Your browser doesn't support javascript.
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 214
Filtrar
1.
Rev Chilena Infectol ; 35(4): 386-394, 2018 Aug.
Artigo em Espanhol | MEDLINE | ID: mdl-30534925

RESUMO

BACKGROUND: The Argentine Hemorrhagic Fever (AHF) is a zoonotic disease endemic in a wide area of the humid pampa of Argentina. The etiologic agent is the Junin virus that is maintained in the wild by the rodent Calomys musculinus and transmitted to humans, mainly, through aerosols generated from secretions and excretions. AIMS: To characterize and compare the assemblages of small rodent composition and diversity inside the epidemic, historic and non-endemic zone of AHF and to register C. musculinus abundance in each zone and in each area within each zone, registering the prevalence of infection in rodent populations. METHOD: One central and two peripheral areas were delimited to sample rodents in each zone with different incidence of AHF. Thus, 18 localities were selected to do the sampling in two years. Host abundance between zones and among areas inside each zone and among nearby areas between zones were compared applying nested ANOVA's. RESULTS: In each zone, the rodent assemblage showed differences in composition, diversity and numeric representation of C. musculinus. The epidemic zone was the richest of the three, registering also great host abundance; meanwhile in the historic zone, A. azarae was the dominant numeric species with less number of other species. Regarding the non-endemic zone, the assemblage composition and C. musculinus abundance varied respect the sampled year. Junin virus infection was only detected in C. musculinus individuals corresponding to the epidemic zone, with a prevalence of 2.7 and 1.1% for the years 2007 and 2008, respectively. CONCLUSION: In this system, the abundance of C. musculinus could be impacting over the pathogen dynamic, rather than the assemblage diversity or the A. azarae presence.


Assuntos
Reservatórios de Doenças/classificação , Febre Hemorrágica Americana/epidemiologia , Vírus Junin/isolamento & purificação , Roedores/virologia , Animais , Argentina/epidemiologia , Reservatórios de Doenças/virologia , Febre Hemorrágica Americana/transmissão , Humanos , Incidência , Densidade Demográfica , Prevalência , Roedores/classificação , Análise Espacial
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 , Cercopithecus aethiops , 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(1)2018 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-29070682

RESUMO

The Candid#1 strain of Junín virus was developed using a conventional attenuation strategy of serial passage in nonhost animals and cultured cells. The live-attenuated Candid#1 vaccine is used in Argentina to protect at-risk individuals against Argentine hemorrhagic fever, but it has not been licensed in the United States. Recent studies have revealed that Candid#1 attenuation is entirely dependent on a phenylalanine-to-isoleucine substitution at position 427 in the fusion subunit (GP2) of the viral envelope glycoprotein complex (GPC), thereby raising concerns regarding the potential for reversion to virulence. In this study, we report the identification and characterization of an intragenic epistatic interaction between the attenuating F427I mutation in GP2 and a lysine-to-serine mutation at position 33 in the stable signal peptide (SSP) subunit of GPC, and we demonstrate the utility of this interaction in creating an evolutionary barrier against reversion to the pathogenic genotype. In the presence of the wild-type F427 residue, the K33S mutation abrogates the ability of ectopically expressed GPC to mediate membrane fusion at endosomal pH. This defect is rescued by the attenuating F427I mutation. We show that the recombinant Candid#1 (rCan) virus bearing K33S GPC is viable and retains its attenuated genotype under cell culture conditions that readily select for reversion in the parental rCan virus. If back-mutation to F427 offers an accessible pathway to increase fitness in rCan, reversion in K33S-GPC rCan is likely to be lethal. The epistatic interaction between K33S and F427I thus may minimize the likelihood of reversion and enhance safety in a second-generation Candid#1 vaccine.IMPORTANCE The live-attenuated Candid#1 vaccine strain of Junín virus is used to protect against Argentine hemorrhagic fever. Recent findings that a single missense mutation in the viral envelope glycoprotein complex (GPC) is responsible for attenuation raise the prospect of facile reversion to pathogenicity. Here, we characterize a genetic interaction between GPC subunits that evolutionarily forces retention of the attenuating mutation. By incorporating this secondary mutation into Candid#1 GPC, we hope to minimize the likelihood of reversion and enhance safety in a second-generation Candid#1 vaccine. A similar approach may guide the design of live-attenuated vaccines against Lassa and other arenaviral hemorrhagic fevers.


