RESUMEN
Efficient virus replication in Aedes vector mosquitoes is essential for the transmission of arboviral diseases such as dengue virus (DENV) in human populations. Like in vertebrates, virus-host protein-protein interactions are essential for viral replication and immune evasion in the mosquito vector. Here, 79 mosquito host proteins interacting with DENV non-structural proteins NS1 and NS5 were identified by label-free mass spectrometry, followed by a functional screening. We confirmed interactions with host factors previously observed in mammals, such as the oligosaccharyltransferase complex, and we identified protein-protein interactions that seem to be specific for mosquitoes. Among the interactors, the double-stranded RNA (dsRNA) binding protein Loquacious (Loqs), an RNA interference (RNAi) cofactor, was found to be essential for efficient replication of DENV and Zika virus (ZIKV) in mosquito cells. Loqs did not affect viral RNA stability or translation of a DENV replicon and its proviral activity was independent of its RNAi regulatory activity. Interestingly, Loqs colocalized with DENV dsRNA replication intermediates in infected cells and directly interacted with high affinity with DENV RNA in the 3' untranslated region in vitro (KD = 48-62 nM). Our study provides an interactome for DENV NS1 and NS5 and identifies Loqs as a key proviral host factor in mosquitoes. We propose that DENV hijacks a factor of the RNAi mechanism for replication of its own RNA.
Asunto(s)
Aedes , Arbovirus , Virus del Dengue , Dengue , Infección por el Virus Zika , Virus Zika , Regiones no Traducidas 3' , Animales , Arbovirus/genética , Virus del Dengue/genética , Humanos , Mamíferos , Mosquitos Vectores , ARN Bicatenario/metabolismo , Replicación Viral/genética , Virus Zika/genéticaRESUMEN
The mRNA-based BNT162b2 protects against severe disease and mortality caused by SARS-CoV-2 via induction of specific antibody and T-cell responses. Much less is known about its broad effects on immune responses against other pathogens. Here, we investigated the adaptive immune responses induced by BNT162b2 vaccination against various SARS-CoV-2 variants and its effects on the responsiveness of immune cells upon stimulation with heterologous stimuli. BNT162b2 vaccination induced effective humoral and cellular immunity against SARS-CoV-2 that started to wane after six months. We also observed long-term transcriptional changes in immune cells after vaccination. Additionally, vaccination with BNT162b2 modulated innate immune responses as measured by inflammatory cytokine production after stimulation - higher IL-1/IL-6 release and decreased IFN-α production. Altogether, these data expand our knowledge regarding the overall immunological effects of this new class of vaccines and underline the need for additional studies to elucidate their effects on both innate and adaptive immune responses.
RESUMEN
In the germline of animals, PIWI interacting (pi)RNAs protect the genome against the detrimental effects of transposon mobilization. In Drosophila, piRNA-mediated cleavage of transposon RNA triggers the production of responder piRNAs via ping-pong amplification. Responder piRNA 3' end formation by the nuclease Zucchini is coupled to the production of downstream trailer piRNAs, expanding the repertoire of transposon piRNA sequences. In Aedes aegypti mosquitoes, piRNAs are generated from viral RNA, yet, it is unknown how viral piRNA 3' ends are formed and whether viral RNA cleavage gives rise to trailer piRNA production. Here we report that in Ae. aegypti, virus- and transposon-derived piRNAs have sharp 3' ends, and are biased for downstream uridine residues, features reminiscent of Zucchini cleavage of precursor piRNAs in Drosophila. We designed a reporter system to study viral piRNA 3' end formation and found that targeting viral RNA by abundant endogenous piRNAs triggers the production of responder and trailer piRNAs. Using this reporter, we identified the Ae. aegypti orthologs of Zucchini and Nibbler, two nucleases involved in piRNA 3' end formation. Our results furthermore suggest that autonomous piRNA production from viral RNA can be triggered and expanded by an initial cleavage event guided by genome-encoded piRNAs.
