RESUMEN
The recent emergence and rapid response to severe acute respiratory syndrome coronavirus 2 was enabled by prototype pathogen and vaccine platform approaches, driven by the preemptive application of RNA vaccine technology to the related Middle East respiratory syndrome coronavirus. Recently, the National Institutes of Allergy and Infectious Diseases identified nine virus families of concern, eight enveloped virus families and one nonenveloped virus family, for which vaccine generation is a priority. Although RNA vaccines have been described for a variety of enveloped viruses, a roadmap for their use against nonenveloped viruses is lacking. Enterovirus D68 was recently designated a prototype pathogen within the family Picornaviridae of nonenveloped viruses because of its rapid evolution and respiratory route of transmission, coupled with a lack of diverse anti-enterovirus vaccine approaches in development. Here, we describe a proof-of-concept approach using a clinical stage RNA vaccine platform that induced robust enterovirus D68-neutralizing antibody responses in mice and nonhuman primates and prevented upper and lower respiratory tract infections and neurological disease in mice. In addition, we used our platform to rapidly characterize the antigenic diversity within the six genotypes of enterovirus D68, providing the necessary data to inform multivalent vaccine compositions that can elicit optimal breadth of neutralizing responses. These results demonstrate that RNA vaccines can be used as tools in our pandemic-preparedness toolbox for nonenveloped viruses.
Asunto(s)
Anticuerpos Neutralizantes , Enterovirus Humano D , Infecciones por Enterovirus , Animales , Infecciones por Enterovirus/prevención & control , Infecciones por Enterovirus/inmunología , Infecciones por Enterovirus/virología , Enterovirus Humano D/inmunología , Enterovirus Humano D/genética , Anticuerpos Neutralizantes/inmunología , Ratones , Vacunas Virales/inmunología , Modelos Animales de Enfermedad , Humanos , Vacunas de ARNm , Anticuerpos Antivirales/inmunología , FemeninoRESUMEN
In December 2023, we observed through hospital-based surveillance a severe outbreak of enterovirus D68 infection in pediatric inpatients in Dakar, Senegal. Molecular characterization revealed that subclade B3, the dominant lineage in outbreaks worldwide, was responsible for the outbreak. Enhanced surveillance in inpatient settings, including among patients with neurologic illnesses, is needed.
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Brotes de Enfermedades , Enterovirus Humano D , Infecciones por Enterovirus , Infecciones del Sistema Respiratorio , Humanos , Senegal/epidemiología , Enterovirus Humano D/genética , Enterovirus Humano D/clasificación , Enterovirus Humano D/aislamiento & purificación , Infecciones por Enterovirus/epidemiología , Infecciones por Enterovirus/virología , Infecciones por Enterovirus/diagnóstico , Infecciones del Sistema Respiratorio/epidemiología , Infecciones del Sistema Respiratorio/virología , Preescolar , Lactante , Niño , Filogenia , Masculino , Femenino , Enfermedad Aguda/epidemiología , Adolescente , Hospitales , Historia del Siglo XXIRESUMEN
Enterovirus D68 (EV-D68) is an emerging agent for which data on the susceptible adult population is scarce. We performed a 6-year analysis of respiratory samples from influenza-like illness (ILI) admitted during 2014-2020 in 4-10 hospitals in the Valencia Region, Spain. EV-D68 was identified in 68 (3.1%) among 2210 Enterovirus (EV)/Rhinovirus (HRV) positive samples. Phylogeny of 59 VP1 sequences showed isolates from 2014 clustering in B2 (6/12), B1 (5/12), and A2/D1 (1/12) subclades; those from 2015 (n = 1) and 2016 (n = 1) in B3 and A2/D1, respectively; and isolates from 2018 in A2/D3 (42/45), and B3 (3/45). B1 and B2 viruses were mainly detected in children (80% and 67%, respectively); B3 were equally distributed between children and adults; whereas A2/D1 and A2/D3 were observed only in adults. B3 viruses showed up to 16 amino acid changes at predicted antigenic sites. In conclusion, two EV-D68 epidemics linked to ILI hospitalized cases occurred in the Valencia Region in 2014 and 2018, with three fatal outcomes and one ICU admission. A2/D3 strains from 2018 were associated with severe respiratory infection in adults. Because of the significant impact of non-polio enteroviruses in ILI and the potential neurotropism, year-round surveillance in respiratory samples should be pursued.
