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1.
Recurso na Internet em Português | LIS - Localizador de Informação em Saúde, LIS-bvsms | ID: lis-LISBR1.1-46946

RESUMO

Portal do Ministério da Saúde- Publicado: Segunda, 20 de Janeiro de 2020, 21h28 Apresenta um vídeo com esclarecimentos sobre o Arenavírus, informa sobre a situação atual do virús em São Paulo e disponibiliza o Boletim epidemiológico


Assuntos
Arenavirus/patogenicidade , Febre Hemorrágica Americana/virologia , Evolução Fatal
2.
Virol J ; 15(1): 99, 2018 06 07.
Artigo em Inglês | MEDLINE | ID: mdl-29879985

RESUMO

BACKGROUND: Machupo virus (MACV) is a member of the Mammarenavirus genus, Arenaviridae family and is the etiologic agent of Bolivian hemorrhagic fever, which causes small outbreaks or sporadic cases. Several other arenaviruses in South America Junín virus (JUNV) in Argentina, Guanarito in Venezuela, Sabiá in Brazil and Chapare in Bolivia, also are responsible for human hemorrhagic fevers. Among these arenaviruses, JUNV caused thousands of human cases until 1991, when the live attenuated Candid #1 vaccine, was used. Other than Candid #1 vaccine, few other therapeutic or prophylactic treatments exist. Therefore, new strategies for production of safe countermeasures with broad spectrum activity are needed. FINDINGS: We tested a tri-segmented MACV, a potential vaccine candidate with several mutations, (r3MACV). In cell culture, r3MACV showed a 2-log reduction in infectious virus particle production and the MACV inhibition of INF-1ß was removed from the construct and produced by infected cells. Furthermore, in an animal experiment, r3MACV was able to protect 50% of guinea pigs from a simultaneous lethal JUNV challenge. Protected animals didn't display clinical symptoms nor were virus particles found in peripheral blood (day 14) or in organs (day 28 post-inoculation). The r3MACV provided a higher protection than the Candid #1 vaccine. CONCLUSIONS: The r3MACV provides a potential countermeasure against two South America arenaviruses responsible of human hemorrhagic fever.


Assuntos
Arenavirus do Novo Mundo/imunologia , Febre Hemorrágica Americana/imunologia , Febre Hemorrágica Americana/prevenção & controle , Vacinas de Partículas Semelhantes a Vírus/imunologia , Animais , Peso Corporal , Linhagem Celular , Modelos Animais de Doenças , Cobaias , Febre Hemorrágica Americana/virologia , Humanos , Vírus Junin/imunologia , Dose Letal Mediana , Taxa de Sobrevida , Vacinação , Vacinas Atenuadas/imunologia , Células Vero , Carga Viral , Viremia/prevenção & controle , Viremia/virologia
3.
Nat Commun ; 9(1): 1884, 2018 05 14.
Artigo em Inglês | MEDLINE | ID: mdl-29760382

RESUMO

While five arenaviruses cause human hemorrhagic fevers in the Western Hemisphere, only Junin virus (JUNV) has a vaccine. The GP1 subunit of their envelope glycoprotein binds transferrin receptor 1 (TfR1) using a surface that substantially varies in sequence among the viruses. As such, receptor-mimicking antibodies described to date are type-specific and lack the usual breadth associated with this mode of neutralization. Here we isolate, from the blood of a recipient of the live attenuated JUNV vaccine, two antibodies that cross-neutralize Machupo virus with varying efficiency. Structures of GP1-Fab complexes explain the basis for efficient cross-neutralization, which involves avoiding receptor mimicry and targeting a conserved epitope within the receptor-binding site (RBS). The viral RBS, despite its extensive sequence diversity, is therefore a target for cross-reactive antibodies with activity against New World arenaviruses of public health concern.


Assuntos
Anticorpos Neutralizantes/química , Anticorpos Antivirais/química , Arenavirus do Novo Mundo/imunologia , Febre Hemorrágica Americana/prevenção & controle , Fragmentos Fab das Imunoglobulinas/química , Vírus Junin/imunologia , Proteínas do Envelope Viral/química , Sequência de Aminoácidos , Anticorpos Neutralizantes/isolamento & purificação , Anticorpos Antivirais/isolamento & purificação , Antígenos CD/química , Antígenos CD/genética , Antígenos CD/imunologia , Antígenos Virais/química , Antígenos Virais/genética , Antígenos Virais/imunologia , Arenavirus do Novo Mundo/genética , Sítios de Ligação de Anticorpos , Reações Cruzadas , Epitopos/química , Epitopos/genética , Epitopos/imunologia , Células HEK293 , Febre Hemorrágica Americana/imunologia , Febre Hemorrágica Americana/virologia , Humanos , Soros Imunes/química , Fragmentos Fab das Imunoglobulinas/isolamento & purificação , Vírus Junin/genética , Modelos Moleculares , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Estrutura Terciária de Proteína , Subunidades Proteicas/química , Subunidades Proteicas/genética , Subunidades Proteicas/imunologia , Receptores da Transferrina/química , Receptores da Transferrina/genética , Receptores da Transferrina/imunologia , Receptores Virais/química , Receptores Virais/genética , Receptores Virais/imunologia , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Proteínas do Envelope Viral/genética , Proteínas do Envelope Viral/imunologia , Vacinas Virais/administração & dosagem
4.
J Virol ; 92(4)2018 02 15.
Artigo em Inglês | MEDLINE | ID: mdl-29187543

