RESUMEN
Venezuelan equine encephalitis virus (VEEV) is an alphavirus transmitted by mosquitos that can cause a febrile illness and induce severe neurological complications in humans and equine populations. Currently there are no FDA approved vaccines or antiviral treatments to combat VEEV. Proteomic techniques were utilized to create an interactome of the E1 fusion glycoprotein of VEEV. VEEV E1 interacted with a number of cellular chaperone proteins including protein disulfide isomerase family A member 6 (PDIA6). PDI inhibition through LOC14 and/or nitazoxanide treatment effectively decreased production of VEEV and other alphaviruses in vitro, including eastern equine encephalitis virus, Sindbis virus, and chikungunya virus. Decreased oxidoreductive capabilities of PDIs through LOC14 or nitazoxanide treatment impacted both early and late events in viral replication, including the production of non-infectious virions and decreased VEEV E1 disulfide bond formation. Results from this study identified PDIs as critical regulators of alphavirus replication and potential therapeutic targets.
Asunto(s)
Alphavirus , Virus Chikungunya , Virus de la Encefalitis Equina Venezolana , Encefalomielitis Equina Venezolana , Humanos , Animales , Caballos , Proteómica , Línea Celular , Replicación Viral , Encefalomielitis Equina Venezolana/tratamiento farmacológico , Proteína Disulfuro Isomerasas/farmacología , Proteína Disulfuro Isomerasas/uso terapéuticoRESUMEN
The innate immune protection provided by cationic antimicrobial peptides (CAMPs) has been shown to extend to antiviral activity, with putative mechanisms of action including direct interaction with host cells or pathogen membranes. The lack of therapeutics available for the treatment of viruses such as Venezuelan equine encephalitis virus (VEEV) underscores the urgency of novel strategies for antiviral discovery. American alligator plasma has been shown to exhibit strong in vitro antibacterial activity, and functionalized hydrogel particles have been successfully employed for the identification of specific CAMPs from alligator plasma. Here, a novel bait strategy in which particles were encapsulated in membranes from either healthy or VEEV-infected cells was implemented to identify peptides preferentially targeting infected cells for subsequent evaluation of antiviral activity. Statistical analysis of peptide identification results was used to select five candidate peptides for testing, of which one exhibited a dose-dependent inhibition of VEEV and also significantly inhibited infectious titers. Results suggest our bioprospecting strategy provides a versatile platform that may be adapted for antiviral peptide identification from complex biological samples.
Asunto(s)
Caimanes y Cocodrilos , Virus de la Encefalitis Equina Venezolana , Encefalomielitis Equina Venezolana , Animales , Caballos , Virus de la Encefalitis Equina Venezolana/fisiología , Antivirales/farmacología , Antivirales/uso terapéutico , Encefalomielitis Equina Venezolana/tratamiento farmacológico , Encefalomielitis Equina Venezolana/prevención & control , Bioprospección , Replicación Viral , PéptidosRESUMEN
The blood brain barrier (BBB) is a multicellular microenvironment that plays an important role in regulating bidirectional transport to and from the central nervous system (CNS). Infections by many acutely infectious viruses such as alphaviruses and flaviviruses are known to impact the integrity of the endothelial lining of the BBB. Infection by Venezuelan Equine Encephalitis Virus (VEEV) through the aerosol route causes significant damage to the integrity of the BBB, which contributes to long-term neurological sequelae. An effective therapeutic intervention strategy should ideally not only control viral load in the host, but also prevent and/or reverse deleterious events at the BBB. Two dimensional monocultures, including trans-well models that use endothelial cells, do not recapitulate the intricate multicellular environment of the BBB. Complex in vitro organ-on-a-chip models (OOC) provide a great opportunity to introduce human-like experimental models to understand the mechanistic underpinnings of the disease state and evaluate the effectiveness of therapeutic candidates in a highly relevant manner. Here we demonstrate the utility of a neurovascular unit (NVU) in analyzing the dynamics of infection and proinflammatory response following VEEV infection and therapeutic effectiveness of omaveloxolone to preserve BBB integrity and decrease viral and inflammatory load.
