RESUMO
Recent biological terrorism threats and outbreaks of microbial pathogens clearly emphasize the need for biosensors that can quickly and accurately identify infectious agents. The majority of rapid biosensors generate detectable signals when a molecular probe in the detector interacts with an analyte of interest. Analytes may be whole bacterial or fungal cells, virus particles, or specific molecules, such as chemicals or protein toxins, produced by the infectious agent. Peptides and nucleic acids are most commonly used as probes in biosensors because of their versatility in forming various tertiary structures. The interaction between the probe and the analyte can be detected by various sensor platforms, including quartz crystal microbalances, surface acoustical waves, surface plasmon resonance, amperometrics, and magnetoelastics. The field of biosensors is constantly evolving to develop devices that have higher sensitivity and specificity, and are smaller, portable, and cost-effective. This mini review discusses recent advances in peptide-dependent rapid biosensors and their applications as well as relative advantages and disadvantages of each technology.
Assuntos
Técnicas Biossensoriais/métodos , Bioterrorismo , Técnicas de Sonda Molecular , Animais , Técnicas Biossensoriais/economia , Humanos , Técnicas de Sonda Molecular/economia , Peptídeos/genética , Peptídeos/imunologiaRESUMO
Using site-directed mutagenesis and retroviral vector pseudotyping of the wild type or mutated glycoprotein of Zaire ebolavirus (ZEBOV), we analyzed 15 conserved residues in the N-terminus of the filovirus glycoprotein 1 (GP1) in order to identify residues critical for cell entry. Results from infectivity assays and Western blot analyses identified two phenylalanine residues at positions 88 and 159 that appear to be critical for ZEBOV entry in vitro. We extended this observation by introduction of alanines at either position 88 or 159 of Ivory Coast Ebolavirus (CIEBOV) and observed the same phenotype. Further, we showed that introduction of each of the two mutations in a recombinant full-length clone of ZEBOV (Mayinga strain) that also carried the coding sequence for GFP could not be rescued, suggesting the mutants rendered the virus non-infectious. The two phenylalanines that are critical for both ZEBOV and CIEBOV entry are found in two linear domains of GP1 that are highly conserved among filoviruses, and thus could provide a target for rational development of broadly cross-protective vaccines or antiviral therapies.
Assuntos
Ebolavirus/fisiologia , Doença pelo Vírus Ebola/virologia , Fenilalanina/fisiologia , Proteínas do Envelope Viral/fisiologia , Sequência de Aminoácidos , Animais , Western Blotting , Linhagem Celular , Humanos , Dados de Sequência Molecular , Fenilalanina/genética , Mutação Puntual , Estrutura Terciária de Proteína/fisiologia , Alinhamento de Sequência , Proteínas do Envelope Viral/genética , Replicação ViralRESUMO
The VP35 protein of Zaire Ebola virus is an essential component of the viral RNA polymerase complex and also functions to antagonize the cellular type I interferon (IFN) response by blocking activation of the transcription factor IRF-3. We previously mapped the IRF-3 inhibitory domain within the C terminus of VP35. In the present study, we show that mutations that disrupt the IRF-3 inhibitory function of VP35 do not disrupt viral transcription/replication, suggesting that the two functions of VP35 are separable. Second, using reverse genetics, we successfully recovered recombinant Ebola viruses containing mutations within the IRF-3 inhibitory domain. Importantly, we show that the recombinant viruses were attenuated for growth in cell culture and that they activated IRF-3 and IRF-3-inducible gene expression at levels higher than that for Ebola virus containing wild-type VP35. In the context of Ebola virus pathogenesis, VP35 may function to limit early IFN-beta production and other antiviral signals generated from cells at the primary site of infection, thereby slowing down the host's ability to curb virus replication and induce adaptive immunity.
Assuntos
Vacinas contra Ebola/genética , Ebolavirus/genética , Fator Regulador 3 de Interferon/genética , Vacinas Atenuadas/genética , Proteínas Virais/genética , Replicação Viral/genética , Animais , Antivirais/imunologia , Antivirais/metabolismo , Antivirais/farmacologia , Chlorocebus aethiops , Vacinas contra Ebola/imunologia , Vacinas contra Ebola/metabolismo , Ebolavirus/imunologia , Ebolavirus/metabolismo , Doença pelo Vírus Ebola/genética , Doença pelo Vírus Ebola/imunologia , Doença pelo Vírus Ebola/metabolismo , Humanos , Fator Regulador 3 de Interferon/antagonistas & inibidores , Fator Regulador 3 de Interferon/metabolismo , Interferon beta/imunologia , Interferon beta/metabolismo , Interferon beta/farmacologia , Estrutura Terciária de Proteína/genética , Transcrição Gênica/genética , Transcrição Gênica/imunologia , Vacinas Atenuadas/imunologia , Células Vero , Proteínas Virais/imunologia , Proteínas Virais/metabolismo , Proteínas Virais Reguladoras e Acessórias , Replicação Viral/efeitos dos fármacos , Replicação Viral/imunologiaRESUMO
Zaire ebolavirus causes large outbreaks of severe and usually fatal hemorrhagic disease in humans for which there is no effective treatment or cure. To facilitate examination of early critical events in viral pathogenesis and to identify antiviral compounds, a recombinant Zaire ebolavirus was engineered to express a foreign protein, eGFP, to provide a rapid and sensitive means to monitor virus replication in infected cells. This genetically engineered virus represents the first insertion of a foreign gene into ebolavirus. We show that Ebola-eGFP virus (EboZ-eGFP) infects known early targets of human infections and serves as an ideal model to screen antiviral compounds in less time than any previously published assay.