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Enterovirus Humano D , Infecciones por Enterovirus , Hospitalización , Gripe Humana , Filogenia , Humanos , España/epidemiología , Infecciones por Enterovirus/epidemiología , Infecciones por Enterovirus/virología , Enterovirus Humano D/genética , Enterovirus Humano D/clasificación , Enterovirus Humano D/aislamiento & purificación , Niño , Adulto , Preescolar , Masculino , Adolescente , Femenino , Persona de Mediana Edad , Lactante , Anciano , Adulto Joven , Hospitalización/estadística & datos numéricos , Gripe Humana/epidemiología , Gripe Humana/virología , Infecciones del Sistema Respiratorio/epidemiología , Infecciones del Sistema Respiratorio/virología , Estaciones del Año , Anciano de 80 o más Años , Costo de Enfermedad , Recién NacidoRESUMEN
Objective: To summarize the clinical manifestations, diagnosis, treatment and prognosis of acute flaccid myelitis (AFM) in children. Methods: Clinical characteristics of 4 AFM cases from Department of Neurology, Children's Hospital Affiliated to Capital Institute of Pediatrics, from September 2018 to November 2022, were analyzed retrospectively. Results: The age of 4 children with AFM was 7 years, 4 years and 3 months, 7 years and 1 month, 6 years and 5 months, respectively. There were 2 boys and 2 girls. Prodromal infection status showed 3 children of respiratory tract infection and 1 child of digestive tract infection. The main manifestation was asymmetrical limb weakness after infection, and the affected limb range was from monoplegia to quadriplegia. Cranial nerve injury was involved in 1 child, no encephalopathy. Magnetic resonance imaging in the spinal cord of all 4 children showed long T1 and T2 signals, mainly involving gray matter. Cerebrospinal fluid cell-protein separation was observed in 2 children. Pathogen detected in 1 child pharyngeal swab was enterovirus D68. Antibody IgM to adenovirus was positive in the blood of 1 child. Antibody IgG against Echo and Coxsackie B virus were positive in the blood of another child. After glucocorticoid, human immunoglobulin or simple symptomatic treatment and at the same time under later rehabilitation training, muscle strength recovered to different degrees, but there were disabilities left in 3 children. Conclusions: AFM should be considered in children with acute and asymmetrical flaccid paralysis accompanied by abnormal magnetic resonance imaging signal in the central region of spinal cord, especially post-infection. The effective treatment is limited and the prognosis is poor.
Asunto(s)
Enfermedades Virales del Sistema Nervioso Central , Imagen por Resonancia Magnética , Mielitis , Enfermedades Neuromusculares , Humanos , Mielitis/diagnóstico , Mielitis/virología , Masculino , Femenino , Niño , Preescolar , Estudios Retrospectivos , Enfermedades Virales del Sistema Nervioso Central/diagnóstico , Enfermedades Neuromusculares/diagnóstico , Enterovirus Humano D/aislamiento & purificación , Pronóstico , Médula Espinal/patología , Infecciones por Enterovirus/diagnóstico , Cuadriplejía/etiología , Cuadriplejía/diagnóstico , Infecciones del Sistema Respiratorio/diagnósticoRESUMEN
Enterovirus D68 (EV-D68) is an emerging pathogen that can cause severe respiratory and neurologic disease [acute flaccid myelitis (AFM)]. Intramuscular (IM) injection of neonatal Swiss Webster (SW) mice with US/IL/14-18952 (IL52), a clinical isolate from the 2014 EV-D68 epidemic, results in many of the pathogenic features of human AFM, including viral infection of the spinal cord, death of motor neurons, and resultant progressive paralysis. In distinction, CA/14-4231 (CA4231), another clinical isolate from the 2014 EV-D68 outbreak, does not cause paralysis in mice, does not grow in the spinal cord, and does not cause motor neuron loss following IM injection. A panel of chimeric viruses containing sequences from IL52 and CA4231 was used to demonstrate that VP1 is the main determinant of EV-D68 neurovirulence following IM injection of neonatal SW mice. VP1 contains four amino acid differences between IL52 and CA4231. Mutations resulting in substituting these four amino acids (CA4231 residues into the IL52 polyprotein) completely abolished neurovirulence. Conversely, mutations resulting in substituting VP1 IL52 amino acid residues into the CA4231 polyprotein created a virus that induced paralysis to the same degree as IL52. Neurovirulence following infection of neonatal SW mice with parental and chimeric viruses was associated with viral growth in the spinal cord. IMPORTANCE: Emerging viruses allow us to investigate mutations leading to increased disease severity. Enterovirus D68 (EV-D68), once the cause of rare cases of respiratory illness, recently acquired the ability to cause severe respiratory and neurologic disease. Chimeric viruses were used to demonstrate that viral structural protein VP1 determines growth in the spinal cord, motor neuron loss, and paralysis following intramuscular (IM) injection of neonatal Swiss Webster (SW) mice with EV-D68. These results have relevance for predicting the clinical outcome of future EV-D68 epidemics as well as targeting retrograde transport as a potential strategy for treating virus-induced neurologic disease.