RESUMO

Arenaviruses are negative-strand, enveloped RNA viruses that cause significant human disease. In particular, Junín mammarenavirus (JUNV) is the etiologic agent of Argentine hemorrhagic fever. At present, little is known about the cellular proteins that the arenavirus matrix protein (Z) hijacks to accomplish its various functions, including driving the process of virus release. Furthermore, there is little knowledge regarding host proteins incorporated into arenavirus particles and their importance for virion function. To address these deficiencies, we used mass spectrometry to identify human proteins that (i) interact with the JUNV matrix protein inside cells or within virus-like particles (VLPs) and/or (ii) are incorporated into bona fide JUNV strain Candid#1 particles. Bioinformatics analyses revealed that multiple classes of human proteins were overrepresented in the data sets, including ribosomal proteins, Ras superfamily proteins, and endosomal sorting complex required for transport (ESCRT) proteins. Several of these proteins were required for the propagation of JUNV (ADP ribosylation factor 1 [ARF1], ATPase, H+ transporting, lysosomal 38-kDa, V0 subunit d1 [ATP6V0D1], and peroxiredoxin 3 [PRDX3]), lymphocytic choriomeningitis mammarenavirus (LCMV) (Rab5c), or both viruses (ATP synthase, H+ transporting, mitochondrial F1 complex, beta polypeptide [ATP5B] and IMP dehydrogenase 2 [IMPDH2]). Furthermore, we show that the release of infectious JUNV particles, but not LCMV particles, requires a functional ESCRT pathway and that ATP5B and IMPDH2 are required for JUNV budding. In summary, we have provided a large-scale map of host machinery that associates with JUNV and identified key human proteins required for its propagation. This data set provides a resource for the field to guide antiviral target discovery and to better understand the biology of the arenavirus matrix protein and the importance of host proteins for virion function.IMPORTANCE Arenaviruses are deadly human pathogens for which there are no U.S. Food and Drug Administration-approved vaccines and only limited treatment options. Little is known about the host proteins that are incorporated into arenavirus particles or that associate with its multifunctional matrix protein. Using Junín mammarenavirus (JUNV), the causative agent of Argentine hemorrhagic fever, as a model organism, we mapped the human proteins that are incorporated into JUNV particles or that associate with the JUNV matrix protein. Functional analysis revealed host machinery that is required for JUNV propagation, including the cellular ESCRT pathway. This study improves our understanding of critical arenavirus-host interactions and provides a data set that will guide future studies to better understand arenavirus pathogenesis and identify novel host proteins that can be therapeutically targeted.


Assuntos
Febre Hemorrágica Americana/virologia , Interações Hospedeiro-Patógeno , Vírus Junin/patogenicidade , Proteoma/metabolismo , Proteômica/métodos , Replicação Viral , Células HEK293 , Febre Hemorrágica Americana/metabolismo , Humanos , Vírus Junin/isolamento & purificação , Proteoma/análise , Proteínas da Matriz Viral/metabolismo , Liberação de Vírus
5.
Virology ; 514: 216-229, 2018 01 15.
Artigo em Inglês | MEDLINE | ID: mdl-29202415

RESUMO

Junín arenavirus infections are associated with high levels of interferons in both severe and fatal cases. Upon Junín virus (JUNV) infection a cell signaling cascade initiates, that ultimately attempts to limit viral replication and prevent infection progression through the expression of host antiviral proteins. The interferon stimulated gene (ISG) viperin has drawn our attention as it has been highlighted as an important antiviral protein against several viral infections. The studies of the mechanistic actions of viperin have described important functional domains relating its antiviral and immune-modulating actions through cellular lipid structures. In line with this, through silencing and overexpression approaches, we have identified viperin as an antiviral ISG against JUNV. In addition, we found that lipid droplet structures are modulated during JUNV infection, suggesting its relevance for proper virus multiplication. Furthermore, our confocal microscopy images, bioinformatics and functional results also revealed viperin-JUNV protein interactions that might be participating in this antiviral pathway at lipid droplet level. Altogether, these results will help to better understand the factors mediating innate immunity in arenavirus infection and may lead to the development of pharmacological agents that can boost their effectiveness thereby leading to new treatments for this viral disease.


Assuntos
Febre Hemorrágica Americana/imunologia , Vírus Junin/fisiologia , Gotículas Lipídicas/virologia , Proteínas/imunologia , Febre Hemorrágica Americana/genética , Febre Hemorrágica Americana/virologia , Humanos , Interferons/genética , Interferons/imunologia , Vírus Junin/química , Vírus Junin/genética , Vírus Junin/imunologia , Gotículas Lipídicas/imunologia , Nucleoproteínas/química , Nucleoproteínas/genética , Nucleoproteínas/imunologia , Domínios Proteicos , Proteínas/química , Proteínas/genética , Replicação Viral
6.
Rev Soc Bras Med Trop ; 50(1): 3-8, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28327796