Asunto(s)
Elementos Transponibles de ADN/genética , Densovirinae/genética , Proteínas de Drosophila/genética , Endorribonucleasas/genética , ARN Interferente Pequeño/genética , ARN Viral/genética , Aedes/genética , Aedes/virología , Animales , Proteínas Argonautas/genética , Densovirinae/patogenicidad , Drosophila melanogaster/genética , Drosophila melanogaster/virología , Células Germinativas/virología , División del ARN/genéticaRESUMEN
Coevolution of viruses and their hosts may lead to viral strategies to avoid, evade, or suppress antiviral immunity. An example is antiviral RNA interference (RNAi) in insects: the host RNAi machinery processes viral double-stranded RNA into small interfering RNAs (siRNAs) to suppress viral replication, whereas insect viruses encode suppressors of RNAi, many of which inhibit viral small interfering RNA (vsiRNA) production. Yet, many studies have analyzed viral RNAi suppressors in heterologous systems, due to the lack of experimental systems to manipulate the viral genome of interest, raising questions about in vivo functions of RNAi suppressors. To address this caveat, we generated an RNAi suppressor-defective mutant of invertebrate iridescent virus 6 (IIV6), a large DNA virus in which we previously identified the 340R protein as a suppressor of RNAi. Loss of 340R did not affect vsiRNA production, indicating that 340R binds siRNA duplexes to prevent RNA-induced silencing complex assembly. Indeed, vsiRNAs were not efficiently loaded into Argonaute 2 during wild-type IIV6 infection. Moreover, IIV6 induced a limited set of mature microRNAs in a 340R-dependent manner, most notably miR-305-3p, which we attribute to stabilization of the miR-305-5p:3p duplex by 340R. The IIV6 340R deletion mutant did not have a replication defect in cells, but was strongly attenuated in adult Drosophila This in vivo replication defect was completely rescued in RNAi mutant flies, indicating that 340R is a bona fide RNAi suppressor, the absence of which uncovers a potent antiviral immune response that suppresses virus accumulation â¼100-fold. Together, our work indicates that viral RNAi suppressors may completely mask antiviral immunity.
Asunto(s)
Drosophila/genética , Drosophila/virología , Interacciones Huésped-Patógeno/inmunología , Iridovirus/fisiología , Iridovirus/patogenicidad , Animales , Drosophila/inmunología , Regulación de la Expresión Génica , Interacciones Huésped-Patógeno/genética , MicroARNs/genética , Microorganismos Modificados Genéticamente , Mutación , Interferencia de ARN , Estabilidad del ARN , Proteínas Virales/genética , Proteínas Virales/inmunología , Replicación ViralRESUMEN
Thrombocytopenia and platelet dysfunction are commonly observed in patients with dengue virus (DENV) infection and may contribute to complications such as bleeding and plasma leakage. The etiology of dengue-associated thrombocytopenia is multifactorial and includes increased platelet clearance. The binding of the coagulation protein von Willebrand factor (VWF) to the platelet membrane and removal of sialic acid (desialylation) are two well-known mechanisms of platelet clearance, but whether these conditions also contribute to thrombocytopenia in dengue infection is unknown. In two observational cohort studies in Bandung and Jepara, Indonesia, we show that adult patients with dengue not only had higher plasma concentrations of plasma VWF antigen and active VWF, but that circulating platelets had also bound more VWF to their membrane. The amount of platelet-VWF binding correlated well with platelet count. Furthermore, sialic acid levels in dengue patients were significantly reduced as assessed by the binding of Sambucus nigra lectin (SNA) and Maackia amurensis lectin II (MAL-II) to platelets. Sialic acid on the platelet membrane is neuraminidase-labile, but dengue virus has no known neuraminidase activity. Indeed, no detectable activity of neuraminidase was present in plasma of dengue patients and no desialylation was found of plasma transferrin. Platelet sialylation was also not altered by in vitro exposure of platelets to DENV nonstructural protein 1 or cultured DENV. In contrast, induction of binding of VWF to glycoprotein 1b on platelets using the VWF-activating protein ristocetin resulted in the removal of platelet sialic acid by translocation of platelet neuraminidase to the platelet surface. The neuraminidase inhibitor oseltamivir reduced VWF-induced platelet desialylation. Our data demonstrate that excessive binding of VWF to platelets in dengue results in neuraminidase-mediated platelet desialylation and platelet clearance. Oseltamivir might be a novel treatment option for severe thrombocytopenia in dengue infection.
