RESUMEN
Human astrovirus is a positive-sense, single-stranded RNA virus. Astrovirus infection causes gastrointestinal symptoms and can lead to encephalitis in immunocompromised patients. Positive-strand RNA viruses typically utilize host intracellular membranes to form replication organelles, which are potential antiviral targets. Many of these replication organelles are double-membrane vesicles (DMVs). Here, we show that astrovirus infection leads to an increase in DMV formation through a replication-dependent mechanism that requires some early components of the autophagy machinery. Results indicate that the upstream class III phosphatidylinositol 3-kinase (PI3K) complex, but not LC3 conjugation machinery, is utilized in DMV formation. Both chemical and genetic inhibition of the PI3K complex lead to significant reduction in DMVs, as well as viral replication. Elucidating the role of autophagy machinery in DMV formation during astrovirus infection reveals a potential target for therapeutic intervention for immunocompromised patients. IMPORTANCE These studies provide critical new evidence that astrovirus replication requires formation of double-membrane vesicles, which utilize class III phosphatidylinositol 3-kinase (PI3K), but not LC3 conjugation autophagy machinery, for biogenesis. These results are consistent with replication mechanisms for other positive-sense RNA viruses suggesting that targeting PI3K could be a promising therapeutic option for not only astrovirus, but other positive-sense RNA virus infections.
Asunto(s)
Mamastrovirus , Fosfatidilinositol 3-Quinasa , Replicación Viral , Humanos , Autofagia , Fosfatidilinositol 3-Quinasas Clase III/metabolismo , Membranas Intracelulares/metabolismo , Orgánulos , Fosfatidilinositol 3-Quinasa/metabolismo , Virus ARN , Mamastrovirus/fisiología , Transducción de SeñalRESUMEN
Human astroviruses (HAstV), positive sense single-stranded RNA viruses, are one of the leading causes of diarrhea worldwide. Despite their high prevalence, the cellular mechanisms of astrovirus pathogenesis remain ill-defined. Previous studies showed HAstV increased epithelial barrier permeability by causing a re-localization of the tight junction protein, occludin. In these studies, we demonstrate that HAstV replication induces epithelial-mesenchymal transition (EMT), by upregulating the transcription of EMT-related genes within 8 hours post-infection (hpi), followed by the loss of cell-cell contacts and disruption of polarity by 24 hpi. While multiple classical HAstV serotypes, including clinical isolates, induce EMT, the non-classical genotype HAstV-VA1 and two strains of reovirus are incapable of inducing EMT. Unlike the re-localization of tight junction proteins, HAstV-induced EMT requires productive replication and is dependent transforming growth factor-ß (TGF-ß) activity. Finally, inhibiting TGF-ß signaling and EMT reduces viral replication, highlighting its importance in the viral life cycle. This finding puts classical strains of HAstV-1 in an exclusive group of non-oncogenic viruses triggering EMT.
Asunto(s)
Infecciones por Astroviridae , Mamastrovirus , Transición Epitelial-Mesenquimal , Humanos , Mamastrovirus/genética , Factor de Crecimiento Transformador beta , Replicación ViralRESUMEN
Astroviruses (AstV) are a leading cause of diarrhea, especially in the very young, the elderly, and immunocompromised populations. Despite their significant impact on public health, no drug therapies for astrovirus have been identified. In this study, we fill this gap in knowledge and demonstrate that the FDA-approved broad-spectrum anti-infective drug nitazoxanide (NTZ) blocks astrovirus replication in vitro with a 50% effective concentration (EC50) of approximately 1.47 µM. It can be administered up to 8 h postinfection and is effective against multiple human astrovirus serotypes, including clinical isolates. Most importantly, NTZ reduces viral shedding in vivo, exhibiting its potential as a future clinical therapeutic.IMPORTANCE Human astroviruses (HAstV) are thought to cause between 2 and 9% of acute, nonbacterial diarrhea cases in children worldwide. HAstV infection can be especially problematic in immunocompromised people and infants, where the virus has been associated with necrotizing enterocolitis and severe and persistent diarrhea, as well as rare instances of systemic and fatal disease. And yet, no antivirals have been identified to treat astrovirus infection. Our study provides the first evidence that nitazoxanide may be an effective therapeutic strategy against astrovirus disease.
Asunto(s)
Infecciones por Astroviridae/tratamiento farmacológico , Mamastrovirus/efectos de los fármacos , Tiazoles/antagonistas & inhibidores , Replicación Viral/efectos de los fármacos , Animales , Infecciones por Astroviridae/virología , Células CACO-2 , Supervivencia Celular/efectos de los fármacos , Diarrea/virología , Enterocolitis Necrotizante/tratamiento farmacológico , Enterocolitis Necrotizante/virología , Humanos , Mamastrovirus/inmunología , Nitrocompuestos , Aves de Corral , Replicación Viral/fisiologíaRESUMEN
Obesity, and the associated metabolic syndrome, is a risk factor for increased disease severity with a variety of infectious agents, including influenza virus. Yet, the mechanisms are only partially understood. As the number of people, particularly children, living with obesity continues to rise, it is critical to understand the role of host status on disease pathogenesis. In these studies, we use a diet-induced obese ferret model and tools to demonstrate that, like humans, obesity resulted in notable changes to the lung microenvironment, leading to increased clinical disease and viral spread to the lower respiratory tract. The decreased antiviral responses also resulted in obese animals shedding higher infectious virus for a longer period, making them more likely to transmit to contacts. These data suggest that the obese ferret model may be crucial to understanding obesity's impact on influenza disease severity and community transmission and a key tool for therapeutic and intervention development for this high-risk population.