RESUMO

Emerging infectious diseases are a global threat. In countries like Brazil, where biodiversity is high and public health conditions in terms of infrastructure and medical care are often precarious, emerging diseases are particularly worrisome. The lack of monitoring strategies to identify pathogens with the potential to cause outbreaks or epidemics is another problem in Brazil and other developing countries. In this article, we present the history of the Sabiá virus (SABV), a pathogen that was described in the 1990s in Brazil. Several aspects of the biology and ecology of the SABV remain unknown. The SABV has the potential to cause hemorrhagic fever in humans. To date, four cases of human infections have been reported worldwide; two were naturally acquired (both in Brazil), whereas the other two were linked to occupational exposure in the laboratory environment (one in Brazil and one in the USA). In this review, we summarize the basic biological and ecological characteristics of the SABV. This is the first work to gather all available data on the historical aspects involving the cases of SABV infection along with an update on its characteristic features.


Assuntos
Acidentes de Trabalho , Arenavirus do Novo Mundo , Febre Hemorrágica Americana/virologia , Adulto , Brasil , Humanos , Pessoal de Laboratório , Masculino
7.
Artigo em Inglês | MEDLINE | ID: mdl-28220142

RESUMO

Junin virus (JUNV), a highly pathogenic New World arenavirus, is the causative agent of Argentine hemorrhagic fever (AHF). The live-attenuated Candid #1 (Can) strain currently serves as a vaccine for at-risk populations. We have previously shown that the Can glycoprotein (GPC) gene is the primary gene responsible for attenuation in a guinea pig model of AHF. However, the mechanisms through which the GPC contributes to the attenuation of the Can strain remain unknown. A more complete understanding of the mechanisms underlying the attenuation and immunogenicity of the Can strain will potentially allow for the rational design of additional safe and novel vaccines. Here, we provide a detailed comparison of both RNA and protein expression profiles between both inter- and intra-segment chimeric JUNV recombinant clones expressing combinations of genes from the Can strain and the pathogenic Romero (Rom) strain. The recombinant viruses that express Can GPC, which were shown to be attenuated in guinea pigs, displayed different RNA levels and GPC processing patterns as determined by Northern and Western blot analyses, respectively. Analysis of recombinant viruses containing amino acid substitutions selected at different mouse brain passages during the generation of Can revealed that altered Can GPC processing was primarily due to the T168A substitution within G1, which eliminates an N-linked glycosylation motif. Incorporation of the T168A substitution in the Rom GPC resulted in a Can-like processing pattern of Rom GPC. In addition, JUNV GPCs containing T168A substitution were retained within the endoplasmic reticulum (ER) and displayed significantly lower cell surface expression than wild-type Rom GPC. Interestingly, the reversion A168T in Can GPC significantly increased GPC expression at the cell surface. Our results demonstrate that recombinant JUNV (rJUNV) expressing Can GPC display markedly different protein expression and elevated genomic RNA expression when compared to viruses expressing Rom GPC. Additionally, our findings indicate that the N-linked glycosylation motif at amino acid positions 166-168 is important for trafficking of JUNV GPC to the cell surface, and the elimination of this motif interferes with the GPC release from the ER.


Assuntos
Motivos de Aminoácidos , Arenavirus do Novo Mundo/imunologia , Glicoproteínas/genética , Glicoproteínas/metabolismo , Febre Hemorrágica Americana , Vacinas Virais , Animais , Arenavirus do Novo Mundo/genética , Linhagem Celular , Células Cultivadas , Cricetinae , Retículo Endoplasmático/metabolismo , Estresse do Retículo Endoplasmático , Expressão Gênica , Regulação Viral da Expressão Gênica , Glicoproteínas/química , Glicoproteínas/imunologia , Glicosilação , Febre Hemorrágica Americana/imunologia , Febre Hemorrágica Americana/metabolismo , Febre Hemorrágica Americana/prevenção & controle , Febre Hemorrágica Americana/virologia , Humanos , Processamento de Proteína Pós-Traducional , Transporte Proteico , Transcrição Genética , Vacinas Virais/genética , Vacinas Virais/imunologia , Virulência
8.
J Virol ; 90(9): 4494-4510, 2016 May.
Artigo em Inglês | MEDLINE | ID: mdl-26912630