Asunto(s)
Plaquetas/metabolismo , Ácido N-Acetilneuramínico/metabolismo , Factor de von Willebrand/fisiología , Adolescente , Adulto , Factores de Coagulación Sanguínea , Plaquetas/fisiología , Estudios de Cohortes , Dengue/metabolismo , Femenino , Fibrinógeno , Humanos , Indonesia , Cinética , Masculino , Proteínas Proteolipídicas Asociadas a Mielina y Linfocito , Neuraminidasa/metabolismo , Lectinas de Plantas , Glicoproteínas de Membrana Plaquetaria/metabolismo , Proteínas Inactivadoras de Ribosomas , Trombocitopenia , Adulto Joven , Factor de von Willebrand/metabolismoRESUMEN
The genus Alphavirus harbours mostly insect-transmitted viruses that cause severe disease in humans, livestock and wildlife. Thus far, only three alphaviruses with a host range restricted to insects have been found in mosquitoes from the Old World, namely Eilat virus (EILV), Taï Forest alphavirus (TALV) and Mwinilunga alphavirus (MWAV). In this study, we found a novel alphavirus in one Culex declarator mosquito sampled in Panama. The virus was isolated in C6/36 mosquito cells, and full genome sequencing revealed an 11â468 nt long genome with maximum pairwise nucleotide identity of 62.7â% to Sindbis virus. Phylogenetic analyses placed the virus as a solitary deep rooting lineage in a basal relationship to the Western equine encephalitis antigenic complex and to the clade comprising EILV, TALV and MWAV, indicating the detection of a novel alphavirus, tentatively named Agua Salud alphavirus (ASALV). No growth of ASALV was detected in vertebrate cell lines, including cell lines derived from ectothermic animals, and replication of ASALV was strongly impaired above 31 °C, suggesting that ASALV represents the first insect-restricted alphavirus of the New World.
Asunto(s)
Alphavirus/genética , Culicidae/virología , Especificidad del Huésped/genética , Virus de Insectos/genética , Animales , Línea Celular , Panamá , Filogenia , ARN Viral/genética , Vertebrados/virología , Replicación Viral/genéticaRESUMEN
Interactions between the insect immune system and RNA viruses have been extensively studied in Drosophila, in which RNA interference, NF-κB, and JAK-STAT pathways underlie antiviral immunity. In response to RNA interference, insect viruses have convergently evolved suppressors of this pathway that act by diverse mechanisms to permit viral replication. However, interactions between the insect immune system and DNA viruses have received less attention, primarily because few Drosophila-infecting DNA virus isolates are available. In this study, we used a recently isolated DNA virus of Drosophila melanogaster, Kallithea virus (KV; family Nudiviridae), to probe known antiviral immune responses and virus evasion tactics in the context of DNA virus infection. We found that fly mutants for RNA interference and immune deficiency (Imd), but not Toll, pathways are more susceptible to Kallithea virus infection. We identified the Kallithea virus-encoded protein gp83 as a potent inhibitor of Toll signalling, suggesting that Toll mediates antiviral defense against Kallithea virus infection but that it is suppressed by the virus. We found that Kallithea virus gp83 inhibits Toll signalling through the regulation of NF-κB transcription factors. Furthermore, we found that gp83 of the closely related Drosophila innubila nudivirus (DiNV) suppresses D. melanogaster Toll signalling, suggesting an evolutionarily conserved function of Toll in defense against DNA viruses. Together, these results provide a broad description of known antiviral pathways in the context of DNA virus infection and identify the first Toll pathway inhibitor in a Drosophila virus, extending the known diversity of insect virus-encoded immune inhibitors.IMPORTANCE Coevolution of multicellular organisms and their natural viruses may lead to an intricate relationship in which host survival requires effective immunity and virus survival depends on evasion of such responses. Insect antiviral immunity and reciprocal virus immunosuppression tactics have been well studied in Drosophila melanogaster, primarily during RNA, but not DNA, virus infection. Therefore, we describe interactions between a recently isolated Drosophila DNA virus (Kallithea virus [KV]) and immune processes known to control RNA viruses, such as RNA interference (RNAi) and Imd pathways. We found that KV suppresses the Toll pathway and identified gp83 as a KV-encoded protein that underlies this suppression. This immunosuppressive ability is conserved in another nudivirus, suggesting that the Toll pathway has conserved antiviral activity against DNA nudiviruses, which have evolved suppressors in response. Together, these results indicate that DNA viruses induce and suppress NF-κB responses, and they advance the application of KV as a model to study insect immunity.
