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/genéticaRESUMO
UNLABELLED: The arenavirus family includes several important pathogens that cause severe and sometimes fatal diseases in humans. The highly pathogenic Old World (OW) arenavirus Lassa fever virus (LASV) is the causative agent of Lassa fever (LF) disease in humans. LASV infections in severe cases are generally immunosuppressive without stimulating interferon (IFN) induction, a proinflammatory response, or T cell activation. However, the host innate immune responses to highly pathogenic New World (NW) arenaviruses are not well understood. We have previously shown that the highly pathogenic NW arenavirus, Junin virus (JUNV), induced an IFN response in human A549 cells. Here, we report that Machupo virus (MACV), another highly pathogenic NW arenavirus, also induces an IFN response. Importantly, both pathogenic NW arenaviruses, in contrast to the OW highly pathogenic arenavirus LASV, readily elicited an IFN response in human primary dendritic cells and A549 cells. Coinfection experiments revealed that LASV could potently inhibit MACV-activated IFN responses even at 6 h after MACV infection, while the replication levels of MACV and LASV were not affected by virus coinfection. Our results clearly demonstrated that although all viruses studied herein are highly pathogenic to humans, the host IFN responses toward infections with the NW arenaviruses JUNV and MACV are quite different from responses to infections with the OW arenavirus LASV, a discovery that needs to be further investigated in relevant animal models. This finding might help us better understand various interplays between the host immune system and highly pathogenic arenaviruses as well as distinct mechanisms underlying viral pathogenesis. IMPORTANCE: Infections of humans with the highly pathogenic OW LASV are accompanied by potent suppression of interferon or proinflammatory cytokine production. In contrast, infections with the highly pathogenic NW arenavirus JUNV are associated with high levels of IFNs and cytokines in severe and fatal cases. Arenaviruses initially target macrophages and dendritic cells, which are potent IFN/cytokine-producers. In human macrophages, JUNV reportedly does not trigger IFN responses. We here demonstrated that JUNV activated IFN responses in human dendritic cells. MACV, another highly pathogenic NW arenavirus, also activated IFN responses. LASV did not induce detectable IFN responses, in spite of higher replication levels, and blocked the MACV-triggered IFN response in a coinfection assay. Although these viruses are highly pathogenic to humans, our study highlights distinct innate immune responses to infections with the NW arenaviruses JUNV and MACV and to infection with the OW arenavirus LASV and provides important insights into the virus-host interaction and pathogenesis.
Assuntos
Arenavirus do Novo Mundo/imunologia , Células Dendríticas/imunologia , Células Epiteliais/imunologia , Interferons/biossíntese , Vírus Junin/imunologia , Arenavirus do Novo Mundo/fisiologia , Células Cultivadas , Células Dendríticas/virologia , Células Epiteliais/virologia , Humanos , Vírus Junin/fisiologia , Replicação ViralRESUMO
Machupo virus (MACV) is the etiological agent of Bolivian hemorrhagic fever (BHF), a reemerging and neglected tropical disease associated with high mortality. The prototypical strain of MACV, Carvallo, was isolated from a human patient in 1963, but minimal in vitro and in vivo characterization has been reported. To this end, we utilized reverse genetics to rescue a pathogenic MACV from cloned cDNAs. The recombinant MACV (rMACV) had in vitro growth properties similar to those of the parental MACV. Both viruses caused similar disease development in alpha/beta and gamma interferon receptor knockout mice, including neurological disease development and high mortality. In addition, we have identified a novel murine model with mortality and neurological disease similar to BHF disease reported in humans and nonhuman primates.
Assuntos
Arenavirus do Novo Mundo/genética , DNA Complementar/genética , Modelos Animais de Doenças , Febre Hemorrágica Americana/genética , Análise de Variância , Animais , Sequência de Bases , Linhagem Celular , Chlorocebus aethiops , Cricetinae , Primers do DNA/genética , Técnicas Histológicas , Camundongos , Camundongos Knockout , Dados de Sequência Molecular , Plasmídeos/genética , Receptor de Interferon alfa e beta/deficiência , Receptor de Interferon alfa e beta/genética , Receptores de Interferon/deficiência , Receptores de Interferon/genética , Genética Reversa/métodos , Análise de Sequência de RNA , Células Vero , Receptor de Interferon gamaRESUMO
BACKGROUND: Arenavirus Junin is the causative agent of Argentine hemorrhagic fever. Limited information is available concerning the pathogenesis of this human disease, especially the pathogenesis of acute and late neurological symptoms. METHODS: In our study we present for the first time cDNA microarray profile of human astrocytes infected with the virulent strain of Junin virus. Transcriptional profiling was confirmed by quantitative real-time RT-PCR and cytokine/chemokine/growth factor assay. RESULTS: We demonstrated the impact of virus infection on immune/inflammatory response/interferon signaling and apoptosis. Pro-apoptotic response and amplification with time of pro-inflammatory cascade of human astrocytes suggested neurodegenerative dysfunctional reactive astrogliosis in response to Junin virus infection. CONCLUSION: Our results suggest potential pathogenic role of astroglial cells in the development of neurological symptoms and late neurological syndrome during Argentine hemorrhagic fever.