RESUMO

UNLABELLED: Virus entry into cells is a multistep process that often requires the subversion of subcellular machineries. A more complete understanding of these steps is necessary to develop new antiviral strategies. While studying the potential role of the actin network and one of its master regulators, the small GTPase Cdc42, during Junin virus (JUNV) entry, we serendipitously uncovered the small molecule ZCL278, reported to inhibit Cdc42 function as an entry inhibitor for JUNV and for vesicular stomatitis virus, lymphocytic choriomeningitis virus, and dengue virus but not for the nonenveloped poliovirus. Although ZCL278 did not interfere with JUNV attachment to the cell surface or virus particle internalization into host cells, it prevented the release of JUNV ribonucleoprotein cores into the cytosol and decreased pH-mediated viral fusion with host membranes. We also identified SVG-A astroglial cell-derived cells to be highly permissive for JUNV infection and generated new cell lines expressing fluorescently tagged Rab5c or Rab7a or lacking Cdc42 using clustered regularly interspaced short palindromic repeat (CRISPR)-caspase 9 (Cas9) gene-editing strategies. Aided by these tools, we uncovered that perturbations in the actin cytoskeleton or Cdc42 activity minimally affect JUNV entry, suggesting that the inhibitory effect of ZCL278 is not mediated by ZCL278 interfering with the activity of Cdc42. Instead, ZCL278 appears to redistribute viral particles from endosomal to lysosomal compartments. ZCL278 also inhibited JUNV replication in a mouse model, and no toxicity was detected. Together, our data suggest the unexpected antiviral activity of ZCL278 and highlight its potential for use in the development of valuable new tools to study the intracellular trafficking of pathogens. IMPORTANCE: The Junin virus is responsible for outbreaks of Argentine hemorrhagic fever in South America, where 5 million people are at risk. Limited options are currently available to treat infections by Junin virus or other viruses of the Arenaviridae, making the identification of additional tools, including small-molecule inhibitors, of great importance. How Junin virus enters cells is not yet fully understood. Here we describe new cell culture models in which the cells are susceptible to Junin virus infection and to which we applied CRISPR-Cas9 genome engineering strategies to help characterize early steps during virus entry. We also uncovered ZCL278 to be a new antiviral small molecule that potently inhibits the cellular entry of the Junin virus and other enveloped viruses. Moreover, we show that ZCL278 also functions in vivo, thereby preventing Junin virus replication in a mouse model, opening the possibility for the discovery of ZCL278 derivatives of therapeutic potential.


Assuntos
Antivirais/farmacologia , Benzamidas/farmacologia , Descoberta de Drogas , Tioureia/análogos & derivados , Internalização do Vírus/efeitos dos fármacos , Actinas/metabolismo , Animais , Linhagem Celular , Células Cultivadas , Clatrina/metabolismo , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Endocitose/efeitos dos fármacos , Endossomos/efeitos dos fármacos , Endossomos/virologia , Técnicas de Inativação de Genes , Febre Hemorrágica Americana/genética , Febre Hemorrágica Americana/metabolismo , Febre Hemorrágica Americana/virologia , Humanos , Vírus Junin/efeitos dos fármacos , Vírus Junin/fisiologia , Camundongos , Ligação Proteica , Transporte Proteico , Proteólise , Ribonucleoproteínas/metabolismo , Tioureia/farmacologia , Carga Viral , Proteínas Virais/metabolismo , Ligação Viral/efeitos dos fármacos , Replicação Viral/efeitos dos fármacos , Proteína cdc42 de Ligação ao GTP/genética , Proteína cdc42 de Ligação ao GTP/metabolismo
9.
Vet Pathol ; 53(1): 190-9, 2016 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-26139838

RESUMO

Machupo virus, the cause of Bolivian hemorrhagic fever, is a highly lethal viral hemorrhagic fever with no Food and Drug Administration-approved vaccines or therapeutics. This study evaluated the guinea pig as a model using the Machupo virus-Chicava strain administered via aerosol challenge. Guinea pigs (Cavia porcellus) were serially sampled to evaluate the temporal progression of infection, gross and histologic lesions, and sequential changes in serum chemistry and hematology. The incubation period was 5 to 12 days, and complete blood counts revealed leukopenia with lymphopenia and thrombocytopenia. Gross pathologic findings included congestion and hemorrhage of the gastrointestinal mucosa and serosa, noncollapsing lungs with fluid exudation, enlarged lymph nodes, and progressive pallor and friability of the liver. Histologic lesions consisted of foci of degeneration and cell death in the haired skin, liver, pancreas, adrenal glands, lymph nodes, tongue, esophagus, salivary glands, renal pelvis, small intestine, and large intestine. Lymphohistiocytic interstitial pneumonia was also present. Inflammation within the central nervous system, interpreted as nonsuppurative encephalitis, was histologically apparent approximately 16 days postexposure and was generally progressive. Macrophages in the tracheobronchial lymph node, on day 5 postexposure, were the first cells to demonstrate visible viral antigen. Viral antigen was detected throughout the lymphoid system by day 9 postexposure, followed by prominent spread within epithelial tissues and then brain. This study provides insight into the course of Machupo virus infection and supports the utility of guinea pigs as an additional animal model for vaccine and therapeutic development.


Assuntos
Arenavirus do Novo Mundo/patogenicidade , Modelos Animais de Doenças , Cobaias , Febre Hemorrágica Americana/patologia , Glândulas Suprarrenais/patologia , Aerossóis , Animais , Epitélio/patologia , Feminino , Febre Hemorrágica Americana/virologia , Humanos , Fígado/patologia , Pulmão/patologia , Linfonodos/patologia , Masculino , Pâncreas/patologia
10.
Sci Rep ; 5: 15990, 2015 Nov 09.
Artigo em Inglês | MEDLINE | ID: mdl-26549784

RESUMO

Many viruses have evolved strategies of so-called "superinfection exclusion" to prevent re-infection of a cell that the same virus has already infected. Although Old World arenavirus infection results in down-regulation of its viral receptor and thus superinfection exclusion, whether New World arenaviruses have evolved such a mechanism remains unclear. Here we show that acute infection by the New World Junin virus (JUNV) failed to down-regulate the transferrin receptor and did not induce superinfection exclusion. We observed that Vero cells infected by a first round of JUNV (Candid1 strain) preserve an ability to internalize new incoming JUNV particles that is comparable to that of non-infected cells. Moreover, we developed a dual infection assay with the wild-type Candid1 JUNV and a recombinant JUNV-GFP virus to discriminate between first and second infections at the transcriptional and translational levels. We found that Vero and A549 cells already infected by JUNV were fully competent to transcribe viral RNA from a second round of infection. Furthermore, flow cytometry analysis of viral protein expression indicated that viral translation was normal, regardless of whether cells were previously infected or not. We conclude that in acutely infected cells, Junin virus lacks a superinfection exclusion mechanism.