Asunto(s)
Virus de la Encefalitis Equina Venezolana , Encefalomielitis Equina Venezolana , Humanos , Animales , Caballos , Virus de la Encefalitis Equina Venezolana/fisiología , Barrera Hematoencefálica , Encefalomielitis Equina Venezolana/tratamiento farmacológico , Encefalomielitis Equina Venezolana/prevención & control , Células Endoteliales , Sistemas MicrofisiológicosRESUMEN
Venezuelan equine encephalitis virus (VEEV) is an alphavirus that is endemic to the Americas. VEEV outbreaks occur periodically and cause encephalitis in both humans and equids. There are currently no therapeutics or vaccines for treatment of VEEV in humans. Our group has previously reported on the development of a benzamidine VEEV inhibitor, ML336, which shows potent antiviral activity in both in vitro and in vivo models of infection. In cell culture experiments, ML336 inhibits viral RNA synthesis when added 2-4 h post-infection, and mutations conferring resistance occur within the viral nonstructural proteins (nsP2 and nsP4). We hypothesized that ML336 targets an activity of the viral replicase complex and inhibits viral RNA synthesis. To test this hypothesis, we employed various biochemical and cellular assays. Using structural analogues of ML336, we demonstrate that the cellular antiviral activity of these compounds correlates with their inhibition of viral RNA synthesis. For instance, the IC50 of ML336 for VEEV RNA synthesis inhibition was determined as 1.1 nM, indicating potent anti-RNA synthesis activity in the low nanomolar range. While ML336 efficiently inhibited VEEV RNA synthesis, a much weaker effect was observed against the Old World alphavirus Chikungunya virus (IC50 > 4 µM), agreeing with previous data from a cell based assay. Using a tritium incorporation assay, we demonstrated that there was no significant inhibition of cellular transcription. With a combination of fluorography, strand-specific qRT-PCR, and tritium incorporation, we demonstrated that ML336 inhibits the synthesis of the positive sense genomic, negative sense template, and subgenomic RNAs of VEEV. Based on these results, we propose that the mechanism of action for this class of antiviral compounds is inhibition of viral RNA synthesis through interaction with the viral replicase complex.
Asunto(s)
Antivirales/farmacología , Benzamidas/farmacología , Virus de la Encefalitis Equina Venezolana/efectos de los fármacos , Inhibidores de la Síntesis del Ácido Nucleico/farmacología , Piperazinas/farmacología , ARN Viral/antagonistas & inhibidores , Replicación Viral/efectos de los fármacos , Animales , Línea Celular , Chlorocebus aethiops , Cricetinae , Encefalomielitis Equina Venezolana/tratamiento farmacológico , Encefalomielitis Equina Venezolana/virología , Caballos , Interacciones Microbiota-Huesped/efectos de los fármacos , Concentración 50 Inhibidora , Riñón/citología , ARN Viral/biosíntesis , Células VeroRESUMEN
Venezuelan equine encephalitis virus (VEEV) is a category B select agent pathogen that can be aerosolized. Infections in murine models and humans can advance to an encephalitic phenotype which may result in long-term neurological complications or death. No specific FDA-approved treatments or vaccines are available for the treatment or prevention of VEEV infection. Neurotropic viral infections have two damaging components: neuronal death caused by viral replication, and damage from the subsequent inflammatory response. Reducing the level of inflammation may lessen neurological tissue damage that often arises following VEEV infection. In this study, three commercially available anti-inflammatory drugs, Celecoxib, Rolipram, and Tofacitinib, were evaluated for antiviral activity in an astrocyte and a microglial model of VEEV infection. The inhibitors were tested against the vaccine strain VEEV TC-83, as well as the wild-type VEEV Trinidad donkey strain. Celecoxib, Tofacitinib, and Rolipram significantly decreased viral titers both after pre-treatment and post-treatment of infected cells. VEEV Trinidad Donkey (TrD) titers were reduced 6.45-fold in cells treated with 50 µM of Celecoxib, 2.45-fold when treated with 50 µM of Tofacitinib, and 1.81-fold when treated with 50 µM of Rolipram. Celecoxib was also shown to decrease inflammatory gene expression in the context of TC-83 infection. Overall, Celecoxib demonstrated potency as a countermeasure strategy that slowed VEEV infection and infection-induced inflammation in an in vitro model.
Asunto(s)
Antiinflamatorios/farmacología , Antivirales/farmacología , Reposicionamiento de Medicamentos , Virus de la Encefalitis Equina Venezolana/efectos de los fármacos , Encefalomielitis Equina Venezolana/tratamiento farmacológico , Encefalomielitis Equina Venezolana/virología , Replicación Viral/efectos de los fármacos , Animales , Astrocitos/efectos de los fármacos , Línea Celular , Supervivencia Celular/efectos de los fármacos , Citocinas/metabolismo , Aprobación de Drogas , Humanos , Microglía/efectos de los fármacos , Estados Unidos , United States Food and Drug AdministrationRESUMEN
The New World alphaviruses Venezuelan, Eastern, and Western equine encephalitis viruses (VEEV, EEEV and WEEV, respectively) commonly cause a febrile disease that can progress to meningoencephalitis, resulting in significant morbidity and mortality. To address the need for a therapeutic agent for the treatment of Alphavirus infections, we identified and pursued preclinical characterization of a ribonucleoside analog EIDD-1931 (ß-D-N4-hydroxycytidine, NHC), which has shown broad activity against alphaviruses in vitro and has a very high genetic barrier for development of resistance. To be truly effective as a therapeutic agent for VEEV infection a drug must penetrate the blood brain barrier and arrest virus replication in the brain. High plasma levels of EIDD-1931 are rapidly achieved in mice after oral dosing. Once in the plasma EIDD-1931 is efficiently distributed into organs, including brain, where it is rapidly converted to its active 5'-triphosphate. EIDD-1931 showed a good safety profile in mice after 7-day repeated dosing with up to 1000â¯mg/kg/day doses. In mouse model studies, EIDD-1931 was 90-100% effective in protecting mice against lethal intranasal infection when therapeutic treatment was started as late as 24â¯h post-infection, and partial protection was achieved when treatment was delayed for 48â¯h post-infection. These results support further preclinical development of EIDD-1931 as a potential anti-alphavirus drug.