Assuntos
Epistasia Genética , Glicoproteínas/genética , Vírus Junin/genética , Vírus Junin/imunologia , Proteínas do Envelope Viral/genética , Vacinas Virais/genética , Animais , Cercopithecus aethiops , Evolução Molecular , Genótipo , Febre Hemorrágica Americana/prevenção & controle , Humanos , Vírus Junin/metabolismo , Vírus Junin/patogenicidade , Fusão de Membrana , Mutação , Vacinas Atenuadas/genética , Células Vero , Virulência , Internalização do Vírus
5.
Methods Mol Biol ; 1604: 209-215, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-28986836

RESUMO

Independent expression of the VP40 or Z matrix proteins of filoviruses (marburgviruses and ebolaviruses) and arenaviruses (Lassa fever and Junín), respectively, gives rise to the production and release of virus-like particles (VLPs) that are morphologically identical to infectious virions. We can detect and quantify VLP production and egress in mammalian cells by transient transfection, SDS-PAGE, Western blotting, and live cell imaging techniques such as total internal reflection fluorescence (TIRF) microscopy. Since the VLP budding assay accurately mimics budding of infectious virus, this BSL-2 assay is safe and useful for the interrogation of both viral and host determinants required for budding and can be used as an initial screen to identify and validate small molecule inhibitors of virus release and spread.


Assuntos
Doença pelo Vírus Ebola/metabolismo , Liberação de Vírus/fisiologia , Animais , Arenavirus/genética , Arenavirus/metabolismo , Western Blotting , Ebolavirus/genética , Ebolavirus/metabolismo , Eletroforese em Gel de Poliacrilamida , Filoviridae/genética , Filoviridae/metabolismo , Doença pelo Vírus Ebola/genética , Humanos , Vírus Junin/genética , Vírus Junin/metabolismo , Febre Lassa/virologia , Marburgvirus/genética , Marburgvirus/metabolismo , Proteínas da Matriz Viral/genética , Proteínas da Matriz Viral/metabolismo , Liberação de Vírus/genética
6.
Methods Mol Biol ; 1604: 305-329, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-28986845

RESUMO

Argentinian hemorrhagic Fever (AHF) is a febrile, acute disease caused by Junín virus (JUNV), a member of the Arenaviridae. Different approaches to obtain an effective antigen to prevent AHF using complete live or inactivated virus, as well as molecular constructs, have reached diverse development stages. This chapter refers to JUNV live attenuated vaccine strain Candid #1, currently used in Argentina to prevent AHF. A general standardized protocol used at Instituto Nacional de Enfermedades Virales Humanas (Pergamino, Pcia. Buenos Aires, Argentina) to manufacture the tissue culture derived Candid #1 vaccine is described. Intermediate stages like viral seeds and cell culture bank management, bulk vaccine manufacture, and finished product processing are also separately presented in terms of Production and Quality Control/Quality Assurance requirements, under the Adminitracion Nacional de Medicamentos, Alimentos y Tecnología Medica (ANMAT), the Argentine national regulatory authority.


Assuntos
Febre Hemorrágica Americana/imunologia , Febre Hemorrágica Americana/prevenção & controle , Animais , Anticorpos Antivirais/imunologia , Humanos , Vírus Junin/imunologia , Vírus Junin/patogenicidade , Vacinas Atenuadas/imunologia , Vacinas Atenuadas/uso terapêutico , Vacinas Virais/imunologia , Vacinas Virais/uso terapêutico
7.
Methods Mol Biol ; 1604: 351-370, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-28986848

RESUMO

Host restriction factors are cellular components that interfere with viral multiplication. They are up-regulated and expressed upon viral infection and in consequence their activity is specific. So far several important restriction factors have been described against diverse viruses. The cellular antiviral mechanisms defined include proteins with the ability to interfere with early steps of viral replication and others that have been shown to block viral morphogenesis. However, other strategies by which the antiviral action is exerted still remain elusive. An additional interesting matter is how viruses also developed ways to by-pass these host-specific obstacles. Thus, unusual cell localization or re-localization represents a frequent virus choice to evade the cellular surveillance. In the present chapter, we summarize methods to identify cell restriction factors, their antiviral activity, and possible subcellular locations where their activity can take place.