Assuntos
Febre Hemorrágica Americana/genética , Vírus Junin/genética , Receptores da Transferrina/biossíntese , Proteínas Virais/biossíntese , Animais , Regulação Viral da Expressão Gênica , Febre Hemorrágica Americana/virologia , Humanos , Vírus Junin/patogenicidade , RNA Viral/biossíntese , Superinfecção/genética , Células Vero
11.
Infect Genet Evol ; 33: 242-5, 2015 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-25975978

RESUMO

Clade C, of the New World Arenaviruses, is composed of only the Latino and Oliveros viruses and, besides the geographic range of their rodent reservoirs, the distribution of these viruses has been restricted to Bolivia and Argentina. In this study, the genetic detection and phylogenetic analysis of the complete S segment sequences of sympatric arenaviruses from Brazil revealed a new geographic distribution of clade C arenaviruses, as well as the association of Oliveros virus with a new rodent reservoir.


Assuntos
Arenavirus do Novo Mundo/genética , Genótipo , Febre Hemorrágica Americana/epidemiologia , Febre Hemorrágica Americana/virologia , Animais , Arenavirus do Novo Mundo/classificação , Reservatórios de Doenças/virologia , Febre Hemorrágica Americana/transmissão , Interações Hospedeiro-Patógeno , Humanos , Dados de Sequência Molecular , Filogenia , RNA Viral , Roedores , América do Sul/epidemiologia , Análise Espaço-Temporal
12.
J Virol ; 89(11): 5949-56, 2015 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-25810546

RESUMO

UNLABELLED: The New World arenavirus Junin virus (JUNV) is the causative agent of Argentine hemorrhagic fever (AHF), a potentially deadly disease endemic to central regions of Argentina. The live-attenuated Candid #1 (Can) strain of JUNV is currently used to vaccinate the human population at risk. However, the mechanism of attenuation of this strain is still largely unknown. Therefore, the identification and functional characterization of viral genetic determinants dictating JUNV virulence or attenuation would significantly improve the understanding of the mechanisms underlying AHF and facilitate the development of novel, more effective, and safer vaccines. Here, we utilized a reverse genetics approach to generate recombinant JUNV (rJUNV) strains encoding different gene combinations of the pathogenic Romero (Rom) and attenuated Can strains of JUNV. All strains of rJUNV exhibited in vitro growth kinetics similar to those of their parental counterparts. Analysis of virulence of the rJUNV in a guinea pig model of lethal infection that closely reproduces the features of AHF identified the envelope glycoproteins (GPs) as the major determinants of pathogenesis and attenuation of JUNV. Accordingly, rJUNV strains expressing the full-length GPs of Rom and Can exhibited virulent and attenuated phenotypes, respectively, in guinea pigs. Mutation F427I in the transmembrane region of JUNV envelope glycoprotein GP2 has been shown to attenuate the neurovirulence of JUNV in suckling mice. We document that in the guinea pig model of AHF, mutation F427I in GP2 is also highly attenuating but insufficient to prevent virus dissemination and development of mild clinical and pathological symptoms, indicating that complete attenuation of JUNV requires additional mutations present in Can glycoprotein precursor (GPC). IMPORTANCE: Development of antiviral strategies against viral hemorrhagic fevers, including AHF, is one of the top priorities within the Implementation Plan of the U.S. Department of Health and Human Services Public Health Emergency Medical Countermeasures Enterprise. Live-attenuated Candid #1 strain, derived from the 44th mouse brain passage of the prototype XJ strain of JUNV, has been demonstrated to be safe, immunogenic, and highly protective and is currently licensed for human use in Argentina. However, the bases for the attenuated phenotype of Candid #1 have not been established. Therefore, the identification and functional characterization of viral genetic factors implicated in JUNV pathogenesis and attenuation would significantly improve the understanding of the molecular mechanisms underlying AHF and facilitate the development of novel antiviral strategies.