Asunto(s)
Virus ADN/inmunología , Drosophila melanogaster/metabolismo , Inmunidad Innata/inmunología , FN-kappa B/metabolismo , Proteínas Virales/metabolismo , Replicación Viral/inmunología , Animales , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/crecimiento & desarrollo , Drosophila melanogaster/virología , Femenino , FN-kappa B/antagonistas & inhibidores , FN-kappa B/genética , Interferencia de ARN , Transducción de Señal , Receptores Toll-Like/genética , Receptores Toll-Like/metabolismo , Proteínas Virales/genéticaRESUMEN
Little is known about the tolerance mechanisms that reduce the negative effects of microbial infection on host fitness. Here, we demonstrate that the histone H3 lysine 9 methyltransferase G9a regulates tolerance to virus infection by shaping the response of the evolutionary conserved Jak-Stat pathway in Drosophila. G9a-deficient mutants are more sensitive to RNA virus infection and succumb faster to infection than wild-type controls, which was associated with strongly increased Jak-Stat dependent responses, but not with major differences in viral load. Genetic experiments indicate that hyperactivated Jak-Stat responses are associated with early lethality in virus-infected flies. Our results identify an essential epigenetic mechanism underlying tolerance to virus infection.
Asunto(s)
Drosophila melanogaster/virología , Epigénesis Genética , Regulación de la Expresión Génica/inmunología , N-Metiltransferasa de Histona-Lisina/inmunología , Tolerancia Inmunológica/inmunología , Infecciones por Virus ARN/inmunología , Animales , Inmunoprecipitación de Cromatina , Drosophila melanogaster/enzimología , Drosophila melanogaster/inmunología , Virus ARN , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Despite the enormous disease burden associated with dengue virus infections, a licensed antiviral drug is lacking. Here, we show that the paracetamol (acetaminophen) metabolite AM404 inhibits dengue virus replication. Moreover, we find that mutations in NS4B that were previously found to confer resistance to the antiviral compounds NITD-618 and SDM25N also render dengue virus insensitive to AM404. Our work provides further support for NS4B as a direct or indirect target for antiviral drug development.
Asunto(s)
Antivirales/farmacología , Ácidos Araquidónicos/farmacología , Virus del Dengue/efectos de los fármacos , Farmacorresistencia Viral Múltiple/genética , Mutación , Proteínas no Estructurales Virales/genética , Acetaminofén/metabolismo , Acetaminofén/farmacología , Ácidos Araquidónicos/metabolismo , Biotransformación , Virus del Dengue/genética , Virus del Dengue/crecimiento & desarrollo , Virus del Dengue/metabolismo , Expresión Génica , Genes Reporteros , Células HeLa , Humanos , Luciferasas/genética , Luciferasas/metabolismo , Pruebas de Sensibilidad Microbiana , Naltrexona/análogos & derivados , Naltrexona/farmacología , Ribavirina/farmacología , Proteínas no Estructurales Virales/metabolismo , Replicación Viral/efectos de los fármacosRESUMEN
The ongoing conflict between viruses and their hosts can drive the co-evolution between host immune genes and viral suppressors of immunity. It has been suggested that an evolutionary 'arms race' may occur between rapidly evolving components of the antiviral RNAi pathway of Drosophila and viral genes that antagonize it. We have recently shown that viral protein 1 (VP1) of Drosophila melanogaster Nora virus (DmelNV) suppresses Argonaute-2 (AGO2)-mediated target RNA cleavage (slicer activity) to antagonize antiviral RNAi. Here we show that viral AGO2 antagonists of divergent Nora-like viruses can have host specific activities. We have identified novel Nora-like viruses in wild-caught populations of D. immigrans (DimmNV) and D. subobscura (DsubNV) that are 36% and 26% divergent from DmelNV at the amino acid level. We show that DimmNV and DsubNV VP1 are unable to suppress RNAi in D. melanogaster S2 cells, whereas DmelNV VP1 potently suppresses RNAi in this host species. Moreover, we show that the RNAi suppressor activity of DimmNV VP1 is restricted to its natural host species, D. immigrans. Specifically, we find that DimmNV VP1 interacts with D. immigrans AGO2, but not with D. melanogaster AGO2, and that it suppresses slicer activity in embryo lysates from D. immigrans, but not in lysates from D. melanogaster. This species-specific interaction is reflected in the ability of DimmNV VP1 to enhance RNA production by a recombinant Sindbis virus in a host-specific manner. Our results emphasize the importance of analyzing viral RNAi suppressor activity in the relevant host species. We suggest that rapid co-evolution between RNA viruses and their hosts may result in host species-specific activities of RNAi suppressor proteins, and therefore that viral RNAi suppressors could be host-specificity factors.
