RESUMEN
BACKGROUND: Lassa virus and Marburg virus are two causative agents of viral hemorrhagic fever. Their diagnosis is difficult because patients infected with either pathogen present similar nonspecific symptoms early after infection. Current diagnostic tests are based on detecting viral proteins or nucleic acids in the blood, but these cannot be found during the early stages of disease, before the virus starts replicating in the blood. Using the transcriptional response of the host during infection can lead to earlier diagnoses compared to those of traditional methods. RESULTS: In this study, we use RNA sequencing to obtain a high-resolution view of the in vivo transcriptional dynamics of peripheral blood mononuclear cells (PBMCs) throughout both types of infection. We report a subset of host mRNAs, including heat-shock proteins like HSPA1B, immunoglobulins like IGJ, and cell adhesion molecules like SIGLEC1, whose differences in expression are strong enough to distinguish Lassa infection from Marburg infection in non-human primates. We have validated these infection-specific expression differences by using microarrays on a larger set of samples, and by quantifying the expression of individual genes using RT-PCR. CONCLUSIONS: These results suggest that host transcriptional signatures are correlated with specific viral infections, and that they can be used to identify highly pathogenic viruses during the early stages of disease, before standard detection methods become effective.
Asunto(s)
Fiebres Hemorrágicas Virales/veterinaria , Interacciones Huésped-Patógeno/genética , Virus Lassa , Marburgvirus , Enfermedades de los Monos/genética , Transcripción Genética , Animales , Femenino , Perfilación de la Expresión Génica , Regulación de la Expresión Génica/efectos de los fármacos , Interferón Tipo I/farmacología , Leucocitos Mononucleares/metabolismo , Leucocitos Mononucleares/virología , Macaca fascicularis , Masculino , Enfermedades de los Monos/diagnóstico , Enfermedades de los Monos/virología , Reproducibilidad de los Resultados , Análisis de Secuencia de ARNRESUMEN
There is a clear need for novel, effective therapeutic approaches to hemorrhagic fever due to filoviruses. Ebola virus hemorrhagic fever is associated with robust interferon (IFN)-α production, with plasma concentrations of IFN-α that greatly (60- to 100-fold) exceed those seen in other viral infections, but little IFN-ß production. While all of the type I IFNs signal through the same receptor complex, both quantitative and qualitative differences in biological activity are observed after stimulation of the receptor complex with different type I IFNs. Taken together, this suggested potential for IFN-ß therapy in filovirus infection. Here we show that early postexposure treatment with IFN-ß significantly increased survival time of rhesus macaques infected with a lethal dose of Ebola virus, although it failed to alter mortality. Early treatment with IFN-ß also significantly increased survival time after Marburg virus infection. IFN-ß may have promise as an adjunctive postexposure therapy in filovirus infection.
Asunto(s)
Fiebre Hemorrágica Ebola/tratamiento farmacológico , Interferón beta/farmacología , Enfermedad del Virus de Marburg/tratamiento farmacológico , Marburgvirus/efectos de los fármacos , Animales , Ebolavirus/efectos de los fármacos , Femenino , Fiebre Hemorrágica Ebola/virología , Humanos , Macaca mulatta , Masculino , Enfermedad del Virus de Marburg/virología , Proteínas Recombinantes/farmacologíaRESUMEN
The mechanisms of Ebola (EBOV) pathogenesis are only partially understood, but the dysregulation of normal host immune responses (including destruction of lymphocytes, increases in circulating cytokine levels, and development of coagulation abnormalities) is thought to play a major role. Accumulating evidence suggests that much of the observed pathology is not the direct result of virus-induced structural damage but rather is due to the release of soluble immune mediators from EBOV-infected cells. It is therefore essential to understand how the candidate therapeutic may be interrupting the disease process and/or targeting the infectious agent. To identify genetic signatures that are correlates of protection, we used a DNA microarray-based approach to compare the host genome-wide responses of EBOV-infected nonhuman primates (NHPs) responding to candidate therapeutics. We observed that, although the overall circulating immune response was similar in the presence and absence of coagulation inhibitors, surviving NHPs clustered together. Noticeable differences in coagulation-associated genes appeared to correlate with survival, which revealed a subset of distinctly differentially expressed genes, including chemokine ligand 8 (CCL8/MCP-2), that may provide possible targets for early-stage diagnostics or future therapeutics. These analyses will assist us in understanding the pathogenic mechanisms of EBOV infection and in identifying improved therapeutic strategies.