Assuntos
Astrócitos/metabolismo , Astrócitos/virologia , Gliose/etiologia , Febre Hemorrágica Americana/complicações , Febre Hemorrágica Americana/genética , Vírus Junin/fisiologia , Transcriptoma , Animais , Apoptose/genética , Linhagem Celular , Análise por Conglomerados , Citocinas/biossíntese , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Humanos , Inflamação/genética , Inflamação/imunologia , Inflamação/metabolismo , Transdução de Sinais , Replicação ViralRESUMO
Junin virus (JUNV) causes a highly lethal human disease, Argentine hemorrhagic fever. Previous work has demonstrated the requirement for human transferrin receptor 1 for virus entry, and the absence of the receptor was proposed to be a major cause for the resistance of laboratory mice to JUNV infection. In this study, we present for the first time in vivo evidence that the disruption of interferon signaling is sufficient to generate a disease-susceptible mouse model for JUNV infection. After peripheral inoculation with virulent JUNV, adult mice lacking alpha/beta and gamma interferon receptors developed disseminated infection and severe disease.
Assuntos
Infecções por Arenaviridae/patologia , Infecções por Arenaviridae/virologia , Suscetibilidade a Doenças , Vírus Junin/patogenicidade , Receptor de Interferon alfa e beta/fisiologia , Receptores de Interferon/fisiologia , Animais , Infecções por Arenaviridae/genética , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Replicação Viral , Receptor de Interferon gamaRESUMO
Intranasal infection with vaccine strain of Venezuelan equine encephalitis virus (TC83) caused persistent viral infection in the brains of mice without functional αß T-cells (αß-TCR -/-). Remarkably, viral kinetics, host response gene transcripts and symptomatic disease are similar between αß-TCR -/- and wild-type C57BL/6 (WT) mice during acute phase of infection [0-13 days post-infection (dpi)]. While WT mice clear infectious virus in the brain by 13 dpi, αß-TCR -/- maintain infectious virus in the brain to 92 dpi. Persistent brain infection in αß-TCR -/- correlated with inflammatory infiltrates and elevated cytokine protein levels in the brain at later time points. Persistent brain infection of αß-TCR -/- mice provides a novel model to study prolonged alphaviral infection as well as the effects and biomarkers of long-term viral inflammation in the brain.
RESUMO
The new world arenavirus Junín virus (JUNV) is the causative agent of Argentine hemorrhagic fever, a lethal human infectious disease. Adult laboratory mice are generally resistant to peripheral infection by JUNV. The mechanism underlying the mouse resistance to JUNV infection is largely unknown. We have reported that interferon receptor knockout mice succumb to JUNV infection, indicating the critical role of interferon in restricting JUNV infection in mice. Here we report that the pathogenic and vaccine strains of JUNV were highly sensitive to interferon in murine primary cells. Treatment with low concentrations of interferon abrogated viral NP protein expression in murine cells. The replication of both JUNVs was enhanced in IRF3/IRF7 deficient cells. In addition, the vaccine strain of JUNV displayed impaired growth in primary murine cells. Our data suggested a direct and potent role of host interferon response in restricting JUNV replication in mice. The defect in viral growth for vaccine JUNV might also partially explain its attenuation in mice.