Asunto(s)
Proteínas de la Cápside , Enfermedades Virales del Sistema Nervioso Central , Modelos Animales de Enfermedad , Enterovirus Humano D , Infecciones por Enterovirus , Mielitis , Enfermedades Neuromusculares , Animales , Enterovirus Humano D/patogenicidad , Enterovirus Humano D/genética , Enterovirus Humano D/fisiología , Mielitis/virología , Ratones , Infecciones por Enterovirus/virología , Infecciones por Enterovirus/patología , Enfermedades Neuromusculares/virología , Enfermedades Neuromusculares/patología , Proteínas de la Cápside/genética , Proteínas de la Cápside/metabolismo , Enfermedades Virales del Sistema Nervioso Central/virología , Enfermedades Virales del Sistema Nervioso Central/patología , Humanos , Médula Espinal/virología , Médula Espinal/patología , Neuronas Motoras/virología , Neuronas Motoras/patología , Animales Recién Nacidos , Virulencia , Parálisis/virologíaRESUMEN
Enterovirus D68 (EV-D68) is a picornavirus associated with severe respiratory illness and a paralytic disease called acute flaccid myelitis in infants. Currently, no protective vaccines or antivirals are available to combat this virus. Like other enteroviruses, EV-D68 uses components of the cellular autophagy pathway to rewire membranes for its replication. Here, we show that transcription factor EB (TFEB), the master transcriptional regulator of autophagy and lysosomal biogenesis, is crucial for EV-D68 infection. Knockdown of TFEB attenuated EV-D68 genomic RNA replication but did not impact viral binding or entry into host cells. The 3C protease of EV-D68 cleaves TFEB at the N-terminus at glutamine 60 (Q60) immediately post-peak viral RNA replication, disrupting TFEB-RagC interaction and restricting TFEB transport to the surface of the lysosome. Despite this, TFEB remained mostly cytosolic during EV-D68 infection. Overexpression of a TFEB mutant construct lacking the RagC-binding domain, but not the wild-type construct, blocks autophagy and increases EV-D68 nonlytic release in H1HeLa cells but not in autophagy-defective ATG7 KO H1HeLa cells. Our results identify TFEB as a vital host factor regulating multiple stages of the EV-D68 lifecycle and suggest that TFEB could be a promising target for antiviral development against EV-D68. IMPORTANCE: Enteroviruses are among the most significant causes of human disease. Some enteroviruses are responsible for severe paralytic diseases such as poliomyelitis or acute flaccid myelitis. The latter disease is associated with multiple non-polio enterovirus species, including enterovirus D68 (EV-D68), enterovirus 71, and coxsackievirus B3 (CVB3). Here, we demonstrate that EV-D68 interacts with a host transcription factor, transcription factor EB (TFEB), to promote viral RNA(vRNA) replication and regulate the egress of virions from cells. TFEB was previously implicated in the viral egress of CVB3, and the viral protease 3C cleaves TFEB during infection. Here, we show that EV-D68 3C protease also cleaves TFEB after the peak of vRNA replication. This cleavage disrupts TFEB interaction with the host protein RagC, which changes the localization and regulation of TFEB. TFEB lacking a RagC-binding domain inhibits autophagic flux and promotes virus egress. These mechanistic insights highlight how common host factors affect closely related, medically important viruses differently.
Asunto(s)
Autofagia , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice , Enterovirus Humano D , Infecciones por Enterovirus , Replicación Viral , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/metabolismo , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/genética , Humanos , Enterovirus Humano D/fisiología , Enterovirus Humano D/metabolismo , Enterovirus Humano D/genética , Infecciones por Enterovirus/metabolismo , Infecciones por Enterovirus/virología , Proteasas Virales 3C/metabolismo , Lisosomas/metabolismo , ARN Viral/metabolismo , ARN Viral/genética , Proteínas Virales/metabolismo , Proteínas Virales/genética , Mielitis/metabolismo , Mielitis/virología , Unión Proteica , Células HEK293 , Enfermedades Neuromusculares , Enfermedades Virales del Sistema Nervioso CentralRESUMEN
The globally reemerging respiratory pathogen enterovirus D68 (EV-D68) is implicated in outbreaks of severe respiratory illness and associated with acute flaccid myelitis. However, there remains a lack of effective treatments for EV-D68 infection. In this work, we found that the host Toll-like receptor 7 (TLR7) proteins, which function as powerful innate immune sensors, were selectively elevated in expression in response to EV-D68 infection. Subsequently, we investigated the impact of Vesatolimod (GS-9620), a Toll-like receptor 7 agonist, on EV-D68 replication. Our findings revealed that EV-D68 infection resulted in increased mRNA levels of TLR7. Treatment with Vesatolimod significantly inhibited EV-D68 replication [half maximal effective concentration (EC50) = 0.1427 µM] without inducing significant cytotoxicity at virucidal concentrations. Although Vesatolimod exhibited limited impact on EV-D68 attachment, it suppressed RNA replication and viral protein synthesis after virus entry. Vesatolimod broadly inhibited the replication of circulating isolated strains of EV-D68. Furthermore, our findings demonstrated that treatment with Vesatolimod conferred resistance to both respiratory and neural cells against EV-D68 infection. Overall, these results present a promising strategy for drug development by pharmacologically activating TLR7 to initiate an antiviral state in EV-D68-infected cells selectively.IMPORTANCEConventional strategies for antiviral drug development primarily focus on directly targeting viral proteases or key components, as well as host proteins involved in viral replication. In this study, based on our intriguing discovery that enterovirus D68 (EV-D68) infection specifically upregulates the expression of immune sensor Toll-like receptor 7 (TLR7) protein, which is either absent or expressed at low levels in respiratory cells, we propose a potential antiviral approach utilizing TLR7 agonists to activate EV-D68-infected cells into an anti-viral defense state. Notably, our findings demonstrate that pharmacological activation of TLR7 effectively suppresses EV-D68 replication in respiratory tract cells through a TLR7/MyD88-dependent mechanism. This study not only presents a promising drug candidate and target against EV-D68 dissemination but also highlights the potential to exploit unique alterations in cellular innate immune responses induced by viral infections, selectively inducing a defensive state in infected cells while safeguarding uninfected normal cells from potential adverse effects associated with therapeutic interventions.