Assuntos
Glicoproteínas/metabolismo , Febre Hemorrágica Americana/virologia , Vírus Junin/fisiologia , Proteínas do Envelope Viral/metabolismo , Animais , Modelos Animais de Doenças , Glicoproteínas/genética , Cobaias , Febre Hemorrágica Americana/patologia , Vírus Junin/genética , Genética Reversa , Proteínas do Envelope Viral/genética , Virulência , Fatores de Virulência
13.
Mol Cell Proteomics ; 14(3): 646-57, 2015 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-25573744

RESUMO

The Syrian golden hamster has been increasingly used to study viral hemorrhagic fever (VHF) pathogenesis and countermeasure efficacy. As VHFs are a global health concern, well-characterized animal models are essential for both the development of therapeutics and vaccines as well as for increasing our understanding of the molecular events that underlie viral pathogenesis. However, the paucity of reagents or platforms that are available for studying hamsters at a molecular level limits the ability to extract biological information from this important animal model. As such, there is a need to develop platforms/technologies for characterizing host responses of hamsters at a molecular level. To this end, we developed hamster-specific kinome peptide arrays to characterize the molecular host response of the Syrian golden hamster. After validating the functionality of the arrays using immune agonists of defined signaling mechanisms (lipopolysaccharide (LPS) and tumor necrosis factor (TNF)-α), we characterized the host response in a hamster model of VHF based on Pichinde virus (PICV(1)) infection by performing temporal kinome analysis of lung tissue. Our analysis revealed key roles for vascular endothelial growth factor (VEGF), interleukin (IL) responses, nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) signaling, and Toll-like receptor (TLR) signaling in the response to PICV infection. These findings were validated through phosphorylation-specific Western blot analysis. Overall, we have demonstrated that hamster-specific kinome arrays are a robust tool for characterizing the species-specific molecular host response in a VHF model. Further, our results provide key insights into the hamster host response to PICV infection and will inform future studies with high-consequence VHF pathogens.


Assuntos
Febre Hemorrágica Americana/virologia , Pulmão/enzimologia , Vírus Pichinde/fisiologia , Proteínas Quinases/isolamento & purificação , Proteoma/análise , Animais , Modelos Animais de Doenças , Feminino , Febre Hemorrágica Americana/enzimologia , Interleucinas/isolamento & purificação , Pulmão/virologia , Mesocricetus , NF-kappa B/isolamento & purificação , Fosforilação , Transdução de Sinais , Especificidade da Espécie , Receptores Toll-Like/isolamento & purificação , Fator A de Crescimento do Endotélio Vascular/isolamento & purificação
14.
Artigo em Russo | MEDLINE | ID: mdl-26950993

RESUMO

AIM: Experience of study and possible ways of elimination of false positive and false negative results during execution of polymerase chain reaction on an example of Junin virus RNA detection. MATERIALSS AND METHODS: Junin virus--causative agent of Argentine hemorrhagic fever (AHF) strain XJpR37/5787 was obtained from the State collection of pathogenicity group I causative agents of the 48th Central Research Institute. Reagent kit for detection of Junin virus RNA by RT-PCR was developed in the Institute and consists of 4 sets: for isolation of RNA, execution of reverse-transcription reaction, execution of PCR and electrophoretic detection of PCR products. RT-PCR was carried out by a standard technique. Continuous cell cultures of African green monkey Vero B, GMK-AH-1(D) were obtained from the museum of cell culture department of the Centre. RESULTS: An experimental study of the effect of various factors of impact on the sample under investigation ("thawing-freezing", presence of formaldehyde, heparin) on the obtaining of false negative results during Junin virus RNA detection by using RT-PCR was studied. Addition of 0.01% heparin to the samples was shown to completely inhibit PCR. Addition of 0.05% formaldehyde significantly reduces sensitivity of the method. A possibility of reduction of analysis timeframe from 15 to 5 days was shown during detection of the causative agent in samples with low concentration of the latter by growing the samples and subsequent analysis of the material obtained by using RT-PCR. CONCLUSION: During detection of causative agent by using RT-PCR false negative results could appear in the presence of formaldehyde and heparin in the sample. A possibility of elimination of false negative PCR results due to concentration of the causative agent in the sample under investigation at a level below sensitivity threshold was shown on the example of Junin virus RNA detection by using growing of the pathogen in appropriate accumulation system with subsequent analysis of the material obtained using PCR.


Assuntos
Formaldeído/química , Febre Hemorrágica Americana/diagnóstico , Heparina/química , Vírus Junin/genética , RNA Viral/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa/normas , Animais , Reações Falso-Negativas , Reações Falso-Positivas , Febre Hemorrágica Americana/sangue , Febre Hemorrágica Americana/virologia , Humanos , Vírus Junin/isolamento & purificação , RNA Viral/isolamento & purificação , Kit de Reagentes para Diagnóstico/normas , Células Vero
15.
Arch Virol ; 160(2): 469-75, 2015 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-25488290

RESUMO

In previous work, we demonstrated that the arenavirus Junín virus (JUNV) is able to activate Akt by means of the phosphatidylinositol-3-kinase (PI3K) survival pathway during virus entry. This work extends our study, emphasizing the relevance of this pathway in the establishment and maintenance of persistent infection in vitro. During the course of infection, JUNV-infected Vero cells showed a typical cytopathic effect that may be ascribed to apoptotic cell death. Treatment of infected cultures with Ly294002, an inhibitor of the PI3K/Akt pathway, produced an apoptotic response similar to that observed for uninfected cells treated with the drug. This result suggests that virus-induced activation of the PI3K/Akt pathway does not deliver a strong enough anti-apoptotic signal to explain the low proportion of apoptotic cells observed during infection. Also, inhibition of the PI3K/Akt pathway during the acute stage of infection did not prevent the establishment of persistence. Furthermore, treatment of persistently JUNV-infected cells with Ly294002 did not alter viral protein expression. These findings indicate that despite the positive modulation of the PI3/Akt pathway during Junín virus entry, this would not play a critical role in the establishment and maintenance of JUNV persistence in Vero cells.