Asunto(s)
Antivirales/farmacología , Virus de la Encefalitis Equina Venezolana/efectos de los fármacos , Encefalomielitis Equina Venezolana/virología , Ribonucleósidos/farmacología , Administración Oral , Animales , Antivirales/administración & dosificación , Antivirales/química , Antivirales/farmacocinética , Línea Celular , Cromatografía Liquida , Modelos Animales de Enfermedad , Encefalomielitis Equina Venezolana/tratamiento farmacológico , Caballos , Ratones , Estructura Molecular , Ribonucleósidos/administración & dosificación , Ribonucleósidos/química , Ribonucleósidos/farmacocinética , Espectrometría de Masas en Tándem , Distribución Tisular , Replicación Viral/efectos de los fármacosRESUMEN
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.
Asunto(s)
Antivirales/farmacología , Bencilaminas/farmacología , Virus de la Encefalitis Equina Venezolana/efectos de los fármacos , Encefalomielitis Equina Venezolana/virología , Animales , Antivirales/química , Bencilaminas/química , Línea Celular , Chlorocebus aethiops , Relación Dosis-Respuesta a Droga , Encefalomielitis Equina Venezolana/tratamiento farmacológico , Ensayos Analíticos de Alto Rendimiento , Humanos , Estructura Molecular , Bibliotecas de Moléculas Pequeñas/química , Bibliotecas de Moléculas Pequeñas/farmacología , Bibliotecas de Moléculas Pequeñas/uso terapéutico , Células Vero , Carga Viral/efectos de los fármacos , Replicación Viral/efectos de los fármacosRESUMEN
Currently, there are no licensed human vaccines or antivirals for treatment of or prevention from infection with encephalitic alphaviruses. Because epidemics are sporadic and unpredictable, and endemic disease is common but rarely diagnosed, it is difficult to identify all populations requiring vaccination; thus, an effective post-exposure treatment method is needed to interrupt ongoing outbreaks. To address this public health need, we have continued development of ML336 to deliver a molecule with prophylactic and therapeutic potential that could be relevant for use in natural epidemics or deliberate release scenario for Venezuelan equine encephalitis virus (VEEV). We report findings from in vitro assessments of four analogs of ML336, and in vivo screening of three of these new derivatives, BDGR-4, BDGR-69 and BDGR-70. The optimal dosing for maximal protection was observed at 12.5â¯mg/kg/day, twice daily for 8 days. BDGR-4 was tested further for prophylactic and therapeutic efficacy in mice challenged with VEEV Trinidad Donkey (TrD). Mice challenged with VEEV TrD showed 100% and 90% protection from lethal disease when treated at 24 and 48â¯h post-infection, respectively. We also measured 90% protection for BDGR-4 in mice challenged with Eastern equine encephalitis virus. In additional assessments of BDGR-4 in mice alone, we observed no appreciable toxicity as evaluated by clinical chemistry indicators up to a dose of 25â¯mg/kg/day over 4 days. In these same mice, we observed no induction of interferon. Lastly, the resistance of VEEV to BDGR-4 was evaluated by next-generation sequencing which revealed specific mutations in nsP4, the viral polymerase.