Asunto(s)
Antivirales , Enterovirus Humano D , Receptor Toll-Like 7 , Replicación Viral , Receptor Toll-Like 7/agonistas , Receptor Toll-Like 7/metabolismo , Humanos , Replicación Viral/efectos de los fármacos , Enterovirus Humano D/efectos de los fármacos , Antivirales/farmacología , Indoles/farmacología , Infecciones por Enterovirus/virología , Inmunidad Innata/efectos de los fármacos , Línea Celular , Internalización del Virus/efectos de los fármacos , PteridinasRESUMEN
Human enteroviruses (EVs) represent a global public health concern due to their association with a range of serious pediatric illnesses. Despite the high morbidity and mortality exerted by EVs, no broad-spectrum antivirals are currently available. Herein, we presented evidence that doxycycline can inhibit in vitro replication of various neurotropic EVs, including enterovirus A71 (EV-A71), enterovirus D68 (EV-D68), and coxsackievirus (CV)-A6, in a dose-dependent manner. Further investigations indicated that the drug primarily acted at the post-entry stage of virus infection in vitro, with inhibitory effects reaching up to 89 % for EV-A71 when administered two hours post-infection. These findings provide valuable insights for the development of antiviral drugs against EV infections.
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Antivirales , Doxiciclina , Enterovirus , Replicación Viral , Humanos , Doxiciclina/farmacología , Replicación Viral/efectos de los fármacos , Antivirales/farmacología , Enterovirus/efectos de los fármacos , Enterovirus/fisiología , Infecciones por Enterovirus/virología , Infecciones por Enterovirus/tratamiento farmacológico , Enterovirus Humano A/efectos de los fármacos , Enterovirus Humano A/fisiología , Línea Celular , Enterovirus Humano D/efectos de los fármacos , Enterovirus Humano D/fisiología , Animales , Internalización del Virus/efectos de los fármacosRESUMEN
Human enteroviruses are the most common human pathogen with over 300 distinct genotypes. Previous work with poliovirus has suggested that it is possible to generate antibody responses in humans and animals that can recognize members of multiple enterovirus species. However, cross protective immunity across multiple enteroviruses is not observed epidemiologically in humans. Here we investigated whether immunization of mice or baboons with inactivated poliovirus or enterovirus virus-like-particles (VLPs) vaccines generates antibody responses that can recognize enterovirus D68 or A71. We found that mice only generated antibodies specific for the antigen they were immunized with, and repeated immunization failed to generate cross-reactive antibody responses as measured by both ELISA and neutralization assay. Immunization of baboons with IPV failed to generate neutralizing antibody responses against enterovirus D68 or A71. These results suggest that a multivalent approach to enterovirus vaccination is necessary to protect against enterovirus disease in vulnerable populations.
Asunto(s)
Anticuerpos Antivirales , Reacciones Cruzadas , Infecciones por Enterovirus , Vacuna Antipolio de Virus Inactivados , Animales , Ratones , Reacciones Cruzadas/inmunología , Anticuerpos Antivirales/inmunología , Infecciones por Enterovirus/inmunología , Infecciones por Enterovirus/prevención & control , Infecciones por Enterovirus/virología , Vacuna Antipolio de Virus Inactivados/inmunología , Vacuna Antipolio de Virus Inactivados/administración & dosificación , Vacunas de Partículas Similares a Virus/inmunología , Anticuerpos Neutralizantes/inmunología , Papio/inmunología , Humanos , Poliovirus/inmunología , Femenino , Formación de Anticuerpos/inmunología , Enterovirus/inmunología , Ratones Endogámicos BALB C , Enterovirus Humano D/inmunologíaRESUMEN
Enterovirus D68 (EV-D68), a pathogen that causes respiratory symptoms, mainly in children, has been implicated in acute flaccid myelitis, which is a poliomyelitis-like paralysis. Currently, there are no licensed vaccines or treatments for EV-D68 infections. Here, we investigated the optimal viral inactivation reagents, vaccine adjuvants, and route of vaccination in mice to optimize an inactivated whole-virion (WV) vaccine against EV-D68. We used formalin, ß-propiolactone (BPL), and hydrogen peroxide as viral inactivation reagents and compared their effects on antibody responses. Use of any of these three viral inactivation reagents effectively induced neutralizing antibodies. Moreover, the antibody response induced by the BPL-inactivated WV vaccine was enhanced when adjuvanted with cytosine phosphoguanine oligodeoxynucleotide (CpG ODN) or AddaVax (MF59-like adjuvant), but not with aluminum hydroxide (alum). Consistent with the antibody response results, the protective effect of the inactivated WV vaccine against the EV-D68 challenge was enhanced when adjuvanted with CpG ODN or AddaVax, but not with alum. Further, while the intranasal inactivated WV vaccine induced EV-D68-specific IgA antibodies in the respiratory tract, it was less protective against EV-D68 challenge than the injectable vaccine. Thus, an injectable inactivated EV-D68 WV vaccine prepared with appropriate viral inactivation reagents and an optimal adjuvant is a promising EV-D68 vaccine.