Assuntos
Cromonas/farmacologia , Inibidores Enzimáticos/farmacologia , Febre Hemorrágica Americana/virologia , Vírus Junin/efeitos dos fármacos , Morfolinas/farmacologia , Fosfatidilinositol 3-Quinases/farmacologia , Proteínas Proto-Oncogênicas c-akt/fisiologia , Internalização do Vírus/efeitos dos fármacos , Replicação Viral/efeitos dos fármacos , Animais , Apoptose , Linhagem Celular , Febre Hemorrágica Americana/tratamento farmacológico , Vírus Junin/crescimento & desenvolvimento , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Células Vero , Proteínas Virais/biossíntese
16.
Vet Pathol ; 52(1): 18-20, 2015 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-25352204

RESUMO

The reports for Ebola virus Zaire (EBOV), Nipah virus, and Machupo virus (MACV) pathogenesis, in this issue of Veterinary Pathology, are timely considering recent events, both nationally and internationally. EBOV, Nipah virus, and MACV cause highly lethal infections for which no Food and Drug Administration (FDA) licensed vaccines or therapies exist. Not only are there concerns that these agents could be used by those with malicious intent, but shifts in ecological distribution of viral reservoirs due to climate change or globalization could lead to more frequent infections within remote regions than previously seen as well as outbreaks in more populous areas. The current EBOV epidemic shows no sign of abating across 3 West African nations (as of October 2014), including densely populated areas, far outpacing infection rates of previous outbreaks. A limited number of cases have also arisen in the United States and Europe. With few treatment options for these deadly viruses, development of animal models reflective of human disease is paramount to combat these diseases. As an example of this potential, a new treatment compound, ZMapp, that had demonstrated efficacy against EBOV infection in nonhuman primates (NHPs) received an emergency compassionate use exception from the FDA for the treatment of 2 American medical workers infected with EBOV, and they are currently virus free and recovering.


Assuntos
Arenavirus do Novo Mundo/fisiologia , Modelos Animais de Doenças , Ebolavirus/fisiologia , Febre Hemorrágica Americana/epidemiologia , Doença pelo Vírus Ebola/epidemiologia , Infecções por Henipavirus/epidemiologia , Vírus Nipah/fisiologia , Animais , Arenavirus do Novo Mundo/efeitos dos fármacos , Mudança Climática , Ensaios de Uso Compassivo , Surtos de Doenças , Ebolavirus/efeitos dos fármacos , Epidemias , Europa (Continente)/epidemiologia , Febre Hemorrágica Americana/tratamento farmacológico , Febre Hemorrágica Americana/virologia , Doença pelo Vírus Ebola/tratamento farmacológico , Doença pelo Vírus Ebola/virologia , Infecções por Henipavirus/tratamento farmacológico , Infecções por Henipavirus/virologia , Humanos , Internacionalidade , Vírus Nipah/efeitos dos fármacos , Estados Unidos/epidemiologia , United States Food and Drug Administration
17.
Vet Pathol ; 52(1): 26-37, 2015 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-24990481

RESUMO

Machupo virus, the causative agent of Bolivian hemorrhagic fever (BHF), is a highly lethal viral hemorrhagic fever of which little is known and for which no Food and Drug Administration-approved vaccines or therapeutics are available. This study evaluated the cynomolgus macaque as an animal model using the Machupo virus, Chicava strain, via intramuscular and aerosol challenge. The incubation period was 6 to 10 days with initial signs of depression, anorexia, diarrhea, mild fever, and a petechial skin rash. These were often followed by neurologic signs and death within an average of 18 days. Complete blood counts revealed leukopenia as well as marked thrombocytopenia. Serum chemistry values identified a decrease in total protein, marked increases in alanine aminotransferase and aspartate aminotransferase, and moderate increases in alkaline phosphatase. Gross pathology findings included a macular rash extending across the axillary and inguinal regions beginning at approximately 10 days postexposure as well as enlarged lymph nodes and spleen, enlarged and friable liver, and sporadic hemorrhages along the gastrointestinal mucosa and serosa. Histologic lesions consisted of foci of degeneration and necrosis/apoptosis in the haired skin, liver, pancreas, adrenal glands, lymph nodes, tongue, esophagus, salivary glands, stomach, small intestine, and large intestine. Lymphohistiocytic interstitial pneumonia was also present. Inflammation within the central nervous system (nonsuppurative encephalitis) was histologically apparent approximately 16 days postexposure and was generally progressive. This study provides insight into the course of Machupo virus infection in cynomolgus macaques and supports the usefulness of cynomolgus macaques as a viable model of human Machupo virus infection.