Asunto(s)
Benzamidas , Benzamidinas , Farmacorresistencia Viral/genética , Virus de la Encefalitis Equina del Este/efectos de los fármacos , Virus de la Encefalitis Equina Venezolana/efectos de los fármacos , Piperazinas , Animales , Antivirales/síntesis química , Antivirales/farmacología , Benzamidas/síntesis química , Benzamidas/farmacología , Benzamidinas/síntesis química , Benzamidinas/farmacología , Línea Celular , Encefalomielitis Equina Oriental/tratamiento farmacológico , Encefalomielitis Equina Oriental/prevención & control , Encefalomielitis Equina Venezolana/tratamiento farmacológico , Encefalomielitis Equina Venezolana/prevención & control , Genes Virales , Ratones , Mutación , Piperazinas/síntesis química , Piperazinas/farmacologíaRESUMEN
Venezuelan equine encephalitis virus (VEEV) is a known biological defense threat. A live-attenuated investigational vaccine, TC-83, is available, but it has a high non-response rate and can also cause severe reactogenicity. We generated two novel VEE vaccine candidates using self-amplifying mRNA (SAM). LAV-CNE is a live-attenuated VEE SAM vaccine formulated with synthetic cationic nanoemulsion (CNE) and carrying the RNA genome of TC-83. IAV-CNE is an irreversibly-attenuated VEE SAM vaccine formulated with CNE, delivering a TC-83 genome lacking the capsid gene. LAV-CNE launches a TC-83 infection cycle in vaccinated subjects but eliminates the need for live-attenuated vaccine production and potentially reduces manufacturing time and complexity. IAV-CNE produces a single cycle of RNA amplification and antigen expression without generating infectious viruses in subjects, thereby creating a potentially safer alternative to live-attenuated vaccine. Here, we demonstrated that mice vaccinated with LAV-CNE elicited immune responses similar to those of TC-83, providing 100% protection against aerosol VEEV challenge. IAV-CNE was also immunogenic, resulting in significant protection against VEEV challenge. These studies demonstrate the proof of concept for using the SAM platform to streamline the development of effective attenuated vaccines against VEEV and closely related alphavirus pathogens such as western and eastern equine encephalitis and Chikungunya viruses.
Asunto(s)
Virus de la Encefalitis Equina Venezolana/inmunología , Encefalomielitis Equina Venezolana/tratamiento farmacológico , Amplificación de Genes , Inmunogenicidad Vacunal , ARN Mensajero/genética , Vacunas Atenuadas/uso terapéutico , Vacunas Virales/uso terapéutico , Células A549 , Animales , Anticuerpos Neutralizantes/inmunología , Anticuerpos Antivirales/inmunología , Modelos Animales de Enfermedad , Emulsiones/química , Encefalomielitis Equina Venezolana/virología , Femenino , Humanos , Ratones , Ratones Endogámicos BALB C , Transfección , Vacunas Virales/farmacología , Replicación ViralRESUMEN
Venezuelan equine encephalitis (VEE) virus causes an acute central nervous system infection in human and animals. Melatonin (MLT), minocycline (MIN) and ascorbic acid (AA) have been shown to have antiviral activities in experimental infections; however, the mechanisms involved are poorly studied. Therefore, the aim of this study was to determine the effects of those compounds on the viral titers, NO production and lipid peroxidation in the brain of mice and neuroblastoma cultures infected by VEE virus. Infected mouse (10 LD50) were treated with MLT (500 µg/kg bw), MIN (50mg/kg bw) or AA (50mg/kg bw). Infected neuroblastoma cultures (MOI: 1); MLT: 0.5, 1, 5mM, MIN: 0.1, 0.2, 2 µM or AA: 25, 50, 75 µM. Brains were obtained at days 1, 3 and 5. In addition, survival rate of infected treated mice was also analyzed. Viral replication was determined by the plaque formation technique. NO and lipid peroxidation were measured by Griess׳ reaction and thiobarbituric acid assay respectively. Increased viral replication, NO production and lipid peroxidation were observed in both, infected brain and neuroblastoma cell cultures compared with uninfected controls. Those effects were diminished by the studied treatments. In addition, increased survival rate (50%) in treated infected animals compared with untreated infected mice (0%) was found. MLT, MIN and AA have an antiviral effect involving their anti-oxidant properties, and suggesting a potential use of these compounds for human VEE virus infection.
Asunto(s)
Antivirales/farmacología , Ácido Ascórbico/farmacología , Encefalomielitis Equina Venezolana/tratamiento farmacológico , Melatonina/farmacología , Minociclina/farmacología , Estrés Oxidativo/efectos de los fármacos , Animales , Encéfalo/efectos de los fármacos , Encéfalo/metabolismo , Línea Celular Tumoral , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Virus de la Encefalitis Equina Venezolana/metabolismo , Encefalomielitis Equina Venezolana/metabolismo , Encefalomielitis Equina Venezolana/mortalidad , Peroxidación de Lípido/efectos de los fármacos , Peroxidación de Lípido/fisiología , Masculino , Ratones , Neuroblastoma/tratamiento farmacológico , Neuroblastoma/metabolismo , Fármacos Neuroprotectores/farmacología , Óxido Nítrico/metabolismo , Estrés Oxidativo/fisiología , Tasa de Supervivencia , Resultado del Tratamiento , Carga ViralRESUMEN
BACKGROUND: Pro-inflammatory and oxidative events during brain Venezuelan equine encephalitis virus infection could lead to apoptosis and induce anti-inflammatory responses (increased expression of CD200). The aim of this study was to determine the effect of melatonin on brain apoptosis, oxidative stress, and CD200 molecule in mice and neuroblastoma cultures infected by Venezuelan equine encephalitis virus. METHODS: Mice were infected with 10 median lethal doses (LD50) of Venezuelan equine encephalitis virus, treated with melatonin (500 µg/kg bw; three days before infection and during all experimental time) and sacrificed on days 1, 3, and 5 postinfection. Brain samples were obtained at those periods of time. In addition, infected neuroblastoma cell cultures (multiplicity of infection [MOI]: 1) were treated with 0, 0.1, 0.5, and 1 mM of melatonin and analyzed at 2, 4, and 6 h. CD200 and apoptosis expressions were analyzed by immunohistochemistry and TUNEL assay, respectively. Nitrites and malondialdehyde were determined by appropriate biochemical methods. RESULTS: Increased brain expression of apoptosis, nitrite, and malondialdehyde productions and CD200 of infected mice were found. Melatonin diminished those expressions. Similarly, high apoptosis expression and nitrite and malondialdehyde productions on infected neuroblastoma cultures were diminished by melatonin. Melatonin increased the survival rate (25%) in Venezuelan equine encephalitis virus-infected animals compared with untreated infected mice (0%). CONCLUSIONS: Neurological damage during brain Venezuelan equine encephalitis virus infection could be mediated by apoptosis and oxidative stress and CD200 molecule could be an important anti-inflammatory response. Melatonin could be beneficial reducing apoptosis and oxidative stress.