Asunto(s)
Proteínas Argonautas , Proteínas de Drosophila , Interferencia de ARN , Virus ARN , Proteínas Virales , Animales , Proteínas Argonautas/antagonistas & inhibidores , Proteínas Argonautas/genética , Proteínas Argonautas/metabolismo , Secuencia de Bases , Proteínas de Drosophila/antagonistas & inhibidores , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster , Datos de Secuencia Molecular , Virus ARN/genética , Virus ARN/metabolismo , Proteínas Virales/genética , Proteínas Virales/metabolismoRESUMEN
RNA interference (RNAi) is a crucial antiviral defense mechanism in insects, including the major mosquito species that transmit important human viruses. To counteract the potent antiviral RNAi pathway, insect viruses encode RNAi suppressors. However, whether mosquito-specific viruses suppress RNAi remains unclear. We therefore set out to study RNAi suppression by Culex Y virus (CYV), a mosquito-specific virus of the Birnaviridae family that was recently isolated from Culex pipiens mosquitoes. We found that the Culex RNAi machinery processes CYV double-stranded RNA (dsRNA) into viral small interfering RNAs (vsiRNAs). Furthermore, we show that RNAi is suppressed in CYV-infected cells and that the viral VP3 protein is responsible for RNAi antagonism. We demonstrate that VP3 can functionally replace B2, the well-characterized RNAi suppressor of Flock House virus. VP3 was found to bind long dsRNA as well as siRNAs and interfered with Dicer-2-mediated cleavage of long dsRNA into siRNAs. Slicing of target RNAs by pre-assembled RNA-induced silencing complexes was not affected by VP3. Finally, we show that the RNAi-suppressive activity of VP3 is conserved in Drosophila X virus, a birnavirus that persistently infects Drosophila cell cultures. Together, our data indicate that mosquito-specific viruses may encode RNAi antagonists to suppress antiviral RNAi.
Asunto(s)
Culex/genética , Drosophila melanogaster/genética , Entomobirnavirus/fisiología , Interferencia de ARN , ARN Bicatenario/metabolismo , ARN Interferente Pequeño/metabolismo , Animales , Células Cultivadas , Culex/virología , Drosophila melanogaster/virología , Entomobirnavirus/genética , Entomobirnavirus/metabolismo , Proteínas Virales/metabolismoRESUMEN
RNA interference (RNAi) is a major antiviral pathway that shapes evolution of RNA viruses. We show here that Nora virus, a natural Drosophila pathogen, is both a target and suppressor of RNAi. We detected viral small RNAs with a signature of Dicer-2 dependent small interfering RNAs in Nora virus infected Drosophila. Furthermore, we demonstrate that the Nora virus VP1 protein contains RNAi suppressive activity in vitro and in vivo that enhances pathogenicity of recombinant Sindbis virus in an RNAi dependent manner. Nora virus VP1 and the viral suppressor of RNAi of Cricket paralysis virus (1A) antagonized Argonaute-2 (AGO2) Slicer activity of RNA induced silencing complexes pre-loaded with a methylated single-stranded guide strand. The convergent evolution of AGO2 suppression in two unrelated insect RNA viruses highlights the importance of AGO2 in antiviral defense.