Asunto(s)
Ebolavirus/inmunología , Predisposición Genética a la Enfermedad , Genoma , Fiebre Hemorrágica Ebola/inmunología , Fiebre Hemorrágica Ebola/terapia , Transcripción Genética , Animales , Coagulación Sanguínea/genética , Inhibidores de Factor de Coagulación Sanguínea/genética , Inhibidores de Factor de Coagulación Sanguínea/metabolismo , Perfilación de la Expresión Génica , Regulación Viral de la Expresión Génica , Fiebre Hemorrágica Ebola/genética , Activación de Linfocitos/genética , Macaca mulatta , Familia de Multigenes , Análisis de Secuencia por Matrices de OligonucleótidosRESUMEN
Ebola virus (EBOV) infection in humans and non-human primates (NHPs) is highly lethal, and there is limited understanding of the mechanisms associated with pathogenesis and survival. Here, we describe a transcriptomic analysis of NHPs that survived lethal EBOV infection, compared to NHPs that did not survive. It has been previously demonstrated that anticoagulant therapeutics increase the survival rate in EBOV-infected NHPs, and that the characteristic transcriptional profile of immune response changes in anticoagulant-treated NHPs. In order to identify transcriptional signatures that correlate with survival following EBOV infection, we compared the mRNA expression profile in peripheral blood mononuclear cells from EBOV-infected NHPs that received anticoagulant treatment, to those that did not receive treatment. We identified a small set of 20 genes that are highly confident predictors and can accurately distinguish between surviving and non-surviving animals. In addition, we identified a larger predictive signature of 238 genes that correlated with disease outcome and treatment; this latter signature was associated with a variety of host responses, such as the inflammatory response, T cell death, and inhibition of viral replication. Notably, among survival-associated genes were subsets of genes that are transcriptionally regulated by (1) CCAAT/enhancer-binding protein alpha, (2) tumor protein 53, and (3) megakaryoblastic leukemia 1 and myocardin-like protein 2. These pathways merit further investigation as potential transcriptional signatures of host immune response to EBOV infection.
Asunto(s)
Anticoagulantes/uso terapéutico , Ebolavirus/patogenicidad , Perfilación de la Expresión Génica , Fiebre Hemorrágica Ebola/patología , Interacciones Huésped-Patógeno , Animales , Modelos Animales de Enfermedad , Fiebre Hemorrágica Ebola/inmunología , Leucocitos Mononucleares/inmunología , Macaca mulatta , Análisis por Micromatrices , Resultado del TratamientoRESUMEN
Lassa virus (LASV) is a significant human pathogen that is endemic to several countries in West Africa. Infection with LASV leads to the development of hemorrhagic fever in a significant number of cases, and it is estimated that thousands die each year from the disease. Little is known about the complex immune mechanisms governing the response to LASV or the genetic determinants of susceptibility and resistance to infection. In the study presented here, we have used a whole-genome, microarray-based approach to determine the temporal host response in the peripheral blood mononuclear cells (PBMCs) of non-human primates (NHP) following aerosol exposure to LASV. Sequential sampling over the entire disease course showed that there are strong transcriptional changes of the immune response to LASV exposure, including the early induction of interferon-responsive genes and Toll-like receptor signaling pathways. However, this increase in early innate responses was coupled with a lack of pro-inflammatory cytokine response in LASV exposed NHPs. There was a distinct lack of cytokines such as IL1ß and IL23α, while immunosuppressive cytokines such as IL27 and IL6 were upregulated. Comparison of IRF/STAT1-stimulated gene expression with the viral load in LASV exposed NHPs suggests that mRNA expression significantly precedes viremia, and thus might be used for early diagnostics of the disease. Our results provide a transcriptomic survey of the circulating immune response to hemorrhagic LASV exposure and provide a foundation for biomarker identification to allow clinical diagnosis of LASV infection through analysis of the host response.