Asunto(s)
Compuestos de Alumbre , Enterovirus Humano D , Infecciones por Enterovirus , Polisorbatos , Escualeno , Humanos , Niño , Animales , Ratones , Anticuerpos Antivirales , Vacunas de Productos Inactivados , Oligodesoxirribonucleótidos , Adyuvantes InmunológicosRESUMEN
Acute flaccid myelitis (AFM) is a serious neurologic condition primarily affecting children; AFM can cause acute respiratory failure and permanent paralysis. AFM is a rare but known complication of various viral infections, particularly those of enteroviruses (EVs). Increases in AFM cases during 2014, 2016, and 2018 were associated with EV-D68 infection. This report examines trends in confirmed AFM cases during 2018-2022 and patients' clinical and laboratory characteristics. The number of AFM cases was low during 2019-2022 (28-47 cases per year); the number of cases remained low in 2022 despite evidence of increased EV-D68 circulation in the United States. Compared with cases during the most recent peak year (2018), fewer cases during 2019-2021 had upper limb involvement, prodromal respiratory or febrile illness, or cerebrospinal fluid pleocytosis, and more were associated with lower limb involvement. It is unclear why EV-D68 circulation in 2022 was not associated with an increase in AFM cases or when the next increase in AFM cases will occur. Nonetheless, clinicians should continue to suspect AFM in any child with acute flaccid limb weakness, especially those with a recent respiratory or febrile illness.
Asunto(s)
Enfermedades Virales del Sistema Nervioso Central , Enterovirus Humano D , Infecciones por Enterovirus , Mielitis , Enfermedades Neuromusculares , Niño , Humanos , Estados Unidos/epidemiología , Enfermedades Neuromusculares/epidemiología , Parálisis , Mielitis/epidemiología , Enfermedades Virales del Sistema Nervioso Central/epidemiología , Infecciones por Enterovirus/epidemiologíaRESUMEN
Surveillance for emerging pathogens is critical for developing early warning systems to guide preparedness efforts for future outbreaks of associated disease. To better define the epidemiology and burden of associated respiratory disease and acute flaccid myelitis (AFM), as well as to provide actionable data for public health interventions, we developed a multimodal surveillance program in Colorado, USA, for enterovirus D68 (EV-D68). Timely local, state, and national public health outreach was possible because prospective syndromic surveillance for AFM and asthma-like respiratory illness, prospective clinical laboratory surveillance for EV-D68 among children hospitalized with respiratory illness, and retrospective wastewater surveillance led to early detection of the 2022 outbreak of EV-D68 among Colorado children. The lessons learned from developing the individual layers of this multimodal surveillance program and how they complemented and informed the other layers of surveillance for EV-D68 and AFM could be applied to other emerging pathogens and their associated diseases.