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Proteínas Argonautas/metabolismo , Proteínas de Drosophila/metabolismo , Evolución Molecular , Silenciador del Gen , Virus de Insectos/metabolismo , Virus ARN/metabolismo , Animales , Proteínas Argonautas/genética , Proteínas de Drosophila/genética , Drosophila melanogaster , Virus de Insectos/genética , Virus ARN/genéticaRESUMEN
After decades of being considered non-pathogenic, Zika virus (ZIKV) emerged as an important threat to human health during the epidemic of 2015-2016. ZIKV infections are usually asymptomatic, but can cause Guillain-Barré syndrome in adults and microcephaly in newborns. As there are currently no approved antiviral drugs against ZIKV, we tested anti-ZIKV activity of compounds from the NIH Clinical Collection for which we previously showed antiviral activity against the related dengue virus. One of the top hits from the screen was lacidipine, a 1,4-dihydropyridine calcium antagonist that is approved as an antihypertensive drug. Our data show that lacidipine is antiviral against ZIKV (strain H/PF/2013) in both Vero cells and induced pluripotent stem cell (iPSC)-derived human neural progenitor cells with IC50 values of 3.0 µM and <50 nM, respectively. The antiviral effect was also observed against four other ZIKV strains from the African and Asian lineages. Time-of-addition and replicon assays indicated that lacidipine acts at the post-entry stage of the viral replication cycle, inhibiting viral genome replication. Lacidipine altered the subcellular distribution of free cholesterol and neutral lipids, suggesting that the antiviral effect of lacidipine is mediated by altered trafficking of lipids. Together, these results identify lacidipine as a novel inhibitor of ZIKV replication that likely disturbs trafficking of lipids needed for replication organelle formation.
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Bloqueadores de los Canales de Calcio , Dihidropiridinas , Células-Madre Neurales , Infección por el Virus Zika , Animales , Antivirales/farmacología , Bloqueadores de los Canales de Calcio/farmacología , Canales de Calcio , Chlorocebus aethiops , Dihidropiridinas/farmacología , Humanos , Recién Nacido , Lípidos , Células-Madre Neurales/efectos de los fármacos , Células-Madre Neurales/virología , Células Madre , Células Vero , Replicación Viral , Virus Zika , Infección por el Virus Zika/tratamiento farmacológicoRESUMEN
We describe a protocol for single-cell RNA sequencing of SARS-CoV-2-infected human induced pluripotent stem cell (iPSC)-derived kidney organoids. After inoculation of kidney organoids with virus, we use mechanical and enzymatic disruption to obtain single cell suspensions. Next, we process the organoid-derived cells into sequencing-ready SARS-CoV-2-targeted libraries. Subsequent sequencing analysis reveals changes in kidney cells after virus infection. The protocol was designed for kidney organoids cultured in a 6-well transwell format but can be adapted to organoids with different organ backgrounds. For complete details on the use and execution of this protocol, please refer to Jansen et al. (2022).
Asunto(s)
COVID-19 , Células Madre Pluripotentes Inducidas , Humanos , Riñón , Organoides , SARS-CoV-2RESUMEN
Repurposing drugs is a promising strategy to identify therapeutic interventions against novel and re-emerging viruses. Posaconazole is an antifungal drug used to treat invasive aspergillosis and candidiasis. Recently, posaconazole and its structural analog, itraconazole were shown to inhibit replication of multiple viruses by modifying intracellular cholesterol homeostasis. Here, we show that posaconazole inhibits replication of the alphaviruses Semliki Forest virus (SFV), Sindbis virus and chikungunya virus with EC50 values ranging from 1.4 µM to 9.5 µM. Posaconazole treatment led to a significant reduction of virus entry in an assay using a temperature-sensitive SFV mutant, but time-of-addition and RNA transfection assays indicated that posaconazole also inhibits post-entry stages of the viral replication cycle. Virus replication in the presence of posaconazole was partially rescued by the addition of exogenous cholesterol. A transferrin uptake assay revealed that posaconazole considerably slowed down cellular endocytosis. A single point mutation in the SFV E2 glycoprotein, H255R, provided partial resistance to posaconazole as well as to methyl-ß-cyclodextrin, corroborating the effect of posaconazole on cholesterol and viral entry. Our results indicate that posaconazole inhibits multiple steps of the alphavirus replication cycle and broaden the spectrum of viruses that can be targeted in vitro by posaconazole, which could be further explored as a therapeutic agent against emerging viruses.