Asunto(s)
Antígenos CD/biosíntesis , Apoptosis/efectos de los fármacos , Encéfalo/efectos de los fármacos , Virus de la Encefalitis Equina Venezolana/patogenicidad , Encefalomielitis Equina Venezolana/tratamiento farmacológico , Encefalomielitis Equina Venezolana/virología , Melatonina/farmacología , Estrés Oxidativo/efectos de los fármacos , Animales , Encéfalo/metabolismo , Encéfalo/patología , Masculino , Melatonina/administración & dosificación , Melatonina/uso terapéutico , Ratones , Pruebas de Sensibilidad Microbiana , Tasa de SupervivenciaRESUMEN
Venezuelan equine encephalitis virus (VEEV) is classified as a Category B Select Agent and potential bioterror weapon for its severe disease course in humans and equines and its potential for aerosol transmission. There are no current FDA licensed vaccines or specific therapies against VEEV, making identification of potential therapeutic targets a priority. With this aim, our research focuses on the interactions of VEEV with host microRNA (miRNA) machinery. miRNAs are small non-coding RNAs that act as master regulators of gene expression by downregulating or degrading messenger RNA, thus suppressing production of the resultant proteins. Recent publications implicate miRNA interactions in the pathogenesis of various viral diseases. To test the importance of miRNA processing for VEEV replication, cells deficient in Ago2, an important component of the RNA-induced silencing complex (RISC), and cells treated with known Ago2 inhibitors, notably acriflavine (ACF), were utilized. Both conditions caused decreased viral replication and capsid expression. ACF treatment promoted increased survival of neuronal cells over a non-treated, infected control and reduced viral titers of fully virulent VEEV as well as Eastern and Western Equine Encephalitis Viruses and West Nile Virus, but not Vesicular Stomatitis Virus. ACF treatment of VEEV TC-83 infected mice resulted in increased in vivo survival, but did not affect survival or viral loads when mice were challenged with fully virulent VEEV TrD. These results suggest that inhibition of Ago2 results in decreased replication of encephalitic alphaviruses in vitro and this pathway may be an avenue to explore for future therapeutic development.
Asunto(s)
Antivirales/farmacología , Proteínas Argonautas/antagonistas & inhibidores , Virus de la Encefalitis Equina Venezolana/efectos de los fármacos , Virus de la Encefalitis Equina Venezolana/fisiología , Inhibidores Enzimáticos/farmacología , Replicación Viral/efectos de los fármacos , Acriflavina/farmacología , Acriflavina/uso terapéutico , Animales , Antivirales/uso terapéutico , Proteínas de la Cápside/biosíntesis , Supervivencia Celular , Modelos Animales de Enfermedad , Encefalomielitis Equina Venezolana/tratamiento farmacológico , Encefalomielitis Equina Venezolana/virología , Inhibidores Enzimáticos/uso terapéutico , Ratones Endogámicos BALB C , Ratones Endogámicos C3H , Neuronas/fisiología , Neuronas/virología , Análisis de Supervivencia , Resultado del Tratamiento , Carga ViralRESUMEN
Venezuelan equine encephalitis virus (VEEV) is an emerging pathogenic alphavirus that can cause significant disease in humans. Given the absence of therapeutic options available and the significance of VEEV as a weaponized agent, an optimization effort was initiated around a quinazolinone screening hit 1 with promising cellular antiviral activity (EC50 = 0.8 µM), limited cytotoxic liability (CC50 > 50 µM), and modest in vitro efficacy in reducing viral progeny (63-fold at 5 µM). Scaffold optimization revealed a novel rearrangement affording amidines, specifically compound 45, which was found to potently inhibit several VEEV strains in the low nanomolar range without cytotoxicity (EC50 = 0.02-0.04 µM, CC50 > 50 µM) while limiting in vitro viral replication (EC90 = 0.17 µM). Brain exposure was observed in mice with 45. Significant protection was observed in VEEV-infected mice at 5 mg kg(-1) day(-1) and viral replication appeared to be inhibited through interference of viral nonstructural proteins.