Asunto(s)
Modelos Animales de Enfermedad , Hurones , Obesidad , Infecciones por Orthomyxoviridae , Animales , Obesidad/virología , Infecciones por Orthomyxoviridae/transmisión , Infecciones por Orthomyxoviridae/virología , Pulmón/virología , Pulmón/patología , Índice de Severidad de la Enfermedad , Dieta , Humanos , Esparcimiento de Virus , Gripe Humana/transmisión , Gripe Humana/virologíaRESUMEN
Metabolic disease is epidemiologically linked to severe complications upon influenza virus infection, thus vaccination is a priority in this high-risk population. Yet, vaccine responses are less effective in these same hosts. Here we examined how the timing of diet switching from a high-fat diet to a control diet affected influenza vaccine efficacy in diet-induced obese mice. Our results demonstrate that the systemic meta-inflammation generated by high-fat diet exposure limited T cell maturation to the memory compartment at the time of vaccination, impacting the recall of effector memory T cells upon viral challenge. This was not improved with a diet switch post-vaccination. However, the metabolic dysfunction of T cells was reversed if weight loss occurred 4 weeks before vaccination, restoring a functional recall response. This corresponded with changes in the systemic obesity-related biomarkers leptin and adiponectin, highlighting the systemic and specific effects of diet on influenza vaccine immunogenicity.
Asunto(s)
Dieta Alta en Grasa , Vacunas contra la Influenza , Obesidad , Infecciones por Orthomyxoviridae , Animales , Ratones , Vacunas contra la Influenza/inmunología , Vacunas contra la Influenza/administración & dosificación , Dieta Alta en Grasa/efectos adversos , Obesidad/inmunología , Obesidad/metabolismo , Infecciones por Orthomyxoviridae/inmunología , Infecciones por Orthomyxoviridae/prevención & control , Ratones Endogámicos C57BL , Vacunación , Ratones Obesos , Leptina/metabolismo , Masculino , Femenino , Adiponectina/metabolismo , Linfocitos T/inmunologíaRESUMEN
Human astrovirus is a positive sense, single stranded RNA virus. Astrovirus infection causes gastrointestinal symptoms and can lead to encephalitis in immunocompromised patients. Positive strand RNA viruses typically utilize host intracellular membranes to form replication organelles, which are potential antiviral targets. Many of these replication organelles are double membrane vesicles (DMVs). Here we show that astrovirus infection leads to an increase in DMV formation, and this process is replication-dependent. Our data suggest that astrovirus infection induces rearrangement of endoplasmic reticulum fragments, which may become the origin for DMV formation. Transcriptional data suggested that formation of DMVs during astrovirus infection requires some early components of the autophagy machinery. Results indicate that the upstream class III phosphatidylinositol 3-kinase (PI3K) complex, but not LC3 conjugation machinery, is utilized in DMV formation. Inhibition of the PI3K complex leads to significant reduction in viral replication and release from cells. Elucidating the role of autophagy machinery in DMV formation during astrovirus infection reveals a potential target for therapeutic intervention for immunocompromised patients. Importance: These studies provide critical new evidence that astrovirus replication requires formation of double membrane vesicles, which utilize class III PI3K, but not LC3 conjugation autophagy machinery for biogenesis. These results are consistent with replication mechanisms for other positive sense RNA viruses. This suggests that targeting PI3K could be a promising therapeutic option for not only astrovirus, but other positive sense RNA virus infections.
RESUMEN
Obesity, and the associated metabolic syndrome, is a risk factor for increased disease severity with a variety of infectious agents, including influenza virus. Yet the mechanisms are only partially understood. As the number of people, particularly children, living with obesity continues to rise, it is critical to understand the role of host status on disease pathogenesis. In these studies, we use a novel diet-induced obese ferret model and new tools to demonstrate that like humans, obesity resulted in significant changes to the lung microenvironment leading to increased clinical disease and viral spread to the lower respiratory tract. The decreased antiviral responses also resulted in obese animals shedding higher infectious virus for longer making them more likely to transmit to contacts. These data suggest the obese ferret model may be crucial to understanding obesity's impact on influenza disease severity and community transmission, and a key tool for therapeutic and intervention development for this high-risk population. Teaser: A new ferret model and tools to explore obesity's impact on respiratory virus infection, susceptibility, and community transmission.