Asunto(s)
Enfermedades Virales del Sistema Nervioso Central , Enterovirus Humano D , Mielitis , Enfermedades Neuromusculares , Enfermedades Respiratorias , Niño , Humanos , Colorado/epidemiología , Estudios Prospectivos , Estudios Retrospectivos , Aguas Residuales , Monitoreo Epidemiológico Basado en Aguas ResidualesRESUMEN
Following the successful control of poliovirus, the re-emergence of respiratory enterovirus D68 (EV-D68), a prominent non-polio enterovirus, has become a serious public health concern worldwide. Host innate immune responses are the primary defense against EV-D68 invasion; however, the mechanism underlying viral evasion of the antiviral activity of interferons (IFN) remains unclear. In this study, we found that EV-D68 inhibited type I IFN signaling by cleaving signal transducer and activator of transcription 1 (STAT1), a crucial factor in cellular responses to interferons and other cytokines. We observed that the prototype and circulating EV-D68 strains conserved their ability to induce STAT1 cleavage and attenuate IFN signal transduction. Further investigation revealed that EV-D68 3C protease cleaves STAT1 at the 131Q residue. Interestingly, not all enterovirus-encoded 3C proteases exhibited this ability. EV-D68 and poliovirus 3C proteases efficiently induced STAT1 cleavage; whereas, 3C proteases from EV-A71, coxsackievirus A16, and echoviruses did not. STAT1 cleavage also abolished the nuclear translocation capacity of STAT1 in response to IFN stimulation to activate downstream signaling elements. Overall, these results suggest that STAT1, targeted by viral protease 3C, is utilized by EV-D68 to subvert the host's innate immune response.IMPORTANCEEnterovirus D68 (EV-D68) has significantly transformed over the past decade, evolving from a rare pathogen to a potential pandemic pathogen. The interferon (IFN) signaling pathway is an important defense mechanism and therapeutic target for the host to resist viral invasion. Previous studies have reported that the EV-D68 virus blocks or weakens immune recognition and IFN production in host cells through diverse strategies; however, the mechanisms of EV-D68 resistance to IFN signaling have not been fully elucidated. Our study revealed that EV-D68 relies on its own encoded protease, 3C, to directly cleave signal transducer and activator of transcription 1 (STAT1), a pivotal transduction component in the IFN signaling pathway, disrupting the IFN-mediated antiviral response. Previous studies on human enteroviruses have not documented direct cleavage of the STAT1 protein to evade cellular immune defenses. However, not all enteroviral 3C proteins can cleave STAT1. These findings highlight the diverse evolutionary strategies different human enteroviruses employ to evade host immunity.
Asunto(s)
Proteasas Virales 3C , Enterovirus Humano D , Interferón Tipo I , Transducción de Señal , Humanos , Proteasas Virales 3C/metabolismo , Antígenos Virales/metabolismo , Antivirales/farmacología , Cisteína Endopeptidasas/metabolismo , Enterovirus Humano D/fisiología , Interacciones Huésped-Patógeno , Evasión Inmune , Inmunidad Innata , Interferón Tipo I/metabolismo , Péptido Hidrolasas/metabolismo , Proteolisis , Factor de Transcripción STAT1/metabolismo , Proteínas Virales/metabolismoRESUMEN
Pyroptosis, a pro-inflammatory programmed cell death, has been implicated in the pathogenesis of coronavirus disease 2019 and other viral diseases. Gasdermin family proteins (GSDMs), including GSDMD and GSDME, are key regulators of pyroptotic cell death. However, the mechanisms by which virus infection modulates pyroptosis remain unclear. Here, we employed a mCherry-GSDMD fluorescent reporter assay to screen for viral proteins that impede the localization and function of GSDMD in living cells. Our data indicated that the main protease NSP5 of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) blocked GSDMD-mediated pyroptosis via cleaving residues Q29 and Q193 of GSDMD. While another SARS-CoV-2 protease, NSP3, cleaved GSDME at residue G370 but activated GSDME-mediated pyroptosis. Interestingly, respiratory enterovirus EV-D68-encoded proteases 3C and 2A also exhibit similar differential regulation on the functions of GSDMs by inactivating GSDMD but initiating GSDME-mediated pyroptosis. EV-D68 infection exerted oncolytic effects on human cancer cells by inducing pyroptotic cell death. Our findings provide insights into how respiratory viruses manipulate host cell pyroptosis and suggest potential targets for antiviral therapy as well as cancer treatment.IMPORTANCEPyroptosis plays a crucial role in the pathogenesis of coronavirus disease 2019, and comprehending its function may facilitate the development of novel therapeutic strategies. This study aims to explore how viral-encoded proteases modulate pyroptosis. We investigated the impact of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and respiratory enterovirus D68 (EV-D68) proteases on host cell pyroptosis. We found that SARS-CoV-2-encoded proteases NSP5 and NSP3 inactivate gasdermin D (GSDMD) but initiate gasdermin E (GSDME)-mediated pyroptosis, respectively. We also discovered that another respiratory virus EV-D68 encodes two distinct proteases 2A and 3C that selectively trigger GSDME-mediated pyroptosis while suppressing the function of GSDMD. Based on these findings, we further noted that EV-D68 infection triggers pyroptosis and produces oncolytic effects in human carcinoma cells. Our study provides new insights into the molecular mechanisms underlying virus-modulated pyroptosis and identifies potential targets for the development of antiviral and cancer therapeutics.