Asunto(s)
Antivirales/química , Antivirales/farmacología , Benzamidas/farmacología , Virus de la Encefalitis Equina Venezolana/efectos de los fármacos , Piperazinas/farmacología , Animales , Benzamidas/química , Evaluación Preclínica de Medicamentos/métodos , Encefalomielitis Equina Venezolana/tratamiento farmacológico , Compuestos Heterocíclicos con 2 Anillos/química , Ratones Endogámicos C3H , Ratones Endogámicos C57BL , Piperazinas/química , Quinazolinonas/química , Bibliotecas de Moléculas Pequeñas/química , Bibliotecas de Moléculas Pequeñas/farmacología , Relación Estructura-Actividad , Replicación Viral/efectos de los fármacosRESUMEN
Alphaviruses present serious health threats as emerging and re-emerging viruses. Venezuelan equine encephalitis virus (VEEV), a New World alphavirus, can cause encephalitis in humans and horses, but there are no therapeutics for treatment. To date, compounds reported as anti-VEEV or anti-alphavirus inhibitors have shown moderate activity. To discover new classes of anti-VEEV inhibitors with novel viral targets, we used a high-throughput screen based on the measurement of cell protection from live VEEV TC-83-induced cytopathic effect to screen a 340,000 compound library. Of those, we identified five novel anti-VEEV compounds and chose a quinazolinone compound, CID15997213 (IC50â=â0.84 µM), for further characterization. The antiviral effect of CID15997213 was alphavirus-specific, inhibiting VEEV and Western equine encephalitis virus, but not Eastern equine encephalitis virus. In vitro assays confirmed inhibition of viral RNA, protein, and progeny synthesis. No antiviral activity was detected against a select group of RNA viruses. We found mutations conferring the resistance to the compound in the N-terminal domain of nsP2 and confirmed the target residues using a reverse genetic approach. Time of addition studies showed that the compound inhibits the middle stage of replication when viral genome replication is most active. In mice, the compound showed complete protection from lethal VEEV disease at 50 mg/kg/day. Collectively, these results reveal a potent anti-VEEV compound that uniquely targets the viral nsP2 N-terminal domain. While the function of nsP2 has yet to be characterized, our studies suggest that the protein might play a critical role in viral replication, and further, may represent an innovative opportunity to develop therapeutic interventions for alphavirus infection.
Asunto(s)
Antivirales/farmacología , Virus de la Encefalitis Equina Venezolana/efectos de los fármacos , Encefalomielitis Equina Venezolana/tratamiento farmacológico , Quinazolinonas/farmacología , Animales , Línea Celular , Chlorocebus aethiops , Cricetinae , Modelos Animales de Enfermedad , Farmacorresistencia Viral/genética , Virus de la Encefalitis Equina Venezolana/genética , Encefalomielitis Equina Venezolana/virología , Ensayos Analíticos de Alto Rendimiento , Ratones , Ratones Endogámicos C3H , Especificidad de la Especie , Relación Estructura-Actividad , Células Vero , Ensayo de Placa Viral , Replicación Viral/efectos de los fármacosRESUMEN
BACKGROUND: Stupor, coma, and other alterations of consciousness are among the most serious life-threatening emergencies faced by the emergency department physician. When a patient arrives with altered mentation from Central or South America, the usual causes that occur in the United States must be considered; however, other unusual tropical disease must be excluded, such as Venezuelan equine encephalitis (VEE). OBJECTIVE: This study aimed to review the clinical features of VEE. CASE: A 17-year-old female traveled to Belize and developed vomiting, diarrhea, fever, headaches, and myalgias. Over the next few hours, she became disoriented and had a generalized seizure. She was given diazepam, 50% dextrose, phenytoin, mannitol, and vitamin K. A computed tomographic scan of the head was unremarkable. Her parents arranged for a medical air transport. After eliminating other possibilities, she was diagnosed with VEE, which was confirmed in the laboratory. Over the next week, her mental status improved back to her normal neurologic baseline. CONCLUSIONS: Venezuelan equine encephalitis is an acute viral disease that causes acute illness in equines and humans, with symptoms ranging from a mild, flu-like syndrome to encephalitis or death. Laboratory abnormalities are common and include elevated hepatic transaminases, lymphocytosis, eosinophilia, and thrombocytopenia. Treatment is supportive, and complete recovery is expected within several weeks in most patients.
