RESUMO
Venezuelan equine encephalitis virus (VEEV) is an encephalitic alphavirus responsible for epidemics of neurological disease across the Americas. Low-density lipoprotein receptor class A domain-containing 3 (LDLRAD3) is a recently reported entry receptor for VEEV. Here, using wild-type and Ldlrad3-deficient mice, we define a critical role for LDLRAD3 in controlling steps in VEEV infection, pathogenesis, and neurotropism. Our analysis shows that LDLRAD3 is required for efficient VEEV infection and pathogenesis prior to and after central nervous system invasion. Ldlrad3-deficient mice survive intranasal and intracranial VEEV inoculation and show reduced infection of neurons in different brain regions. As LDLRAD3 is a determinant of pathogenesis and an entry receptor required for VEEV infection of neurons of the brain, receptor-targeted therapies may hold promise as countermeasures.
Assuntos
Encefalomielite Equina Venezuelana , Receptores de LDL , Animais , Camundongos , Encéfalo/patologia , Sistema Nervoso Central , Vírus da Encefalite Equina Venezuelana/fisiologia , Encefalomielite Equina Venezuelana/patologia , Receptores de LDL/fisiologiaRESUMO
Venezuelan equine encephalitis virus (VEEV) is a disease typically confined to South and Central America, whereby human disease is characterised by a transient systemic infection and occasionally severe encephalitis, which is associated with lethality. Using an established mouse model of VEEV infection, the encephalitic aspects of the disease were analysed to identify biomarkers associated with inflammation. Sequential sampling of lethally challenged mice (infected subcutaneously) confirmed a rapid onset systemic infection with subsequent spread to the brain within 24 h of the challenge. Changes in inflammatory biomarkers (TNF-α, CCL-2, and CCL-5) and CD45+ cell counts were found to correlate strongly to pathology (R>0.9) and present previously unproven biomarkers for disease severity in the model, more so than viral titre. The greatest level of pathology was observed within the olfactory bulb and midbrain/thalamus. The virus was distributed throughout the brain/encephalon, often in areas not associated with pathology. The principal component analysis identified five principal factors across two independent experiments, with the first two describing almost half of the data: (1) confirmation of a systemic Th1-biased inflammatory response to VEEV infection, and (2) a clear correlation between specific inflammation of the brain and clinical signs of disease. Targeting strongly associated biomarkers of deleterious inflammation may ameliorate or even eliminate the encephalitic syndrome of this disease.
Assuntos
Vírus da Encefalite Equina Venezuelana , Encefalomielite Equina Venezuelana , Humanos , Cavalos , Camundongos , Animais , Fator de Necrose Tumoral alfa , Vírus da Encefalite Equina Venezuelana/fisiologia , Encéfalo , Inflamação/patologia , Quimiocinas , LeucócitosRESUMO
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.
Assuntos
Jacarés e Crocodilos , Vírus da Encefalite Equina Venezuelana , Encefalomielite Equina Venezuelana , Animais , Cavalos , Vírus da Encefalite Equina Venezuelana/fisiologia , Antivirais/farmacologia , Antivirais/uso terapêutico , Encefalomielite Equina Venezuelana/tratamento farmacológico , Encefalomielite Equina Venezuelana/prevenção & controle , Bioprospecção , Replicação Viral , PeptídeosRESUMO
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.
Assuntos
Vírus da Encefalite Equina Venezuelana , Encefalomielite Equina Venezuelana , Humanos , Animais , Cavalos , Vírus da Encefalite Equina Venezuelana/fisiologia , Barreira Hematoencefálica , Encefalomielite Equina Venezuelana/tratamento farmacológico , Encefalomielite Equina Venezuelana/prevenção & controle , Células Endoteliais , Sistemas MicrofisiológicosRESUMO
Acutely infectious new world alphaviruses such as Venezuelan Equine Encephalitis Virus (VEEV) pose important challenges to the human population due to a lack of effective therapeutic intervention strategies. Small interfering RNAs that can selectively target the viral genome (vsiRNAs) has been observed to offer survival advantages in several in vitro and in vivo models of acute virus infections, including alphaviruses such as Chikungunya virus and filoviruses such as Ebola virus. In this study, novel vsiRNAs that targeted conserved regions in the nonstructural and structural genes of the VEEV genome were designed and evaluated for antiviral activity in mammalian cells in the context of VEEV infection. The data demonstrate that vsiRNAs were able to effectively decrease the infectious virus titer at earlier time points post infection in the context of the attenuated TC-83 strain and the virulent Trinidad Donkey strain, while the inhibition was overcome at later time points. Depletion of Argonaute 2 protein (Ago2), the catalytic component of the RISC complex, negated the inhibitory effect of the vsiRNAs, underscoring the involvement of the siRNA pathway in the inhibition process. Depletion of the RNAi pathway proteins Dicer, MOV10, TRBP2 and Matrin 3 decreased viral load in infected cells, alluding to an impact of the RNAi pathway in the establishment of a productive infection. Additional studies focused on rational combinations of effective vsiRNAs and delivery strategies to confer better in vivo bioavailability and distribution to key target tissues such as the brain can provide effective solutions to treat encephalitic diseases resulting from alphavirus infections.