Asunto(s)
Perfilación de la Expresión Génica/métodos , Fiebre de Lassa/genética , Virus Lassa/inmunología , Leucocitos Mononucleares/metabolismo , Macaca/inmunología , Macaca/virología , Animales , Femenino , Inmunidad Innata/inmunología , Fiebre de Lassa/virología , Virus Lassa/patogenicidad , Masculino , Receptores Toll-Like/metabolismoRESUMEN
Cells infected with human cytomegalovirus (HCMV) after commencing DNA replication do not initiate viral immediate-early (IE) gene expression and divide before arresting. To determine the nature of this blockade, we examined cells that were infected 24 h after release from G(0) using immunofluorescence, laser scanning cytometry, and fluorescence-activated cell sorting (FACS) analysis. Approximately 40 to 50% of the cells had 2N DNA content, became IE(+) in the first 12 h, and arrested. Most but not all of the cells with >2N DNA content did not express IE antigens until after mitosis. To define the small population of IE(+) cells that gradually accumulated within the S and G(2)/M compartments, cells were pulsed with bromodeoxyuridine (BrdU) just prior to S-phase infection and analyzed at 12 h postinfection for IE gene expression, BrdU positivity, and cell cycle position. Most of the BrdU(+) cells were IE(-) and had progressed into G(2)/M or back to G(1). The majority of the IE(+) cells in S and G(2)/M were BrdU(-). Only a few cells were IE(+) BrdU(+), and they resided in G(2)/M. Multipoint BrdU pulse-labeling revealed that, compared to cells actively synthesizing DNA at the beginning of the infection, a greater percentage of the cells that initiated DNA replication 4 h later could express IE antigens and proceed into S. Synchronization of the cells with aphidicolin also indicated that the blockade to the activation of IE gene expression was established in cells soon after initiation of DNA replication. It appears that a short-lived protein in S-phase cells may be required for IE gene expression, as it is partially restored by treatment with the proteasome inhibitor MG132.
Asunto(s)
Cisteína Endopeptidasas/metabolismo , Citomegalovirus/genética , Expresión Génica , Genes Inmediatos-Precoces , Proteínas Inmediatas-Precoces/genética , Glicoproteínas de Membrana , Complejos Multienzimáticos/metabolismo , Transactivadores , Proteínas del Envoltorio Viral , Proteínas Virales , Células Cultivadas , Inhibidores de Cisteína Proteinasa/farmacología , Citomegalovirus/fisiología , ADN/metabolismo , Replicación del ADN , Fibroblastos/citología , Perfilación de la Expresión Génica , Humanos , Leupeptinas/farmacología , Complejo de la Endopetidasa Proteasomal , Fase de Descanso del Ciclo Celular , Fase S , Factores de TiempoRESUMEN
Using bacterial artificial chromosome (BAC) technology, we have constructed and characterized a human cytomegalovirus recombinant virus with a mutation in the exon specific for the major immediate-early region 2 (IE2) gene product. The resulting IE2 86-kDa protein (IE2 86) has an internal deletion of amino acids 136 to 290 and is fused at the carboxy terminus to enhanced green fluorescent protein (EGFP). The deletion also removes the promoter and initiator methionine for the p40 form of IE2 and initiator methionine for the p60 form of the protein, and therefore, these late gene products are not produced. The mutant virus IE2 86 Delta SX-EGFP is viable but exhibits altered growth characteristics in tissue culture compared with a full-length wild-type (wt) IE2 86-EGFP virus or a revertant virus. When cells are infected with the mutant virus at a low multiplicity of infection (MOI), there is a marked delay in the production of infectious virus. This is associated with slower cell-to-cell spread of the virus. By immunofluorescence and Western blot analyses, we show that the early steps in the replication of the mutant virus are comparable to those for the wt. Although there is significantly less IE2 protein in the cells infected with the mutant, there is only a modest lag in the initial accumulation of IE1 72 and viral early proteins, and viral DNA replication proceeds normally. The mutation also has only a small effect on the synthesis of the viral major capsid protein. The most notable molecular defect in the mutant virus infection is that the steady-state levels of the pp65 (UL83) and pp28 (UL99) matrix proteins are greatly reduced. In the case of UL83, but not UL99, there is also a corresponding decrease in the amount of mRNA present in cells infected with the mutant virus.