Asunto(s)
Virus de la Encefalitis Equina Venezolana/inmunología , Encefalomielitis Equina Venezolana/diagnóstico , Viaje , Adolescente , Antibacterianos/administración & dosificación , Anticuerpos Antivirales/análisis , Belice/etnología , Ceftriaxona/administración & dosificación , Diagnóstico Diferencial , Encefalomielitis Equina Venezolana/tratamiento farmacológico , Encefalomielitis Equina Venezolana/etnología , Ensayo de Inmunoadsorción Enzimática , Femenino , Estudios de Seguimiento , Humanos , Inyecciones Intravenosas , Estados Unidos/epidemiologíaRESUMEN
Alphaviruses, including Venezuelan Equine Encephalitis Virus (VEEV), cause disease in both equine and humans that exhibit overt encephalitis in a significant percentage of cases. Features of the host immune response and tissue-specific responses may contribute to fatal outcomes as well as the development of encephalitis. It has previously been shown that VEEV infection of mice induces transcription of pro-inflammatory cytokines genes (e.g., IFN-γ, IL-6, IL-12, iNOS and TNF-α) within 6 h. GSK-3ß is a host protein that is known to modulate pro-inflammatory gene expression and has been a therapeutic target in neurodegenerative disorders such as Alzheimer's. Hence inhibition of GSK-3ß in the context of encephalitic viral infections has been useful in a neuroprotective capacity. Small molecule GSK-3ß inhibitors and GSK-3ß siRNA experiments indicated that GSK-3ß was important for VEEV replication. Thirty-eight second generation BIO derivatives were tested and BIOder was found to be the most potent inhibitor, with an IC(50) of â¼0.5 µM and a CC(50) of >100 µM. BIOder was a more potent inhibitor of GSK-3ß than BIO, as demonstrated through in vitro kinase assays from uninfected and infected cells. Size exclusion chromatography experiments demonstrated that GSK-3ß is found in three distinct complexes in VEEV infected cells, whereas GSK-3ß is only present in one complex in uninfected cells. Cells treated with BIOder demonstrated an increase in the anti-apoptotic gene, survivin, and a decrease in the pro-apoptotic gene, BID, suggesting that modulation of pro- and anti-apoptotic genes contributes to the protective effect of BIOder treatment. Finally, BIOder partially protected mice from VEEV induced mortality. Our studies demonstrate the utility of GSK-3ß inhibitors for modulating VEEV infection.
Asunto(s)
Virus de la Encefalitis Equina Venezolana/efectos de los fármacos , Encefalomielitis Equina Venezolana/tratamiento farmacológico , Inhibidores Enzimáticos/uso terapéutico , Glucógeno Sintasa Quinasa 3/antagonistas & inhibidores , Animales , Proteína Proapoptótica que Interacciona Mediante Dominios BH3/análisis , Encefalomielitis Equina Venezolana/mortalidad , Femenino , Glucógeno Sintasa Quinasa 3 beta , Proteínas Inhibidoras de la Apoptosis/análisis , Ratones , Ratones Endogámicos C3H , Proteínas Represoras/análisis , Survivin , Replicación Viral/efectos de los fármacosRESUMEN
We recently described a Venezuelan equine encephalitis virus (VEEV)-specific human monoclonal antibody (MAb), F5 nIgG, that recognizes a new neutralization epitope on the VEEV E2 envelope glycoprotein. In this study, we investigated the ability of F5 nIgG given prophylactically or therapeutically to protect mice from subcutaneous or aerosolized VEEV infection. F5 nIgG had potent ability to protect mice from infection by either route when administered 24h before exposure; however, mice treated 24h after aerosol exposure developed central nervous system infections but exhibited no clinical signs of disease. Infectious virus, viral antigen and RNA were detected in brains of both treated and untreated mice 2-6 days after aerosol exposure but were cleared from the brains of treated animals by 14-28 days after infection. This fully human MAb could be useful for prophylaxis or immediate therapy for individuals exposed to VEEV accidentally in the laboratory or during a deliberate release.