Assuntos
Vírus da Encefalite Equina Venezuelana , RNA Interferente Pequeno , Animais , Linhagem Celular , Vírus da Encefalite Equina Venezuelana/fisiologia , Cavalos , Humanos , RNA Helicases , RNA Interferente Pequeno/farmacologia , Replicação ViralRESUMO
Venezuelan equine encephalitis virus (VEEV) is an Alphavirus in the Togaviridae family of positive-strand RNA viruses. The viral genome of positive-strand RNA viruses is infectious, as it produces infectious virus upon introduction into a cell. VEEV is a select agent and samples containing viral RNA are subject to additional regulations due to their infectious nature. Therefore, RNA isolated from cells infected with BSL-3 select agent strains of VEEV or other positive-strand viruses must be inactivated before removal from high-containment laboratories. In this study, we tested the inactivation of the viral genome after RNA fragmentation or cDNA synthesis, using the Trinidad Donkey and TC-83 strains of VEEV. We successfully inactivated VEEV genomic RNA utilizing these two protocols. Our cDNA synthesis method also inactivated the genomic RNA of eastern and western equine encephalitis viruses (EEEV and WEEV). We also tested whether the purified VEEV genomic RNA can produce infectious virions in the absence of transfection. Our result showed the inability of the viral genome to cause infection without being transfected into the cells. Overall, this work introduces RNA fragmentation and cDNA synthesis as reliable methods for the inactivation of samples containing the genomes of positive-strand RNA viruses.
Assuntos
Vírus da Encefalite Equina Venezuelana/genética , Genoma Viral , RNA Viral , Inativação de Vírus , Animais , Células Cultivadas , Chlorocebus aethiops , Efeito Citopatogênico Viral , DNA Complementar/biossíntese , Vírus da Encefalite Equina do Leste/genética , Vírus da Encefalite Equina do Leste/fisiologia , Vírus da Encefalite Equina Venezuelana/fisiologia , Vírus da Encefalite Equina do Oeste/genética , Vírus da Encefalite Equina do Oeste/fisiologia , RNA Viral/química , RNA Viral/fisiologia , Ribonucleases/metabolismo , Células VeroRESUMO
Venezuelan equine encephalitis virus (VEEV) is a neurotropic virus that causes significant disease in both humans and equines. Here we characterized the impact of VEEV on signaling pathways regulating cell death in human primary astrocytes. VEEV productively infected primary astrocytes and caused an upregulation of early growth response 1 (EGR1) gene expression at 9 and 18â¯h post infection. EGR1 induction was dependent on extracellular signal-regulated kinase1/2 (ERK1/2) and protein kinase R (PKR)-like endoplasmic reticulum kinase (PERK), but not on p38 mitogen activated protein kinase (MAPK) or phosphoinositide 3-kinase (PI3K) signaling. Knockdown of EGR1 significantly reduced VEEV-induced apoptosis and impacted viral replication. Knockdown of ERK1/2 or PERK significantly reduced EGR1 gene expression, dramatically reduced viral replication, and increased cell survival as well as rescued cells from VEEV-induced apoptosis. These data indicate that EGR1 activation and subsequent cell death are regulated through ERK and PERK pathways in VEEV infected primary astrocytes.
Assuntos
Morte Celular , Proteína 1 de Resposta de Crescimento Precoce/genética , Vírus da Encefalite Equina Venezuelana/fisiologia , Encefalomielite Equina Venezuelana/virologia , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , eIF-2 Quinase/metabolismo , Apoptose , Astrócitos/metabolismo , Astrócitos/patologia , Astrócitos/virologia , Sobrevivência Celular , Células Cultivadas , Proteína 1 de Resposta de Crescimento Precoce/metabolismo , Vírus da Encefalite Equina Venezuelana/patogenicidade , Encefalomielite Equina Venezuelana/metabolismo , Encefalomielite Equina Venezuelana/patologia , Expressão Gênica , Técnicas de Silenciamento de Genes , Humanos , Proteína Quinase 1 Ativada por Mitógeno/genética , Proteína Quinase 3 Ativada por Mitógeno/genética , Transdução de Sinais , Replicação Viral , eIF-2 Quinase/genéticaRESUMO
The challenges associated with operating electron microscopes (EM) in biosafety level 3 and 4 containment facilities have slowed progress of cryo-EM studies of high consequence viruses. We address this gap in a case study of Venezuelan Equine Encephalitis Virus (VEEV) strain TC-83. Chemical inactivation of viruses may physically distort structure, and hence to verify retention of native structure, we selected VEEV strain TC-83 to develop this methodology as this virus has a 4.8â¯Å resolution cryo-EM structure. In our method, amplified VEEV TC-83 was concentrated directly from supernatant through a 30 % sucrose cushion, resuspended, and chemically inactivated with 1 % glutaraldehyde. A second 30 % sucrose cushion removed any excess glutaraldehyde that might interfere with single particle analyses. A cryo-EM map of fixed, inactivated VEEV was determined to a resolution of 7.9â¯Å. The map retained structural features of the native virus such as the icosahedral symmetry, and the organization of the capsid core and the trimeric spikes. Our results suggest that our strategy can easily be adapted for inactivation of other enveloped, RNA viruses requiring BSL-3 or BSL-4 for cryo-EM. However, the validation of inactivation requires the oversight of Biosafety Committee for each Institution.