Assuntos
Antivirais/farmacologia , Interferons/imunologia , Interferons/farmacologia , Vírus Junin/efeitos dos fármacos , Vírus Junin/imunologia , Animais , Células Cultivadas , Fator Regulador 3 de Interferon/deficiência , Fator Regulador 7 de Interferon/deficiência , Interferons/deficiência , Vírus Junin/crescimento & desenvolvimento , Vírus Junin/fisiologia , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas Virais/biossíntese , Replicação Viral/efeitos dos fármacosRESUMO
Junin virus (JUNV) is the etiological agent of Argentine hemorrhagic fever (AHF), a human disease with a high case-fatality rate. It is widely accepted that arenaviral infections, including JUNV infections, are generally non-cytopathic. In contrast, here we demonstrated apoptosis induction in human lung epithelial carcinoma (A549), human hepatocarcinoma and Vero cells upon infection with the attenuated Candid#1 strain of, JUNV as determined by phosphatidylserine (PS) translocation, Caspase 3 (CASP3) activation, Poly (ADP-ribose) polymerase (PARP) cleavage and/or chromosomal DNA fragmentation. Moreover, as determined by DNA fragmentation, we found that the pathogenic Romero strain of JUNV was less cytopathic than Candid#1 in human hepatocarcinoma and Vero, but more apoptotic in A549 and Vero E6 cells. Additionally, we found that JUNV-induced apoptosis was enhanced by RIG-I signaling. Consistent with the previously reported role of RIG-I like helicase (RLH) signaling in initiating programmed cell death, we showed that cell death or DNA fragmentation of Candid#1-infected A549 cells was decreased upon siRNA or shRNA silencing of components of RIG-I pathway in spite of increased virus production. Similarly, we observed decreased DNA fragmentation in JUNV-infected human hepatocarcinoma cells deficient for RIG-I when compared with that of RIG-I-competent cells. In addition, DNA fragmentation detected upon Candid#1 infection of type I interferon (IFN)-deficient Vero cells suggested a type I IFN-independent mechanism of apoptosis induction in response to JUNV. Our work demonstrated for the first time apoptosis induction in various cells of mammalian origin in response to JUNV infection and partial mechanism of this cell death.
Assuntos
Apoptose/genética , RNA Helicases DEAD-box/metabolismo , Febre Hemorrágica Americana/genética , Interferon Tipo I/genética , Vírus Junin/imunologia , Animais , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/imunologia , Carcinoma Hepatocelular/virologia , Morte Celular/genética , Linhagem Celular Tumoral , Chlorocebus aethiops , Proteína DEAD-box 58 , Fragmentação do DNA , Febre Hemorrágica Americana/imunologia , Febre Hemorrágica Americana/virologia , Humanos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/imunologia , Neoplasias Hepáticas/virologia , RNA Interferente Pequeno/genética , Receptores Imunológicos , Transdução de Sinais/genética , Células VeroRESUMO
Arenaviruses are enveloped, negative-stranded RNA viruses that belong to the family Arenaviridae. This diverse family can be further classified into OW (Old World) and NW (New World) arenaviruses based on their antigenicity, phylogeny, and geographical distribution. Many of the NW arenaviruses are highly pathogenic viruses that cause systemic human infections characterized by hemorrhagic fever and/or neurological manifestations, constituting public health problems in their endemic regions. NW arenavirus infection induces a variety of host innate immune responses, which could contribute to the viral pathogenesis and/or influence the final outcome of virus infection in vitro and in vivo. On the other hand, NW arenaviruses have also developed several strategies to counteract the host innate immune response. We will review current knowledge regarding the interplay between the host innate immune response and NW arenavirus infection in vitro and in vivo, with emphasis on viral-encoded proteins and their effect on the type I interferon response.
Assuntos
Infecções por Arenaviridae/imunologia , Arenavirus do Novo Mundo/imunologia , Imunidade Inata/imunologia , Interferon Tipo I/metabolismo , Proteínas Virais/metabolismo , Animais , Infecções por Arenaviridae/virologia , Arenavirus do Novo Mundo/genética , Arenavirus do Novo Mundo/fisiologia , Arenavirus do Velho Mundo/imunologia , Interações Hospedeiro-Patógeno/imunologia , Humanos , Evasão da Resposta Imune , Interferon Tipo I/genética , Camundongos , Modelos Moleculares , Proteínas Virais/genéticaRESUMO
Junín virus, the etiological agent of Argentine hemorrhagic fever, causes significant morbidity and mortality. The virus is spread through the aerosolization of host rodent excreta and endemic to the humid pampas of Argentina. Recently, significant progress has been achieved with the development of new technologies (e.g. reverse genetics) that have expanded knowledge about the pathogenesis and viral replication of Junín virus. We will review the pathogenesis of Junín virus in various animal models and the role of innate and adaptive immunity during infection. We will highlight current research regarding the role of molecular biology of Junín virus in elucidating virus attenuation. We will also summarize current knowledge on Junín virus pathogenesis focusing on the recent development of vaccines and potential therapeutics.