Asunto(s)
Anticuerpos Monoclonales/uso terapéutico , Anticuerpos Antivirales/uso terapéutico , Virus de la Encefalitis Equina Venezolana/fisiología , Encefalomielitis Equina Venezolana/tratamiento farmacológico , Encefalomielitis Equina Venezolana/prevención & control , Animales , Anticuerpos Monoclonales/inmunología , Anticuerpos Antivirales/inmunología , Línea Celular , Modelos Animales de Enfermedad , Virus de la Encefalitis Equina Venezolana/inmunología , Virus de la Encefalitis Equina Venezolana/patogenicidad , Encefalomielitis Equina Venezolana/inmunología , Encefalomielitis Equina Venezolana/virología , Femenino , Humanos , Masculino , Ratones , Ratones Endogámicos ICR , Pruebas de Neutralización , Profilaxis Posexposición , Proteínas del Envoltorio Viral/inmunología , VirulenciaRESUMEN
The effect of Luzindole (LZ) in mice treated with melatonin (MEL) during the infection with the Venezuelan equine encephalomyelitis (VEE) virus was examined. Melatonin (500 microg/Kg b.w.) was administered daily 3 days before and 5 days after the infection. Luzindole (5 mg/Kg b.w.) was injected intraperitoneally 3 days before (pre-infection) or 5 days after (post-infection) the infection. Mortality rates in the infected mice treated both with MEL and LZ were higher than in those treated with MEL alone in which the lowest brain and serum viral titers were detected. On the third post-infection day, viral titers of the MEL + VEE + LZ (pre-infection) group were higher than those of the remainder groups. On the fifth day, viral titers in infected mice were similar to those of the MEL + VEE + LZ (pre-infection) group, but higher than those detected in the MEL + VEE + LZ (post-infection). In conclusion, the protective effect of MEL in mice infected with VEE virus was inhibited by LZ suggesting that this protection is mediated by MEL receptors.
Asunto(s)
Virus de la Encefalitis Equina Venezolana/aislamiento & purificación , Encefalomielitis Equina Venezolana/tratamiento farmacológico , Melatonina/uso terapéutico , Triptaminas/farmacología , Animales , Encefalomielitis Equina Venezolana/virología , Ensayo de Inmunoadsorción Enzimática , Masculino , Melatonina/antagonistas & inhibidores , RatonesRESUMEN
Venezuelan equine encephalitis virus (VEEV) may cause encephalitis in humans, for which no FDA-approved antiviral treatment is available. Carbocyclic cytosine (carbodine) has broad-spectrum activity but toxicity has limited its utility. It was anticipated that one of the enantiomers of carbodine would show enhanced activity and reduced toxicity. The activity of the d-(-) enantiomer of carbodine [(-)-carbodine] was evaluated by infectious cell culture assay and was found to have a 50% effective concentration (EC50) of 0.2 microg/ml against the TC-83 vaccine strain of VEEV in Vero cells, while the l-(+) enantiomer had no activity. Virus titer inhibition correlated with intracellular cytidine triphosphate reduction after treatment with (-)-carbodine, as determined by HPLC analysis. Pre-treatment with 200 mg/(kgd) resulted in significant improvement in survival, virus load in the brain, weight change, and mean day-to-death in a mouse model of TC-83 VEEV disease. A single dose of (-)-carbodine resulted in a slight extension of mean time to death in mice infected with wild-type VEEV. Post-virus exposure treatment with (-)-carbodine was effective in significantly improving disease parameters in mice infected with TC-83 VEEV when treatment was initiated as late as 4 days post-virus installation (dpi). It is remarkable that (-)-carbodine is effective when initiated after the establishment of brain infection.
Asunto(s)
Antivirales/farmacología , Citidina/análogos & derivados , Virus de la Encefalitis Equina Venezolana/efectos de los fármacos , Encefalomielitis Equina Venezolana/tratamiento farmacológico , Animales , Chlorocebus aethiops , Citidina/farmacología , Citidina Trifosfato/metabolismo , Encefalomielitis Equina Venezolana/virología , Femenino , Humanos , Ratones , Ratones Endogámicos C3H , Ratones Endogámicos ICR , Células VeroRESUMEN
The TC-83 vaccine strain of Venezuelan equine encephalitis virus (VEEV) causes encephalitis and death in C3H/HeN mice infected by intranasal (i.n.) instillation. Since TC-83 is exempt as a select agent, this mouse model was used in the evaluation of antiviral therapies. Virus titers in the brains of infected mice peaked on 4 dpi and persisted at high levels until death at 9.4+/-0.5 dpi. Mouse brains appeared histologically normal on 2 dpi, but developed meningoencephalitis, neuropil vacuolation, and gliosis by 8 dpi. Results from a protein cytokine array showed significant elevations over time in interleukin (IL)-1alpha, IL-1beta, IL-6, IL-12, MCP-1, IFNgamma, TNFalpha, MIP-1alpha, and RANTES in homogenized brain samples of infected mice. Immunohistochemical staining showed a colocalization of viral antigen with neuron markers. Treatment with interferon-alpha B/D or ampligen significantly improved survival, brain virus titer and cytokine levels, mean day-to-death, and weight change in infected mice. The time-course of infection and disease parameters of mice infected with TC-83 VEEV were similar in many ways to disease parameters in mice infected with other VEEV strains. Thus, infection of C3H/HeN mice with TC-83 VEEV may serve as a suitable model for the evaluation of antiviral compounds for the treatment of this viral disease.