Assuntos
Febre Hemorrágica com Síndrome Renal/metabolismo , Animais , Dengue/virologia , Vírus da Dengue/metabolismo , Humanos , Vírus Junin/metabolismo , RNA Interferente Pequeno
8.
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
9.
PLoS Negl Trop Dis ; 11(11): e0006075, 2017 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-29155823

RESUMO

BACKGROUND: We describe the development and evaluation of a novel method for targeted amplification and Next Generation Sequencing (NGS)-based identification of viral hemorrhagic fever (VHF) agents and assess the feasibility of this approach in diagnostics. METHODOLOGY: An ultrahigh-multiplex panel was designed with primers to amplify all known variants of VHF-associated viruses and relevant controls. The performance of the panel was evaluated via serially quantified nucleic acids from Yellow fever virus, Rift Valley fever virus, Crimean-Congo hemorrhagic fever (CCHF) virus, Ebola virus, Junin virus and Chikungunya virus in a semiconductor-based sequencing platform. A comparison of direct NGS and targeted amplification-NGS was performed. The panel was further tested via a real-time nanopore sequencing-based platform, using clinical specimens from CCHF patients. PRINCIPAL FINDINGS: The multiplex primer panel comprises two pools of 285 and 256 primer pairs for the identification of 46 virus species causing hemorrhagic fevers, encompassing 6,130 genetic variants of the strains involved. In silico validation revealed that the panel detected over 97% of all known genetic variants of the targeted virus species. High levels of specificity and sensitivity were observed for the tested virus strains. Targeted amplification ensured viral read detection in specimens with the lowest virus concentration (1-10 genome equivalents) and enabled significant increases in specific reads over background for all viruses investigated. In clinical specimens, the panel enabled detection of the causative agent and its characterization within 10 minutes of sequencing, with sample-to-result time of less than 3.5 hours. CONCLUSIONS: Virus enrichment via targeted amplification followed by NGS is an applicable strategy for the diagnosis of VHFs which can be adapted for high-throughput or nanopore sequencing platforms and employed for surveillance or outbreak monitoring.


Assuntos
Febres Hemorrágicas Virais/diagnóstico , Febres Hemorrágicas Virais/virologia , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Técnicas de Amplificação de Ácido Nucleico/métodos , Adulto , Vírus Chikungunya/genética , Vírus Chikungunya/isolamento & purificação , DNA Viral/genética , Ebolavirus/genética , Ebolavirus/isolamento & purificação , Vírus da Febre Hemorrágica da Crimeia-Congo/genética , Vírus da Febre Hemorrágica da Crimeia-Congo/isolamento & purificação , Humanos , Vírus Junin/genética , Vírus Junin/isolamento & purificação , Vírus da Febre do Vale do Rift/genética , Vírus da Febre do Vale do Rift/isolamento & purificação , Sensibilidade e Especificidade , Análise de Sequência de DNA , Vírus da Febre Amarela/genética , Vírus da Febre Amarela/isolamento & purificação
11.
Rev. med. Rosario ; 83(3): 111-118, sep.-dic. 2017. graf, tab, ilus
Artigo em Espanhol | LILACS | ID: biblio-973314

RESUMO

La fiebre Hemorrágica Argentina es una enfermedad viral aguda grave, de carácter sistémico, con duración de una a dos semanas, con cuadros clínicos de gravedad variable. Su agente transmisor es el virus Junín cuyo reservorio natural es el llamado ratón maicero y su zona endémica de distribución comprende sur de la provincia de Santa Fe, Córdoba, Noroeste de Buenos Aires y La Pampa, en Argentina. La primera medida preventiva para la enfermedad es la vacuna llamada Candid 1. Se realizó una encuesta poblacional para dimensionar en la zona de Venado Tuerto y localidades vecinas el alcance de la vacunación y estimar el conocimiento de la existencia de dicha vacuna, que no es de aplicación obligatoria.


Population survey on vaccination against Argentine Hemorrhagic fever in endemic area in the Province of Santa Fe. Argentine Hemorrhagic Fever is a serious systemic, acute viral disease, with a duration of one or two weeks and of variable gravity. Its transmitting agent is Junín Virus, whose natural reservoir is the corn mouse. Its endemic zone is the south of the province of Santa Fe, Cordoba, northwest of Buenos Aires and La Pampa; in Argentina. The first preventive measure for the disease is the vaccine called Candid 1. A population survey was carried out to measure the extent of vaccination and the knowledge of the vaccine, that is not of compulsory application.