Asunto(s)
Alphavirus/efectos de los fármacos , Antivirales/farmacología , Reposicionamiento de Medicamentos/métodos , Triazoles/farmacología , Replicación Viral/efectos de los fármacos , Alphavirus/clasificación , Animales , Línea Celular , Virus Chikungunya/efectos de los fármacos , Chlorocebus aethiops , Cricetinae , Endocitosis/efectos de los fármacos , Humanos , Virus de los Bosques Semliki/efectos de los fármacos , Virus Sindbis/efectos de los fármacos , Células Vero , Internalización del Virus/efectos de los fármacosRESUMEN
Knowledge about contagiousness is key to accurate management of hospitalized COVID-19 patients. Epidemiological studies suggest that in addition to transmission through droplets, aerogenic SARS-CoV-2 transmission contributes to the spread of infection. However, the presence of virus in exhaled air has not yet been sufficiently demonstrated. In pandemic situations low tech disposable and user-friendly bedside devices are required, while commercially available samplers are unsuitable for application in patients with respiratory distress. We included 49 hospitalized COVID-19 patients and used a disposable modular breath sampler to measure SARS-CoV-2 RNA load in exhaled air samples and compared these to SARS-CoV-2 RNA load of combined nasopharyngeal throat swabs and saliva. Exhaled air sampling using the modular breath sampler has proven feasible in a clinical COVID-19 setting and demonstrated viral detection in 25% of the patients.
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COVID-19 , ARN Viral , COVID-19/diagnóstico , Humanos , Nasofaringe , Faringe , ARN Viral/genética , SARS-CoV-2/genéticaRESUMEN
Kidney failure is frequently observed during and after COVID-19, but it remains elusive whether this is a direct effect of the virus. Here, we report that SARS-CoV-2 directly infects kidney cells and is associated with increased tubule-interstitial kidney fibrosis in patient autopsy samples. To study direct effects of the virus on the kidney independent of systemic effects of COVID-19, we infected human-induced pluripotent stem-cell-derived kidney organoids with SARS-CoV-2. Single-cell RNA sequencing indicated injury and dedifferentiation of infected cells with activation of profibrotic signaling pathways. Importantly, SARS-CoV-2 infection also led to increased collagen 1 protein expression in organoids. A SARS-CoV-2 protease inhibitor was able to ameliorate the infection of kidney cells by SARS-CoV-2. Our results suggest that SARS-CoV-2 can directly infect kidney cells and induce cell injury with subsequent fibrosis. These data could explain both acute kidney injury in COVID-19 patients and the development of chronic kidney disease in long COVID.
Asunto(s)
COVID-19 , SARS-CoV-2 , COVID-19/complicaciones , Fibrosis , Humanos , Riñón , Organoides/patología , Síndrome Post Agudo de COVID-19RESUMEN
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged as a new human pathogen in late 2019 and it has infected over 100 million people in less than a year. There is a clear need for effective antiviral drugs to complement current preventive measures, including vaccines. In this study, we demonstrate that berberine and obatoclax, two broad-spectrum antiviral compounds, are effective against multiple isolates of SARS-CoV-2. Berberine, a plant-derived alkaloid, inhibited SARS-CoV-2 at low micromolar concentrations and obatoclax, which was originally developed as an anti-apoptotic protein antagonist, was effective at sub-micromolar concentrations. Time-of-addition studies indicated that berberine acts on the late stage of the viral life cycle. In agreement, berberine mildly affected viral RNA synthesis, but it strongly reduced infectious viral titers, leading to an increase in the particle-to-pfu ratio. In contrast, obatoclax acted at the early stage of the infection, which is in line with its activity to neutralize the acidic environment in endosomes. We assessed infection of primary human nasal epithelial cells that were cultured on an air-liquid interface and found that SARS-CoV-2 infection induced and repressed expression of specific sets of cytokines and chemokines. Moreover, both obatoclax and berberine inhibited SARS-CoV-2 replication in these primary target cells. We propose berberine and obatoclax as potential antiviral drugs against SARS-CoV-2 that could be considered for further efficacy testing.
