells to undergo viral-induced apoptosis. The ability to undergo apoptosis limited infection in wild-type cells. Xbp1-deficient cells were generally resistant to the intrinsic pathway of apoptosis through an indirect mechanism involving activation of the nuclease IRE1α. We observed an IRE1α-dependent reduction in the abundance of the proapoptotic microRNA miR-125a and a corresponding increase in the amounts of the members of the antiapoptotic Bcl-2 family. The activation of IRE1α by the hepatitis C virus (HCV) protein NS4B in XBP1-proficient cells also conferred apoptosis resistance and promoted viral replication. Furthermore, we found evidence of IRE1α activation and decreased miR-125a abundance in liver biopsies from patients infected with HCV compared to those in the livers of healthy controls. Our results reveal a prosurvival role for IRE1α in virally infected cells and suggest a possible target for IFN-independent antiviral therapy.


Assuntos
Apoptose , Endorribonucleases/metabolismo , Hepatite C/virologia , Herpes Simples/virologia , MicroRNAs/genética , Proteínas Serina-Treonina Quinases/metabolismo , Estomatite Vesicular/virologia , Animais , Estudos de Casos e Controles , Células Cultivadas , Feminino , Hepacivirus/patogenicidade , Hepatite C/metabolismo , Hepatite C/patologia , Herpes Simples/metabolismo , Herpes Simples/patologia , Humanos , Fígado/virologia , Masculino , Camundongos , Camundongos Knockout , Simplexvirus/patogenicidade , Estomatite Vesicular/metabolismo , Estomatite Vesicular/patologia , Vírus da Estomatite Vesicular Indiana/patogenicidade , Proteínas não Estruturais Virais/metabolismo , Replicação Viral , Proteína 1 de Ligação a X-Box/fisiologia
20.
Mol Ther ; 25(8): 1900-1916, 2017 08 02.
Artigo em Inglês | MEDLINE | ID: mdl-28527723

RESUMO

Oncolytic viruses (OVs) offer a promising therapeutic approach to treat multiple types of cancer. In this study, we show that the manipulation of the antioxidant network via transcription factor Nrf2 augments vesicular stomatitis virus Δ51 (VSVΔ51) replication and sensitizes cancer cells to viral oncolysis. Activation of Nrf2 signaling by the antioxidant compound sulforaphane (SFN) leads to enhanced VSVΔ51 spread in OV-resistant cancer cells and improves the therapeutic outcome in different murine syngeneic and xenograft tumor models. Chemoresistant A549 lung cancer cells that display constitutive dominant hyperactivation of Nrf2 signaling are particularly vulnerable to VSVΔ51 oncolysis. Mechanistically, enhanced Nrf2 signaling stimulated viral replication in cancer cells and disrupted the type I IFN response via increased autophagy. This study reveals a previously unappreciated role for Nrf2 in the regulation of autophagy and the innate antiviral response that complements the therapeutic potential of VSV-directed oncolysis against multiple types of OV-resistant or chemoresistant cancer.


Assuntos
Autofagia , Fator 2 Relacionado a NF-E2/metabolismo , Vírus Oncolíticos/fisiologia , Transdução de Sinais , Estomatite Vesicular/metabolismo , Estomatite Vesicular/virologia , Vírus da Estomatite Vesicular Indiana/fisiologia , Animais , Antineoplásicos/farmacologia , Antioxidantes/farmacologia , Autofagia/efeitos dos fármacos , Linhagem Celular , Terapia Combinada , Modelos Animais de Doenças , Interações Hospedeiro-Patógeno/imunologia , Humanos , Imunidade/efeitos dos fármacos , Imunidade Inata/efeitos dos fármacos , Isotiocianatos/farmacologia , Camundongos , Camundongos Knockout , Fator 2 Relacionado a NF-E2/genética , Neoplasias/metabolismo , Neoplasias/mortalidade , Neoplasias/patologia , Neoplasias/terapia , Terapia Viral Oncolítica , Deleção de Sequência , Transdução de Sinais/efeitos dos fármacos , Estomatite Vesicular/imunologia , Vírus da Estomatite Vesicular Indiana/efeitos dos fármacos , Proteínas da Matriz Viral/genética , Replicação Viral/efeitos dos fármacos
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