Assuntos
Humanos , Doenças Endêmicas/prevenção & controle , Febre Hemorrágica Americana/prevenção & controle , Vírus Junin , Vírus Junin , Doenças Endêmicas/prevenção & controle , Viroses/prevenção & controle , Vacinas , Inquéritos Epidemiológicos , Vacinas , Inquéritos Epidemiológicos , Viroses/prevenção & controle , Saúde Pública
12.
Antiviral Res ; 145: 131-135, 2017 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-28780425

RESUMO

A collection of Old and New World arenaviruses are etiologic agents of viral hemorrhagic fever, a syndrome that features hematologic abnormalities, vascular leak, hypovolemia, and multi-organ failure. Treatment is limited to ribavirin for Lassa fever and immune plasma for Argentine hemorrhagic fever. Improved therapeutic options that are safe, more effective and widely available are needed. Here, we show that modification of favipiravir treatment to include a high-dose loading period achieves complete protection in a guinea pig model of Argentine hemorrhagic fever when treatment was initiated two days following challenge with Junin virus (JUNV). This loading dose strategy also protected 50% of animals from lethal disease when treatment was delayed until 5 days post-infection and extended the survival time in those that succumbed. Consistent with the survival data, dramatic reductions in serum and tissue virus loads were observed in animals treated with favipiravir. This is the first report demonstrating complete protection against uniformly lethal JUNV infection in guinea pigs by administration of a small molecule antiviral drug.


Assuntos
Amidas/administração & dosagem , Antivirais/administração & dosagem , Febre Hemorrágica Americana/tratamento farmacológico , Vírus Junin/efeitos dos fármacos , Pirazinas/administração & dosagem , Amidas/uso terapêutico , Animais , Antivirais/uso terapêutico , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Cobaias , Febre Hemorrágica Americana/sangue , Febre Hemorrágica Americana/mortalidade , Pirazinas/uso terapêutico , Análise de Sobrevida , Carga Viral/efeitos dos fármacos
13.
J Virol ; 91(20)2017 10 15.
Artigo em Inglês | MEDLINE | ID: mdl-28794024

RESUMO

The arenavirus family consists of several highly pathogenic viruses, including the Old World (OW) arenavirus Lassa fever virus (LASV) and the New World (NW) Junin virus (JUNV) and Machupo virus (MACV). Host response to infection by these pathogenic arenaviruses is distinct in many aspects. JUNV and MACV infections readily induce an interferon (IFN) response in human cells, while LASV infection usually triggers an undetectable or weak IFN response. JUNV induces an IFN response through RIG-I, suggesting that the host non-self RNA sensor readily detects JUNV viral RNAs (vRNAs) during infection and activates IFN response. Double-stranded-RNA (dsRNA)-activated protein kinase R (PKR) is another host non-self RNA sensor classically known for its vRNA recognition activity. Here we report that infection with NW arenaviruses JUNV and MACV, but not OW LASV, activated PKR, concomitant with elevated phosphorylation of the translation initiation factor α subunit of eukaryotic initiation factor 2 (eIF2α). Host protein synthesis was substantially suppressed in MACV- and JUNV-infected cells but was only marginally reduced in LASV-infected cells. Despite the antiviral activity known for PKR against many other viruses, the replication of JUNV and MACV was not impaired but was slightly augmented in wild-type (wt) cells compared to that in PKR-deficient cells, suggesting that PKR or PKR activation did not negatively affect JUNV and MACV infection. Additionally, we found an enhanced IFN response in JUNV- or MACV-infected PKR-deficient cells, which was inversely correlated with virus replication.IMPORTANCE The detection of viral RNA by host non-self RNA sensors, including RIG-I and MDA5, is critical to the initiation of the innate immune response to RNA virus infection. Among pathogenic arenaviruses, the OW LASV usually does not elicit an interferon response. However, the NW arenaviruses JUNV and MACV readily trigger an IFN response in a RIG-I-dependent manner. Here, we demonstrate for the first time that pathogenic NW arenaviruses JUNV and MACV, but not the OW arenavirus LASV, activated the dsRNA-dependent PKR, another host non-self RNA sensor, during infection. Interestingly, the replication of JUNV and MACV was not restricted but was rather slightly augmented in the presence of PKR. Our data provide new evidence for a distinct interplay between host non-self RNA sensors and pathogenic arenaviruses, which also provides insights into the pathogenesis of arenaviruses and may facilitate the design of vaccines and treatments against arenavirus-caused diseases.