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Antivirales/farmacología , Berberina/farmacología , Indoles/farmacología , Pirroles/farmacología , SARS-CoV-2/efectos de los fármacos , Replicación Viral/efectos de los fármacos , Adolescente , Animales , COVID-19/virología , Células Cultivadas , Chlorocebus aethiops , Células Epiteliales/virología , Humanos , Masculino , ARN Viral/genética , SARS-CoV-2/fisiología , Células VeroRESUMEN
Neutrophil extracellular traps (NETs) are increasingly recognized to play a role in the pathogenesis of viral infections, including dengue. NETs can be formed NADPH oxidase (NOX)-dependently or NOX-independently. NOX-independent NETs can be induced by activated platelets and are very potent in activating the endothelium. Platelet activation with thrombocytopenia and endothelial dysfunction are prominent features of dengue virus infection. We postulated that dengue infection is associated with NOX-independent NET formation, which is related to platelet activation, endothelial perturbation and increased vascular permeability. Using our specific NET assays, we investigated the time course of NET formation in a cohort of Indonesian dengue patients. We found that plasma levels of NETs were profoundly elevated and that these NETs were predominantly NOX-independent NETs. During early recovery phase (7-13 days from fever onset), total NETs correlated negatively with platelet number and positively with platelet P-selectin expression, the binding of von Willebrand factor to platelets and levels of Syndecan-1. Patients with gall bladder wall thickening, an early marker of plasma leakage, had a higher median level of total NETs. Ex vivo, platelets induced NOX-independent NET formation in a dengue virus non-structural protein 1 (NS1)-dependent manner. We conclude that NOX-independent NET formation is enhanced in dengue, which is most likely mediated by NS1 and activated platelets.
Asunto(s)
Plaquetas/metabolismo , Virus del Dengue/patogenicidad , Dengue/enzimología , Trampas Extracelulares/metabolismo , NADPH Oxidasas/metabolismo , Neutrófilos/enzimología , Activación Plaquetaria , Adolescente , Adulto , Plaquetas/inmunología , Plaquetas/virología , Estudios de Casos y Controles , Células Cultivadas , Dengue/sangre , Dengue/inmunología , Dengue/virología , Virus del Dengue/inmunología , Virus del Dengue/metabolismo , Trampas Extracelulares/virología , Femenino , Interacciones Huésped-Patógeno , Humanos , Indonesia , Masculino , Neutrófilos/inmunología , Neutrófilos/virología , Estudios Prospectivos , Proteínas no Estructurales Virales/metabolismo , Adulto JovenRESUMEN
Background: Endothelial hyper-permeability with plasma leakage and thrombocytopenia are predominant features of severe dengue virus infection. It is well established that heparanase, the endothelial glycocalyx degrading enzyme, plays a major role in various diseases with vascular leakage. It is yet to be elucidated whether heparanase activity plays a major role in dengue-associated plasma leakage. Moreover, the major source of heparanase secretion and activation in dengue remains elusive. Since a relatively high amount of heparanase is stored in platelets, we postulate that heparanase released by activated platelets contributes to the increased plasma heparanase activity during dengue virus infection. Methods: Heparanase activity (plasma and urine), and heparan sulfate and syndecan-1 (plasma levels) were measured in dengue patients with thrombocytopenia in acute phase (n=30), during course of disease (n=10) and in convalescent phase (n=25). Associations with clinical parameters and plasma leakage markers were explored. Platelets from healthy donors were stimulated with dengue non-structural protein-1, DENV2 virus and thrombin to evaluate heparanase release and activity ex vivo. Results: Heparanase activity was elevated in acute dengue and normalized during convalescence. Similarly, glycocalyx components, such as heparan sulfate and syndecan-1, were increased in acute dengue and restored during convalescence. Increased heparanase activity correlated with the endothelial dysfunction markers heparan sulfate and syndecan-1, as well as clinical markers of plasma leakage such as ascites, hematocrit concentration and gall-bladder wall thickening. Notably, platelet number inversely correlated with heparanase activity. Ex vivo incubation of platelets with thrombin and live DENV2 virus, but not dengue virus-2-derived non-structural protein 1 induced heparanase release from platelets. Conclusion: Taken together, our findings suggest that the increase of heparanase activity in dengue patients is associated with endothelial glycocalyx degradation and plasma leakage. Furthermore, thrombin or DENV2 activated platelets may be considered as a potential source of heparanase.