Asunto(s)
Endopeptidasas , Enterovirus Humano D , Interacciones Microbiota-Huesped , Virus Oncolíticos , Piroptosis , SARS-CoV-2 , Humanos , Línea Celular Tumoral , COVID-19/metabolismo , COVID-19/terapia , COVID-19/virología , Endopeptidasas/genética , Endopeptidasas/metabolismo , Enterovirus Humano D/enzimología , Enterovirus Humano D/genética , Infecciones por Enterovirus/metabolismo , Infecciones por Enterovirus/virología , Gasderminas/antagonistas & inhibidores , Gasderminas/genética , Gasderminas/metabolismo , Viroterapia Oncolítica , Virus Oncolíticos/enzimología , Virus Oncolíticos/genética , SARS-CoV-2/enzimología , SARS-CoV-2/genética , Proteínas Virales/genética , Proteínas Virales/metabolismoRESUMEN
BACKGROUND: Enterovirus D68 (EV-D68) is an important reemerging pathogen that causes severe acute respiratory infection and acute flaccid paralysis, mainly in children. Since 2014, EV-D68 outbreaks have been reported in the United States, Europe, and east Asia; however, no outbreaks have been reported in southeast Asian countries, including Myanmar, during the previous 10 years. METHODS: EV-D68 was detected in nasopharyngeal swabs from children with acute lower respiratory infections in Myanmar. The samples were previously collected from children aged 1 month to 12 years who had been admitted to the Yankin Children Hospital in Yangon, Myanmar, between May 2017 and January 2019. EV-D68 was detected with a newly developed EV-D68-specific real-time PCR assay. The clade was identified by using a phylogenetic tree created with the Bayesian Markov chain Monte Carlo method. RESULTS: During the study period, nasopharyngeal samples were collected from 570 patients. EV-D68 was detected in 42 samples (7.4 %)-11 samples from 2017 to 31 samples from 2018. The phylogenetic tree revealed that all strains belonged to clade B3, which has been the dominant clade worldwide since 2014. We estimate that ancestors of currently circulating genotypes emerged during the period 1980-2004. CONCLUSIONS: To our knowledge, this is the first report of EV-D68 detection in children with acute lower respiratory infections in Yangon, Myanmar, in 2017-2018. Detection and detailed virologic analyses of EV-D68 in southeast Asia is an important aspect of worldwide surveillance and will likely be useful in better understanding the worldwide epidemiologic profile of EV-D68 infection.
Asunto(s)
Enterovirus Humano D , Infecciones por Enterovirus , Enterovirus , Neumonía , Infecciones del Sistema Respiratorio , Niño , Humanos , Estados Unidos , Enterovirus Humano D/genética , Mianmar/epidemiología , Filogenia , Teorema de Bayes , Neumonía/epidemiología , Brotes de Enfermedades , Enterovirus/genéticaRESUMEN
Enterovirus D68 (EV-D68) is an emerging pathogen that has caused outbreaks of severe respiratory disease worldwide, especially in children. We aim to investigate the prevalence and genetic characteristics of EV-D68 in children from Shanghai. Nasopharyngeal swab or bronchoalveolar lavage fluid samples collected from children hospitalized with community-acquired pneumonia were screened for EV-D68. Nine of 3997 samples were EV-D68-positive. Seven of nine positive samples were sequenced and submitted to GenBank. Based on partial polyprotein gene (3D) or complete sequence analysis, we found the seven strains belong to different clades and subclades, including three D1 (detected in 2013 and 2014), one D2 (2013), one D3 (2019), and two B3 (2014 and 2018). Overall, we show different clades and subclades of EV-D68 spread with low positive rates (0.2%) among children in Shanghai between 2013 and 2020. Amino acid mutations were found in the epitopes of the VP1 BC and DE loops and C-terminus; similarity analysis provided evidence for recombination as an important mechanism of genomic diversification. Both single nucleotide mutations and recombination play a role in evolution of EV-D68. Genetic instability within these clinical strains may indicate large outbreaks could occur following cumulative mutations.
Asunto(s)
Enterovirus Humano D , Infecciones por Enterovirus , Enterovirus , Infecciones del Sistema Respiratorio , Niño , Humanos , Epidemiología Molecular , Enterovirus Humano D/genética , Infecciones del Sistema Respiratorio/epidemiología , Infecciones por Enterovirus/epidemiología , Filogenia , China/epidemiología , Brotes de Enfermedades , Enterovirus/genéticaRESUMEN
Enterovirus D68 (EV-D68) is predominantly associated with mild respiratory infections, but can also cause severe respiratory disease and extra-respiratory complications, including acute flaccid myelitis. Systemic dissemination of EV-D68 is crucial for the development of extra-respiratory diseases, but it is currently unclear how EV-D68 spreads systemically (viremia). We hypothesize that immune cells contribute to the systemic dissemination of EV-D68, as this is a mechanism commonly used by other enteroviruses. Therefore, we investigated the susceptibility and permissiveness of human primary immune cells for different EV-D68 isolates. In human peripheral blood mononuclear cells inoculated with EV-D68, only B cells were susceptible but virus replication was limited. However, in B cell-rich cultures, such as Epstein-Barr virus-transformed B-lymphoblastoid cell line (BLCL) and primary lentivirus-transduced B cells, which better represent lymphoid B cells, were productively infected. Subsequently, we showed that dendritic cells (DCs), particularly immature DCs, are susceptible and permissive for EV-D68 infection and that they can spread EV-D68 to autologous BLCL. Altogether, our findings suggest that immune cells, especially B cells and DCs, could play an important role in the pathogenesis of EV-D68 infection. Infection of these cells may contribute to systemic dissemination of EV-D68, which is an essential step toward the development of extra-respiratory complications.IMPORTANCEEnterovirus D68 (EV-D68) is an emerging respiratory virus that has caused outbreaks worldwide since 2014. EV-D68 infects primarily respiratory epithelial cells resulting in mild respiratory diseases. However, EV-D68 infection is also associated with extra-respiratory complications, including polio-like paralysis. It is unclear how EV-D68 spreads systemically and infects other organs. We hypothesized that immune cells could play a role in the extra-respiratory spread of EV-D68. We showed that EV-D68 can infect and replicate in specific immune cells, that is, B cells and dendritic cells (DCs), and that virus could be transferred from DCs to B cells. Our data reveal a potential role of immune cells in the pathogenesis of EV-D68 infection. Intervention strategies that prevent EV-D68 infection of immune cells will therefore potentially prevent systemic spread of virus and thereby severe extra-respiratory complications.