Assuntos
Arenavirus do Novo Mundo/patogenicidade , Arenavirus do Velho Mundo/patogenicidade , Imunidade Inata , Vírus Junin/patogenicidade , Receptores de Reconhecimento de Padrão/metabolismo , Replicação Viral , eIF-2 Quinase/metabolismo , Células A549 , Arenavirus do Novo Mundo/fisiologia , Arenavirus do Velho Mundo/fisiologia , Interações Hospedeiro-Patógeno , Humanos , Interferons/biossíntese , Interferons/imunologia , Vírus Junin/fisiologia , Fosforilação , Receptores de Reconhecimento de Padrão/genética , Fatores de Transcrição/metabolismo , eIF-2 Quinase/genética
14.
Proc Natl Acad Sci U S A ; 114(27): 7031-7036, 2017 07 03.
Artigo em Inglês | MEDLINE | ID: mdl-28630325

RESUMO

Transmission of hemorrhagic fever New World arenaviruses from their rodent reservoirs to human populations poses substantial public health and economic dangers. These zoonotic events are enabled by the specific interaction between the New World arenaviral attachment glycoprotein, GP1, and cell surface human transferrin receptor (hTfR1). Here, we present the structural basis for how a mouse-derived neutralizing antibody (nAb), OD01, disrupts this interaction by targeting the receptor-binding surface of the GP1 glycoprotein from Junín virus (JUNV), a hemorrhagic fever arenavirus endemic in central Argentina. Comparison of our structure with that of a previously reported nAb complex (JUNV GP1-GD01) reveals largely overlapping epitopes but highly distinct antibody-binding modes. Despite differences in GP1 recognition, we find that both antibodies present a key tyrosine residue, albeit on different chains, that inserts into a central pocket on JUNV GP1 and effectively mimics the contacts made by the host TfR1. These data provide a molecular-level description of how antibodies derived from different germline origins arrive at equivalent immunological solutions to virus neutralization.


Assuntos
Anticorpos Neutralizantes/imunologia , Febre Hemorrágica Americana/imunologia , Testes de Neutralização , Anticorpos Monoclonais/imunologia , Anticorpos Antivirais/imunologia , Sítios de Ligação , Cristalografia por Raios X , Ensaio de Imunoadsorção Enzimática , Epitopos/química , Glicoproteínas/química , Células HEK293 , Humanos , Sistema Imunitário , Vírus Junin , Ligação Proteica , Proteínas Recombinantes/imunologia , Proteínas do Envelope Viral/química
15.
J Virol ; 91(15)2017 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-28539447

RESUMO

Arenaviruses are enveloped negative-strand RNA viruses that cause significant human disease. These viruses encode only four proteins to accomplish the viral life cycle, so each arenavirus protein likely plays unappreciated accessory roles during infection. Here we used immunoprecipitation and mass spectrometry to identify human proteins that interact with the nucleoproteins (NPs) of the Old World arenavirus lymphocytic choriomeningitis virus (LCMV) and the New World arenavirus Junín virus (JUNV) strain Candid #1. Bioinformatic analysis of the identified protein partners of NP revealed that host translation appears to be a key biological process engaged during infection. In particular, NP associates with the double-stranded RNA (dsRNA)-activated protein kinase (PKR), a well-characterized antiviral protein that inhibits cap-dependent protein translation initiation via phosphorylation of eIF2α. JUNV infection leads to increased expression of PKR as well as its redistribution to viral replication and transcription factories. Further, phosphorylation of PKR, which is a prerequisite for its ability to phosphorylate eIF2α, is readily induced by JUNV. However, JUNV prevents this pool of activated PKR from phosphorylating eIF2α, even following exposure to the synthetic dsRNA poly(I·C), a potent PKR agonist. This blockade of PKR function is highly specific, as LCMV is unable to similarly inhibit eIF2α phosphorylation. JUNV's ability to antagonize the antiviral activity of PKR appears to be complete, as silencing of PKR expression has no impact on viral propagation. In summary, we provide a detailed map of the host machinery engaged by arenavirus NPs and identify an antiviral pathway that is subverted by JUNV.IMPORTANCE Arenaviruses are important human pathogens for which FDA-approved vaccines do not exist and effective antiviral therapeutics are needed. Design of antiviral treatment options and elucidation of the mechanistic basis of disease pathogenesis will depend on an increased basic understanding of these viruses and, in particular, their interactions with the host cell machinery. Identifying host proteins critical for the viral life cycle and/or pathogenesis represents a useful strategy to uncover new drug targets. This study reveals, for the first time, the extensive human protein interactome of arenavirus nucleoproteins and uncovers a potent antiviral host protein that is neutralized during Junín virus infection. In so doing, it shows further insight into the interplay between the virus and the host innate immune response and provides an important data set for the field.