Assuntos
Arenavirus do Novo Mundo/fisiologia , Febre Hemorrágica Americana/patologia , Glândulas Suprarrenais/patologia , Aerossóis/administração & dosagem , Animais , Modelos Animais de Doenças , Feminino , Febre Hemorrágica Americana/virologia , Humanos , Injeções Intramusculares , Fígado/patologia , Pulmão/patologia , Linfonodos/patologia , Macaca fascicularis , Masculino , Baço/patologia
18.
PLoS One ; 9(12): e115769, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25536075

RESUMO

Arenaviridae are a family of single stranded RNA viruses of mammals and boid snakes. Twenty-nine distinct mammalian arenaviruses have been identified, many of which cause severe hemorrhagic disease in humans, particularly in parts of sub-Saharan Africa, and in Central and South America. Humans typically become infected with an arenavirus through contact with excreta from infected rodents. Tacaribe virus (TCRV) is an arenavirus that was first isolated from bats and mosquitoes during a rabies surveillance survey conducted in Trinidad from 1956 to 1958. Tacaribe virus is unusual because it has never been associated with a rodent host and since that one time isolation, the virus has not been isolated from any vertebrate or invertebrate hosts. We report the re-isolation of the virus from a pool of 100 host-seeking Amblyomma americanum (lone star ticks) collected in a Florida state park in 2012. TCRV was isolated in two cell lines and its complete genome was sequenced. The tick-derived isolate is nearly identical to the only remaining isolate from Trinidad (TRVL-11573), with 99.6% nucleotide identity across the genome. A quantitative RT-PCR assay was developed to test for viral RNA in host-seeking ticks collected from 3 Florida state parks. Virus RNA was detected in 56/500 (11.2%) of surveyed ticks. As this virus was isolated from ticks that parasitize humans, the ability of the tick to transmit the virus to people should be evaluated. Furthermore, reservoir hosts for the virus need to be identified in order to develop risk assessment models of human infection.


Assuntos
Arenavirus do Novo Mundo/isolamento & purificação , Febre Hemorrágica Americana/virologia , Carrapatos/virologia , Animais , Arenavirus do Novo Mundo/genética , Linhagem Celular , Florida , Febre Hemorrágica Americana/transmissão , Humanos , Filogenia , RNA Viral/genética , RNA Viral/isolamento & purificação
19.
Artigo em Russo | MEDLINE | ID: mdl-25286529

RESUMO

AIM: Study sensitivity of laboratory animals to a causative agent ofArgentine hemorrhagic fever. MATERIALS AND METHODS: Junin virus strain XJ P37 was obtained from the State Collection of Causative Agents of Viral Hemorrhagic Fevers of the Pathogenicity Group I of Scientific Research Center of the 33rd Central Scientific Research Test Institute (SRC of the 33rd CSRTI). Junin virus strain XJ P37 culture with biological activity of 5.2 1g PFU x ml was used in the experiments. Mice (2 - 4 and 7 - 14 days old), guinea pigs (250 - 300 g), 1.8 - 2.5 kg shinshilla breed rabbits, 2.0 - 3.0 kg javanese macaque monkeys were obtained from vivarium of the SRC of the 33rd CSRTI. Vero (B) and GMK-AH-1 (D) cell cultures were obtained from cell culture collection of the SRC of the 33rd CSRTI. Biological activity calculation of Junin virus was carried out by Kerber in I.P. Amsharin modification. RESULTS: Lethality in animals was from 12.5 to 50% after intranasal and intraperitoneal infection of guinea pigs, intramuscular, intraperitoneal and subcutaneous infection of rabbits, intracerebral and intranasal infection of mice at the doses from 0.4 to 1.0 x 10(5) PFU. Death of infected monkeys after intramuscular administration of the virus at 1.0 x 10(4) PFU dose was not observed. In 100% of surviving animals formation of virus-neutralizing antibodies was registered. CONCLUSION: Evaluation of sensitivity of laboratory animals to Junin virus has shown that intracerebrally infected mice may be used to maintain causative agent culture, infected guinea pigs - to prepare virus-containing cultures and modelling infection exacerbation in humans. Intramuscularly infected rabbits may be used to obtain hyper-immune sera.


Assuntos
Arenavirus do Novo Mundo/patogenicidade , Febre Hemorrágica Americana/virologia , Vírus Junin/patogenicidade , Animais , Anticorpos Antivirais/isolamento & purificação , Modelos Animais de Doenças , Cobaias , Febre Hemorrágica Americana/epidemiologia , Febre Hemorrágica Americana/patologia , Humanos , Camundongos , Coelhos
20.
J Virol ; 88(18): 10995-9, 2014 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-25031335

RESUMO

Machupo virus (MACV) is the etiologic agent of Bolivian hemorrhagic fever (BHF). Utilizing a reverse-genetics system recently developed, we report the rescue of a rationally modified recombinant MACV containing a single mutation in the transmembrane region of the glycoprotein. Following challenge of susceptible mice, we identified a significant reduction in virulence in the novel virus. We also identified an instability leading to reversion of the single mutation to a wild-type genotype.


Assuntos
Substituição de Aminoácidos , Arenavirus do Novo Mundo/metabolismo , Arenavirus do Novo Mundo/patogenicidade , Membrana Celular/virologia , Glicoproteínas/genética , Febre Hemorrágica Americana/virologia , Mutação de Sentido Incorreto , Proteínas Virais/química , Proteínas Virais/genética , Sequência de Aminoácidos , Animais , Arenavirus do Novo Mundo/química , Arenavirus do Novo Mundo/genética , Sequência de Bases , Glicoproteínas/química , Glicoproteínas/metabolismo , Humanos , Camundongos , Dados de Sequência Molecular , Estrutura Terciária de Proteína , Proteínas Virais/metabolismo , Virulência
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