RESUMO
Venezuelan equine encephalitis virus (VEEV) is a reemerging alphavirus that can cause encephalitis resulting in severe human morbidity and mortality. Using a high-throughput cell-based screen, we identified a quinolinone compound that protected against VEEV-induced cytopathic effects. Analysis of viral replication in cells identified several quinolinone compounds with potent inhibitory activity against vaccine and virulent strains of VEEV. These quinolinones also displayed inhibitory activity against additional alphaviruses, such as Mayaro virus and Ross River virus, although the potency was greatly reduced. Time-of-addition studies indicated that these compounds inhibit the early-to-mid stage of viral replication. Deep sequencing and reverse genetics studies identified two unique resistance mutations in the nsP2 gene (Y102S/C; stalk domain) that conferred VEEV resistance on this chemical series. Moreover, introduction of a K102Y mutation into the nsP2 gene enhanced the sensitivity of chikungunya virus (CHIKV) to this chemical series. Computational modeling of CHIKV and VEEV nsP2 identified a highly probable docking alignment for the quinolinone compounds that require a tyrosine residue at position 102 within the helicase stalk domain. These studies identified a class of compounds with antiviral activity against VEEV and other alphaviruses and provide further evidence that therapeutics targeting nsP2 may be useful against alphavirus infection.
Assuntos
Vírus Chikungunya , Vírus da Encefalite Equina Venezuelana , Quinolonas , Animais , Antivirais/farmacologia , Vírus da Encefalite Equina Venezuelana/genética , Cavalos , Humanos , Quinolonas/farmacologia , Replicação ViralRESUMO
Alphaviruses are arthropod-transmitted members of the Togaviridae family that can cause severe disease in humans, including debilitating arthralgia and severe neurological complications. Currently, there are no approved vaccines or antiviral therapies directed against the alphaviruses, and care is limited to treating disease symptoms. A phenotypic cell-based high-throughput screen was performed to identify small molecules that inhibit the replication of Venezuelan Equine Encephalitis Virus (VEEV). The compound, 1-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)-N-(3-fluoro-4-methoxybenzyl)ethan-1-amine (1), was identified as a highly active, potent inhibitor of VEEV with an effective concentration for 90% inhibition of virus (EC90) of 0.89 µM and 7.49 log reduction in virus titers at 10 µM concentration. These data suggest that further investigation of compound 1 as an antiviral therapeutic against VEEV, and perhaps other alphaviruses, is warranted. Experiments suggested that the antiviral activity of compound 1 is directed at an early step in the VEEV replication cycle by blocking viral RNA and protein synthesis.
Assuntos
Antivirais/farmacologia , Benzilaminas/farmacologia , Vírus da Encefalite Equina Venezuelana/efeitos dos fármacos , Encefalomielite Equina Venezuelana/virologia , Animais , Antivirais/química , Benzilaminas/química , Linhagem Celular , Chlorocebus aethiops , Relação Dose-Resposta a Droga , Encefalomielite Equina Venezuelana/tratamento farmacológico , Ensaios de Triagem em Larga Escala , Humanos , Estrutura Molecular , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/farmacologia , Bibliotecas de Moléculas Pequenas/uso terapêutico , Células Vero , Carga Viral/efeitos dos fármacos , Replicação Viral/efeitos dos fármacosRESUMO
Zoonotic viruses circulate as swarms in animal reservoirs and can emerge into human populations, causing epidemics that adversely affect public health. Portable, safe, and effective vaccine platforms are needed in the context of these outbreak and emergence situations. In this work, we report the generation and characterization of an alphavirus replicon vaccine platform based on a non-select agent, attenuated Venezuelan equine encephalitis (VEE) virus vaccine, strain 3526 (VRP 3526). Using both noroviruses and coronaviruses as model systems, we demonstrate the utility of the VRP 3526 platform in the generation of recombinant proteins, production of virus-like particles, and in vivo efficacy as a vaccine against emergent viruses. Importantly, packaging under biosafety level 2 (BSL2) conditions distinguishes VRP 3526 from previously reported alphavirus platforms and makes this approach accessible to the majority of laboratories around the world. In addition, improved outcomes in the vulnerable aged models as well as against heterologous challenge suggest improved efficacy compared to that of previously attenuated VRP approaches. Taking these results together, the VRP 3526 platform represents a safe and highly portable system that can be rapidly deployed under BSL2 conditions for generation of candidate vaccines against emerging microbial pathogens.IMPORTANCE While VEE virus replicon particles provide a robust, established platform for antigen expression and vaccination, its utility has been limited by the requirement for high-containment-level facilities for production and packaging. In this work, we utilize an attenuated vaccine strain capable of use at lower biocontainment level but retaining the capacity of the wild-type replicon particle. Importantly, the new replicon platform provides equal protection for aged mice and following heterologous challenge, which distinguishes it from other attenuated replicon platforms. Together, the new system represents a highly portable, safe system for use in the context of disease emergence.
Assuntos
Anticorpos Antivirais/imunologia , Vírus da Encefalite Equina Venezuelana/imunologia , Encefalomielite Equina Venezuelana/imunologia , Síndrome Respiratória Aguda Grave/imunologia , Coronavírus Relacionado à Síndrome Respiratória Aguda Grave/imunologia , Vacinas Atenuadas/imunologia , Vacinas Virais/imunologia , Envelhecimento/imunologia , Animais , Anticorpos Antivirais/sangue , Linhagem Celular , Chlorocebus aethiops , Vírus da Encefalite Equina Venezuelana/genética , Encefalomielite Equina Venezuelana/prevenção & controle , Encefalomielite Equina Venezuelana/virologia , Feminino , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Síndrome Respiratória Aguda Grave/prevenção & controle , Síndrome Respiratória Aguda Grave/virologia , Células Vero , Zoonoses/prevenção & controle , Zoonoses/virologiaRESUMO
Here, we report the complete genome sequences of 15 chikungunya virus strains isolated from human plasma from infected patients in Puerto Rico. The results show that currently circulating chikungunya strains in Puerto Rico are closely related.
RESUMO
Venezuelan equine encephalitis virus (VEEV) is an important human and veterinary pathogen causing sporadic epizootic outbreaks of potentially fatal encephalitis. The type I interferon (IFN) system plays a central role in controlling VEEV and other alphavirus infections, and IFN evasion is likely an important determinant of whether these viruses disseminate and cause disease within their hosts. Alphaviruses are thought to limit the induction of type I IFNs and IFN-stimulated genes by shutting off host cell macromolecular synthesis, which in the case of VEEV is partially mediated by the viral capsid protein. However, more specific strategies by which alphaviruses inhibit type I IFN signaling have not been characterized. Analyses of cells infected with VEEV and VEEV replicon particles (VRP) demonstrate that viral infection rapidly disrupts tyrosine phosphorylation and nuclear translocation of the transcription factor STAT1 in response to both IFN-beta and IFN-gamma. This effect was independent of host shutoff and expression of viral capsid, suggesting that VEEV uses novel mechanisms to interfere with type I and type II IFN signaling. Furthermore, at times when STAT1 activation was efficiently inhibited, VRP infection did not limit tyrosine phosphorylation of Jak1, Tyk2, or STAT2 after IFN-beta treatment but did inhibit Jak1 and Jak2 activation in response to IFN-gamma, suggesting that VEEV interferes with STAT1 activation by the type I and II receptor complexes through distinct mechanisms. Identification of the viral requirements for this novel STAT1 inhibition will further our understanding of alphavirus molecular pathogenesis and may provide insights into effective alphavirus-based vaccine design.