Assuntos
Interações Hospedeiro-Patógeno , Evasão da Resposta Imune , Vírus Junin/patogenicidade , Vírus da Coriomeningite Linfocítica/patogenicidade , Proteínas do Nucleocapsídeo/metabolismo , eIF-2 Quinase/antagonistas & inibidores , Linhagem Celular , Humanos , Imunoprecipitação , Espectrometria de Massas , Mapeamento de Interação de Proteínas
16.
Int J Mol Sci ; 18(5)2017 May 12.
Artigo em Inglês | MEDLINE | ID: mdl-28498311

RESUMO

Some New World (NW) and Old World (OW) mammalian arenaviruses are emerging, zoonotic viruses that can cause lethal hemorrhagic fever (HF) infections in humans. While these are closely related RNA viruses, the infected hosts appear to mount different types of immune responses against them. Lassa virus (LASV) infection, for example, results in suppressed immune function in progressive disease stage, whereas patients infected with Junín virus (JUNV) develop overt pro-inflammatory cytokine production. These viruses have also evolved different molecular strategies to evade host immune recognition and activation. This paper summarizes current progress in understanding the differential immune responses to pathogenic arenaviruses and how the information can be exploited toward the development of vaccines against them.


Assuntos
Febre Hemorrágica Americana/imunologia , Vírus Junin/imunologia , Febre Lassa/imunologia , Vírus Lassa/imunologia , Animais , Febre Hemorrágica Americana/prevenção & controle , Febre Hemorrágica Americana/terapia , Humanos , Evasão da Resposta Imune , Febre Lassa/prevenção & controle , Febre Lassa/terapia , Vacinas Virais/imunologia
17.
J Virol Methods ; 246: 51-57, 2017 08.
Artigo em Inglês | MEDLINE | ID: mdl-28359770

RESUMO

Studies were conducted to determine the performance of four dyes in assessing antiviral activities of compounds against three RNA viruses with differing cytopathogenic properties. Dyes included alamarBlue® measured by absorbance (ALB-A) and fluorescence (ALB-F), neutral red (NR), Viral ToxGlo™ (VTG), and WST-1. Viruses were chikungunya, dengue type 2, and Junin, which generally cause 100, 80-90, and 50% maximal cytopathic effect (CPE), respectively, in Vero or Vero 76 cells Compounds evaluated were 6-azauridine, BCX-4430, 3-deazaguanine, EICAR, favipiravir, infergen, mycophenolic acid (MPA), ribavirin, and tiazofurin. The 50% virus-inhibitory (EC50) values for each inhibitor and virus combination did not vary significantly based on the dye used. However, dyes varied in distinguishing the vitality of virus-infected cultures when not all cells were killed by virus infection. For example, VTG uptake into dengue-infected cells was nearly 50% when visual examination showed only 10-20% cell survival. ALB-A measured infected cell viability differently than ALB-F as follows: 16% versus 32% (dengue-infected), respectively, and 51% versus 72% (Junin-infected), respectively. Cytotoxicity (CC50) assays with dyes in uninfected proliferating cells produced similar CC50 values for EICAR (1.5-8.9µM) and MPA (0.8-2.5µM). 6-Azauridine toxicity was 6.1-17.5µM with NR, VTG, and WST-1, compared to 48-92µM with ALB-A and ALB-F (P<0.001). Curiously, the CC50 values for 3-deazaguanine were 83-93µM with ALB-F versus 2.4-7.0µM with all other dyes including ALB-A (P<0.001). Overall, ALB minimized the toxicities detected with these two inhibitors. Because the choice of dyes affected CC50 values, this impacted on the resulting in vitro selectivity indexes (calculated as CC50/EC50 ratio).