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Enterovirus Humano D , Infecciones por Enterovirus , Infecciones por Virus de Epstein-Barr , Infecciones del Sistema Respiratorio , Humanos , Leucocitos Mononucleares , Herpesvirus Humano 4 , Células DendríticasRESUMEN
Enterovirus D68 (EV-D68), belonging to the genus Enterovirus of the Picornavirus family, is an emerging pathogen that can cause neurological and respiratory diseases in children. However, there is little understanding of the pathogenesis of EV-D68, and no effective vaccine or drug for the prevention or treatment of the diseases caused by this virus is available. Autophagy is a cellular process that targets cytoplasmic proteins or organelles to the lysosomes for degradation. Enteroviruses strategically harness the host autophagy pathway to facilitate the completion of their life cycle. Therefore, we selected an autophagy compound library to screen for autophagy-related compounds that may affect viral growth. By using the neutralization screening assay, we identified a compound, 'licochalcone A' that significantly inhibited EV-D68 replication. To investigate the mechanism by which licochalcone A inhibits EV-D68 replication and to identify the viral life cycle stage it inhibits, the time-of-addition, viral attachment, viral entry, and dual-luciferase reporter assays were performed. The results of the time-of-addition assay showed that licochalcone A, a characteristic chalcone found in liquorice roots and widely used in traditional Chinese medicine, inhibits EV-D68 replication during the early stages of the viral life cycle, while those of the dual-luciferase reporter assay showed that licochalcone A does not regulate viral attachment and entry, but inhibits EV-D68 IRES-dependent translation. Licochalcone A also inhibited enterovirus A71 and coxsackievirus B3 but did not significantly inhibit dengue virus 2 or human coronavirus 229E replication. Licochalcone A regulates IRES translation to inhibit EV-D68 viral replication.
Asunto(s)
Chalconas , Enterovirus Humano D , Infecciones por Enterovirus , Enterovirus , Niño , Humanos , Chalconas/farmacología , Infecciones por Enterovirus/tratamiento farmacológico , Antígenos Virales , Enterovirus Humano D/fisiología , LuciferasasRESUMEN
Enterovirus D68 (EV-D68) primarily spreads through the respiratory tract and causes respiratory symptoms in children and acute flaccid myelitis (AFM). Type III interferons (IFNs) play a critical role in inhibiting viral growth in respiratory epithelial cells. However, the mechanism by which EV-D68 induces type III IFN production is not yet fully understood. In this study, we show that EV-D68 infection stimulates Calu-3 cells to secrete IFN-λ. The transfection of EV-D68 viral RNA (vRNA) stimulated IFN-λ via MDA5. Furthermore, our findings provide evidence that EV-D68 infection also induces MDA5-IRF3/IRF7-mediated IFN-λ. In addition, we discovered that EV-D68 infection downregulated MDA5 expression. Knockdown of MDA5 increased EV-D68 replication in Calu-3 cells. Finally, we demonstrated that the IFN-λ1 and IFN-λ2/3 proteins effectively inhibit EV-D68 infection in respiratory epithelial cells. In summary, our study shows that EV-D68 induces type III IFN production via the activated MDA5-IRF3/IRF7 pathway and that type III IFNs inhibit EV-D68 replication in Calu-3 cells.
Asunto(s)
Enterovirus Humano D , Infecciones por Enterovirus , Enfermedades Neuromusculares , Niño , Humanos , Enterovirus Humano D/genética , Interferón lambda , Sistema RespiratorioRESUMEN
In a 2-year study in Leuven, Belgium, we investigated the use of wastewater sampling to assess community spread of respiratory viruses. Comparison with the number of positive clinical samples demonstrated that wastewater data reflected circulation levels of typical seasonal respiratory viruses, such as influenza, respiratory syncytial virus, and enterovirus D68.