Assuntos
Microscopia Crioeletrônica/métodos , Vírus da Encefalite Equina Venezuelana/fisiologia , Vírus de RNA/fisiologia , Inativação de Vírus , Animais , Capsídeo/química , Proteínas do Capsídeo , Linhagem Celular , Chlorocebus aethiops , Contenção de Riscos Biológicos/métodos , Vírus da Encefalite Equina Venezuelana/genética , Glutaral/química , Glutaral/metabolismo , Cavalos , Células Vero , Virologia/métodos , Replicação ViralRESUMO
Traditional pathogenesis studies of alphaviruses involves monitoring survival, viremia, and pathogen dissemination via serial necropsies; however, molecular imaging shifts this paradigm and provides a dynamic assessment of pathogen infection. Positron emission tomography (PET) with PET tracers targeted to study neuroinflammation (N,N-diethyl-2-[4-phenyl]-5,7-dimethylpyrazolo[1,5-a]pyrimidine-3-acetamide, [18F]DPA-714), apoptosis (caspase-3 substrate, [18F]CP-18), hypoxia (fluormisonidazole, [18F]FMISO), blood-brain barrier (BBB) integrity ([18F]albumin), and metabolism (fluorodeoxyglucose, [18F]FDG) was performed on C3H/HeN mice infected intranasally with 7000 plaque-forming units (PFU) of Venezuelan equine encephalitis virus (VEEV) TC-83. The main findings are as follows: (1) whole-brain [18F]DPA-714 and [18F]CP-18 uptake increased three-fold demonstrating, neuroinflammation and apoptosis, respectively; (2) [18F]albumin uptake increased by 25% across the brain demonstrating an altered BBB; (3) [18F]FMISO uptake increased by 50% across the whole brain indicating hypoxic regions; (4) whole-brain [18F]FDG uptake was unaffected; (5) [18F]DPA-714 uptake in (a) cortex, thalamus, striatum, hypothalamus, and hippocampus increased through day seven and decreased by day 10 post exposure, (b) olfactory bulb increased at day three, peaked day seven, and decreased day 10, and (c) brain stem and cerebellum increased through day 10. In conclusion, intranasal exposure of C3H/HeN mice to VEEV TC-83 results in both time-dependent and regional increases in brain inflammation, apoptosis, and hypoxia, as well as modest decreases in BBB integrity; however, it has no effect on brain glucose metabolism.
Assuntos
Apoptose , Barreira Hematoencefálica/metabolismo , Vírus da Encefalite Equina Venezuelana , Encefalomielite Equina Venezuelana/diagnóstico , Encefalomielite Equina Venezuelana/metabolismo , Hipóxia/metabolismo , Tomografia por Emissão de Pósitrons , Animais , Biomarcadores , Barreira Hematoencefálica/patologia , Modelos Animais de Doenças , Vírus da Encefalite Equina Venezuelana/fisiologia , Encefalomielite Equina Venezuelana/virologia , Cavalos , Processamento de Imagem Assistida por Computador , Camundongos , Tomografia por Emissão de Pósitrons combinada à Tomografia Computadorizada , Tomografia por Emissão de Pósitrons/métodos , Compostos Radiofarmacêuticos/química , Compostos Radiofarmacêuticos/metabolismoRESUMO
Venezuelan equine encephalitis virus (VEEV), a new world alphavirus belonging to the Togaviridae family, causes periodic disease outbreaks in humans and equines with high associated mortality and morbidity. VEEV is highly infectious via the aerosol route and so has been developed as a biological weapon (Hawley and Eitzen, 2001). Despite its current classification as a category B select agent, there are no FDA approved vaccines or therapeutics to counter VEEV infections. Here we utilize a naturally occurring host defense peptide, LL-37, as a therapeutic strategy to inhibit VEEV multiplication in infected cells. LL-37 has previously demonstrated activity against several viruses by directly interacting with viral particles and indirectly by establishing an antiviral state in the host cell. We show that LL-37 exhibited potent antiviral activity against VEEV by inhibiting viral replication. Genomic RNA copies of the TC-83 strain of VEEV and viral titers were significantly reduced compared to non-treated controls. LL-37 also inhibited the virulent Trinidad Donkey (TrD) strain of VEEV. Entry assays revealed a robust reduction of viral RNA copies at the early stages of TC-83 infection. Pre-incubation of cells with LL-37 and TC-83 resulted in a strong inhibitory response, indicating that LL-37 impacts early stages of the infectious process. Confocal and electron microscopy images confirmed the aggregation of viral particles, which potentially accounts for entry prevention and hence reduced viral infection. LL-37 treatment also modulated type I interferon (IFN) expression in infected cells. LL-37 treatment dramatically increased IFNß1 expression in treated cells in a time-dependent manner. Our results establish LL-37 as a relevant and novel potential therapeutic strategy for the treatment of VEEV infections.