Assuntos
Antivirais/farmacologia , Sobrevivência Celular/efeitos dos fármacos , Corantes , Efeito Citopatogênico Viral , Vírus de RNA/efeitos dos fármacos , Vírus/efeitos dos fármacos , Animais , Cercopithecus aethiops , Vírus Chikungunya/efeitos dos fármacos , Vírus Chikungunya/patogenicidade , Vírus Chikungunya/fisiologia , Corantes/química , Vírus da Dengue/efeitos dos fármacos , Vírus da Dengue/patogenicidade , Vírus da Dengue/fisiologia , Vírus Junin/efeitos dos fármacos , Vírus Junin/patogenicidade , Vírus Junin/fisiologia , Oxazinas , Vírus de RNA/patogenicidade , Vírus de RNA/fisiologia , Células Vero , Replicação Viral/efeitos dos fármacos , Xantenos
18.
J Virol ; 91(7)2017 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-28100617

RESUMO

The glycoprotein complex (GPC) of arenaviruses, composed of stable signal peptide, GP1, and GP2, is the only antigen correlated with antibody-mediated neutralization. However, despite strong cross-reactivity of convalescent antisera between related arenavirus species, weak or no cross-neutralization occurs. Two closely related clade B viruses, Machupo virus (MACV) and Junín virus (JUNV), have nearly identical overall GPC architecture and share a host receptor, transferrin receptor 1 (TfR1). Given structural and functional similarities of the GP1 receptor binding site (RBS) of these viruses and the recent demonstration that the RBS is an important target for neutralizing antibodies, it is not clear how these viruses avoid cross-neutralization. To address this, MACV/JUNV chimeric GPCs were assessed for interaction with a group of α-JUNV GPC monoclonal antibodies (MAbs) and mouse antisera against JUNV or MACV GPC. All six MAbs targeted GP1, with those that neutralized JUNV GPC-pseudovirions competing with each other for RBS binding. However, these MAbs were unable to bind to a chimeric GPC composed of JUNV GP1 containing a small disulfide bonded loop (loop 10) unique to MACV GPC, suggesting that this loop may block MAbs interaction with the GP1 RBS. Consistent with this loop causing interference, mouse anti-JUNV GPC antisera that solely neutralized pseudovirions bearing autologous GP1 provided enhanced neutralization of MACV GPC when this loop was removed. Our studies provide evidence that loop 10, which is unique to MACV GP1, is an important impediment to binding of neutralizing antibodies and contributes to the poor cross-neutralization of α-JUNV antisera against MACV.IMPORTANCE Multiple New World arenaviruses can cause severe disease in humans, and some geographic overlap exists among these viruses. A vaccine that protects against a broad range of New World arenaviruses is desirable for purposes of simplicity, cost, and broad protection against multiple National Institute of Allergy and Infectious Disease-assigned category A priority pathogens. In this study, we sought to better understand how closely related arenaviruses elude cross-species neutralization by investigating the structural bases of antibody binding and avoidance. In our studies, we found that neutralizing antibodies against two New World arenaviruses, Machupo virus (MACV) and Junín virus (JUNV), bound to the envelope glycoprotein 1 (GP1) with JUNV monoclonal antibodies targeting the receptor binding site (RBS). We further show that altered structures surrounding the RBS pocket in MACV GP1 impede access of JUNV-elicited antibodies.


Assuntos
Anticorpos Neutralizantes/química , Anticorpos Antivirais/química , Antígenos Virais/imunologia , Vírus Junin/imunologia , Proteínas Virais de Fusão/imunologia , Sequência de Aminoácidos , Animais , Antígenos Virais/química , Sítios de Ligação , Cercopithecus aethiops , Reações Cruzadas , Células HEK293 , Humanos , Camundongos , Ligação Proteica , Especificidade da Espécie , Células Vero , Proteínas Virais de Fusão/química
19.
PLoS Negl Trop Dis ; 10(8): e0004969, 2016 08.
Artigo em Inglês | MEDLINE | ID: mdl-27580122