RESUMEN
Astroviruses cause a spectrum of diseases spanning asymptomatic infections to severe diarrhea, but little is understood about their pathogenesis. We previously determined that small intestinal goblet cells were the main cell type infected by murine astrovirus-1. Here, we focused on the host immune response to infection and inadvertently discovered a role for indoleamine 2,3-dioxygenase 1 (Ido1), a host tryptophan catabolizing enzyme, in the cellular tropism of murine and human astroviruses. We identified that Ido1 expression was highly enriched among infected goblet cells, and spatially corresponded to the zonation of infection. Because Ido1 can act as a negative regulator of inflammation, we hypothesized it could dampen host antiviral responses. Despite robust interferon signaling in goblet cells, as well as tuft cell and enterocyte bystanders, we observed delayed cytokine induction and suppressed levels of fecal lipocalin-2. Although we found Ido-/- animals were more resistant to infection, this was not associated with fewer goblet cells nor could it be rescued by knocking out interferon responses, suggesting that IDO1 instead regulates cell permissivity. We characterized IDO1-/- Caco-2 cells and observed significantly reduced human astrovirus-1 infection. Together this study highlights a role for Ido1 in astrovirus infection and epithelial cell maturation.
Asunto(s)
Infecciones por Astroviridae , Indolamina-Pirrol 2,3,-Dioxigenasa , Animales , Humanos , Ratones , Células CACO-2 , Indolamina-Pirrol 2,3,-Dioxigenasa/genética , Indolamina-Pirrol 2,3,-Dioxigenasa/metabolismo , Interferones , Triptófano/metabolismoRESUMEN
Novel human astroviruses (HAstVs) have recently been implicated as rare causes of fatal encephalitis in immunocompromised patients, for which there is no proven treatment. We report 2 cases from our institution in which HAstV-VA1 was detected in the cerebrospinal fluid by metagenomic next-generation sequencing after the initial evaluation revealed no etiology.
Asunto(s)
Infecciones por Astroviridae , Encefalitis , Mamastrovirus , Neoplasias , Infecciones por Astroviridae/diagnóstico , Niño , Heces , Humanos , Huésped Inmunocomprometido , Mamastrovirus/genética , FilogeniaRESUMEN
Since the 1970s, eight closely related serotypes of classical human astroviruses (HAstV) have been associated with gastrointestinal illness worldwide. In the late 2000s, three genetically unique human astrovirus clades, VA1-VA3, VA2-VA4, and MLB, were described. While the exact disease associated with these clades remains to be defined, VA1 has been associated with central nervous system infections. The discovery that VA1 could be grown in cell culture, supports exciting new studies aimed at understanding viral pathogenesis. Given the association of VA1 with often lethal CNS infections, we tested its susceptibility to the antimicrobial drug, nitazoxanide (NTZ), which we showed could inhibit classical HAstV infections. Our studies demonstrate that NTZ inhibited VA1 replication in Caco2 cells even when added at 12 h post-infection, which is later than in HAstV-1 infection. These data led us to further probe VA1 replication kinetics and cellular responses to infection in Caco-2 cells in comparison to the well-studied HAstV-1 strain. Overall, our studies highlight that VA1 replicates more slowly than HAstV-1 and elicits significantly different cellular responses, including the inability to disrupt cellular junctions and barrier permeability.
Asunto(s)
Astroviridae/genética , Antiinfecciosos/farmacología , Astroviridae/efectos de los fármacos , Infecciones por Astroviridae/tratamiento farmacológico , Infecciones por Astroviridae/virología , Células CACO-2 , Línea Celular Tumoral , Sistema Nervioso Central/virología , Humanos , Nitrocompuestos/farmacología , Filogenia , ARN Viral/genética , Tiazoles/farmacología , Replicación Viral/efectos de los fármacos , Replicación Viral/genéticaRESUMEN
Astroviruses are a major cause of diarrhea in the young, elderly, and the immunocompromised. Since the discovery of human astrovirus type 1 (HAstV-1) in 1975, the family Astroviridae has expanded to include two more human clades and numerous mammalian and avian-specific genotypes. Despite this, there is still little known about pathogenesis. The following review highlights the current knowledge of astrovirus pathogenesis, and outlines the critical steps needed to further astrovirus research, including the development of animal models of cell culture systems.
Asunto(s)
Infecciones por Astroviridae/virología , Astroviridae/fisiología , Interacciones Huésped-Patógeno , Animales , HumanosRESUMEN
Astroviruses are nonenveloped, positive-sense single-stranded RNA viruses that cause gastrointestinal illness. Although a leading cause of pediatric diarrhea, human astroviruses are among the least characterized enteric RNA viruses. However, by using in vitro methods and animal models to characterize virus-host interactions, researchers have discovered several important properties of astroviruses, including the ability of the astrovirus capsid to act as an enterotoxin, disrupting the gut epithelial barrier. Improved animal models are needed to study this phenomenon, along with the pathogenesis of astroviruses, particularly in those strains that can cause extraintestinal disease. Much like for other enteric viruses, the current dogma states that astroviruses infect in a species-specific manner; however, this assumption is being challenged by growing evidence that these viruses have potential to cross species barriers. This review summarizes these remarkable facets of astrovirus biology, highlighting critical steps toward increasing our understanding of this unique enteric pathogen.