Assuntos
Infecções por Arenaviridae/imunologia , Vírus Junin/patogenicidade , Replicação Viral , Imunidade Adaptativa , Animais , Anticorpos Antivirais , Infecções por Arenaviridae/patologia , Infecções por Arenaviridae/prevenção & controle , Infecções por Arenaviridae/virologia , Modelos Animais de Doenças , Transmissão de Doença Infecciosa/prevenção & controle , Humanos , Imunidade Inata , Vírus Junin/imunologia , Vírus Junin/fisiologia , Glicoproteínas de Membrana/imunologia , Roedores/virologia , Vacinas Atenuadas/imunologia , Proteínas do Envelope Viral/imunologia , Vacinas Virais/imunologia , Internalização do VírusRESUMO
TC83 is a human vaccine with investigational new drug status and is used as a prototype Venezuelan equine encephalitis virus for pathogenesis and antiviral research. Differing from other experimental models, the virus causes high titer infection in the brain and 90-100% mortality in the C3H/HeN murine model. To better characterize the susceptibility to disease development in C3H/HeN mice, we have analyzed the gene transcriptomes and cytokine production in the brains of infected mice. Our analysis indicated the potential importance of natural killer cells in the encephalitic disease development. This paper describes for the first time a pathogenic role for natural killer cells in VEEV encephalitis.
Assuntos
Vírus da Encefalite Equina Venezuelana/imunologia , Vírus da Encefalite Equina Venezuelana/patogenicidade , Encefalomielite Equina Venezuelana/imunologia , Encefalomielite Equina Venezuelana/patologia , Células Matadoras Naturais/imunologia , Animais , Encéfalo/patologia , Encéfalo/virologia , Citocinas/metabolismo , Modelos Animais de Doenças , Encefalomielite Equina Venezuelana/mortalidade , Perfilação da Expressão Gênica , Camundongos , Camundongos Endogâmicos C3H , Análise de SobrevidaRESUMO
Junín virus (JUNV), an arenavirus, is the causative agent of Argentine hemorrhagic fever, an infectious human disease with 15-30% case fatality. The pathogenesis of AHF is still not well understood. Elevated levels of interferon and cytokines are reported in AHF patients, which might be correlated to the severity of the disease. However the innate immune response to JUNV infection has not been well evaluated. Previous studies have suggested that the virulent strain of JUNV does not induce IFN in human macrophages and monocytes, whereas the attenuated strain of JUNV was found to induce IFN response in murine macrophages via the TLR-2 signaling pathway. In this study, we investigated the interaction between JUNV and IFN pathway in human epithelial cells highly permissive to JUNV infection. We have determined the expression pattern of interferon-stimulated genes (ISGs) and IFN-ß at both mRNA and protein levels during JUNV infection. Our results clearly indicate that JUNV infection activates the type I IFN response. STAT1 phosphorylation, a downstream marker of activation of IFN signaling pathway, was readily detected in JUNV infected IFN-competent cells. Our studies also demonstrated for the first time that RIG-I was required for IFN production during JUNV infection. IFN activation was detected during infection by either the virulent or attenuated vaccine strain of JUNV. Curiously, both virus strains were relatively insensitive to human IFN treatment. Our studies collectively indicated that JUNV infection could induce host type I IFN response and provided new insights into the interaction between JUNV and host innate immune system, which might be important in future studies on vaccine development and antiviral treatment.
Assuntos
Infecções por Arenaviridae/imunologia , RNA Helicases DEAD-box/imunologia , Células Epiteliais/virologia , Febre Hemorrágica Americana/virologia , Interferon Tipo I/imunologia , Vírus Junin/imunologia , Linhagem Celular , Proteína DEAD-box 58 , Células Epiteliais/imunologia , Perfilação da Expressão Gênica , Humanos , Vírus Junin/patogenicidade , Fosforilação , Receptores Imunológicos , Fator de Transcrição STAT1/metabolismoRESUMO
Virus-host interactions essential for alphavirus pathogenesis are poorly understood. To address this shortcoming, we coupled retrovirus insertional mutagenesis and a cell survival selection strategy to generate clonal cell lines broadly resistant to Sindbis virus (SINV) and other alphaviruses. Resistant cells had significantly impaired SINV production relative to wild-type (WT) cells, although virus binding and fusion events were similar in both sets of cells. Analysis of the retroviral integration sites identified the neurofibromin 1 (NF1) gene as disrupted in alphavirus-resistant cell lines. Subsequent analysis indicated that expression of NF1 was significantly reduced in alphavirus-resistant cells. Importantly, independent down-regulation of NF1 expression in WT HEK 293 cells decreased virus production and increased cell viability during SINV infection, relative to infected WT cells. Additionally, we observed hyperactive RAS signalling in the resistant HEK 293 cells, which was anticipated because NF1 is a negative regulator of RAS. Expression of constitutively active RAS (HRAS-G12V) in a WT HEK 293 cell line resulted in a marked delay in virus production, compared with infected cells transfected with parental plasmid or dominant-negative RAS (HRAS-S17N). This work highlights novel host cell determinants required for alphavirus pathogenesis and suggests that RAS signalling may play an important role in neuronal susceptibility to SINV infection.