RESUMO

Machupo virus (MACV), a New World arenavirus, is the etiological agent of Bolivian hemorrhagic fever (BHF). Junin virus (JUNV), a close relative, causes Argentine hemorrhagic fever (AHF). Previously, we reported that a recombinant, chimeric MACV (rMACV/Cd#1-GPC) expressing glycoprotein from the Candid#1 (Cd#1) vaccine strain of JUNV is completely attenuated in a murine model and protects animals from lethal challenge with MACV. A rMACV with a single F438I substitution in the transmembrane domain (TMD) of GPC, which is equivalent to the F427I attenuating mutation in Cd#1 GPC, was attenuated in a murine model but genetically unstable. In addition, the TMD mutation alone was not sufficient to fully attenuate JUNV, indicating that other domains of the GPC may also contribute to the attenuation. To investigate the requirement of different domains of Cd#1 GPC for successful attenuation of MACV, we rescued several rMACVs expressing the ectodomain of GPC from Cd#1 either alone (MCg1), along with the TMD F438I substitution (MCg2), or with the TMD of Cd#1 (MCg3). All rMACVs exhibited similar growth curves in cultured cells. In mice, the MCg1 displayed significant reduction in lethality as compared with rMACV. The MCg1 was detected in brains and spleens of MCg1-infected mice and the infection was associated with tissue inflammation. On the other hand, all animals survived MCg2 and MCg3 infection without detectable levels of virus in various organs while producing neutralizing antibody against Cd#1. Overall our data suggest the indispensable role of each GPC domain in the full attenuation and immunogenicity of rMACV/Cd#1 GPC.


Assuntos
Vírus Junin/imunologia , Glicoproteínas de Membrana/imunologia , Receptores de Interferon/deficiência , Proteínas do Envelope Viral/imunologia , Vacinas Virais/imunologia , Células A549 , Animais , Anticorpos Neutralizantes/sangue , Anticorpos Antivirais/sangue , Cricetinae , Modelos Animais de Doenças , Haplorrinos , Febre Hemorrágica Americana/prevenção & controle , Vírus Junin/patogenicidade , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Testes de Neutralização , Receptores de Interferon/genética , Proteínas Recombinantes/imunologia , Vacinas Atenuadas/imunologia
20.
J Virol ; 90(18): 8341-50, 2016 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-27412594

RESUMO

UNLABELLED: Arenaviruses are responsible for severe and often fatal hemorrhagic disease. In the absence of effective antiviral therapies and vaccines, these viruses pose serious threats to public health and biodefense. Arenaviruses enter the host cell by fusion of the viral and endosomal membranes, a process mediated by the virus envelope glycoprotein GPC. Unlike other class I viral fusion proteins, GPC retains its stable signal peptide (SSP) as an essential third subunit in the mature complex. SSP spans the membrane twice and is myristoylated at its cytoplasmic N terminus. Mutations that abolish SSP myristoylation have been shown to reduce pH-induced cell-cell fusion activity of ectopically expressed GPC to ∼20% of wild-type levels. In order to examine the role of SSP myristoylation in the context of the intact virus, we used reverse genetics to generate Junín viruses (Candid #1 isolate) in which the critical glycine-2 residue in SSP was either replaced by alanine (G2A) or deleted (ΔG2). These mutant viruses produced smaller foci of infection in Vero cells and showed an ∼5-fold reduction in specific infectivity, commensurate with the defect in cell-cell fusion. However, virus assembly and GPC incorporation into budded virions were unaffected. Our findings suggest that the myristate moiety is cryptically disposed in the prefusion GPC complex and may function late in the fusion process to promote merging of the viral and cellular membranes. IMPORTANCE: Hemorrhagic fever arenaviruses pose significant threats to public health and biodefense. Arenavirus entry into the host cell is promoted by the virus envelope glycoprotein GPC. Unlike other viral envelope glycoproteins, GPC contains a myristoylated stable signal peptide (SSP) as an essential third subunit. Myristoylation has been shown to be important for the membrane fusion activity of recombinantly expressed GPC. Here, we use reverse genetics to study the role of SSP myristoylation in the context of the intact virion. We find that nonmyristoylated GPC mutants of the Candid #1 strain of Junín virus display a commensurate deficiency in their infectivity, albeit without additional defects in virion assembly and budding. These results suggest that SSP myristoylation may function late in the fusion process to facilitate merging of the viral and cellular membranes. Antiviral agents that target this novel aspect of GPC membrane fusion may be useful in the treatment of arenavirus hemorrhagic fevers.


Assuntos
Vírus Junin/fisiologia , Glicoproteínas de Membrana/metabolismo , Ácido Mirístico/metabolismo , Processamento de Proteína Pós-Traducional , Sinais Direcionadores de Proteínas , Proteínas do Envelope Viral/metabolismo , Montagem de Vírus , Internalização do Vírus , Substituição de Aminoácidos , Animais , Cercopithecus aethiops , Glicoproteínas/genética , Glicoproteínas/metabolismo , Vírus Junin/genética , Glicoproteínas de Membrana/genética , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Genética Reversa , Deleção de Sequência , Células Vero , Proteínas do Envelope Viral/genética , Vírion/metabolismo
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA