RESUMEN
CRISPR-Cas systems are host-encoded pathways that protect microbes from viral infection using an adaptive RNA-guided mechanism. Using genome-resolved metagenomics, we find that CRISPR systems are also encoded in diverse bacteriophages, where they occur as divergent and hypercompact anti-viral systems. Bacteriophage-encoded CRISPR systems belong to all six known CRISPR-Cas types, though some lack crucial components, suggesting alternate functional roles or host complementation. We describe multiple new Cas9-like proteins and 44 families related to type V CRISPR-Cas systems, including the Casλ RNA-guided nuclease family. Among the most divergent of the new enzymes identified, Casλ recognizes double-stranded DNA using a uniquely structured CRISPR RNA (crRNA). The Casλ-RNA-DNA structure determined by cryoelectron microscopy reveals a compact bilobed architecture capable of inducing genome editing in mammalian, Arabidopsis, and hexaploid wheat cells. These findings reveal a new source of CRISPR-Cas enzymes in phages and highlight their value as genome editors in plant and human cells.
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Bacteriófagos , Sistemas CRISPR-Cas , Animales , Humanos , Microscopía por Crioelectrón , Edición Génica , Genoma , Bacteriófagos/genética , ADN , ARN , Mamíferos/genéticaRESUMEN
SARS-CoV-2 infection can cause severe respiratory COVID-19. However, many individuals present with isolated upper respiratory symptoms, suggesting potential to constrain viral pathology to the nasopharynx. Which cells SARS-CoV-2 primarily targets and how infection influences the respiratory epithelium remains incompletely understood. We performed scRNA-seq on nasopharyngeal swabs from 58 healthy and COVID-19 participants. During COVID-19, we observe expansion of secretory, loss of ciliated, and epithelial cell repopulation via deuterosomal cell expansion. In mild and moderate COVID-19, epithelial cells express anti-viral/interferon-responsive genes, while cells in severe COVID-19 have muted anti-viral responses despite equivalent viral loads. SARS-CoV-2 RNA+ host-target cells are highly heterogenous, including developing ciliated, interferon-responsive ciliated, AZGP1high goblet, and KRT13+ "hillock"-like cells, and we identify genes associated with susceptibility, resistance, or infection response. Our study defines protective and detrimental responses to SARS-CoV-2, the direct viral targets of infection, and suggests that failed nasal epithelial anti-viral immunity may underlie and precede severe COVID-19.
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COVID-19/inmunología , COVID-19/virología , Inmunidad , SARS-CoV-2/fisiología , Índice de Severidad de la Enfermedad , Adulto , Anciano , Efecto Espectador , COVID-19/genética , Estudios de Cohortes , Femenino , Humanos , Masculino , Persona de Mediana Edad , Nasofaringe/patología , Nasofaringe/virología , ARN Viral/análisis , ARN Viral/genética , Mucosa Respiratoria/patología , Mucosa Respiratoria/virología , Transcripción Genética , Carga ViralRESUMEN
Activation of the cyclic guanosine monophosphate (GMP)-AMP (cGAMP) sensor STING requires its translocation from the endoplasmic reticulum to the Golgi apparatus and subsequent polymerization. Using a genome-wide CRISPR-Cas9 screen to define factors critical for STING activation in cells, we identified proteins critical for biosynthesis of sulfated glycosaminoglycans (sGAGs) in the Golgi apparatus. Binding of sGAGs promoted STING polymerization through luminal, positively charged, polar residues. These residues are evolutionarily conserved, and selective mutation of specific residues inhibited STING activation. Purified or chemically synthesized sGAGs induced STING polymerization and activation of the kinase TBK1. The chain length and O-linked sulfation of sGAGs directly affected the level of STING polymerization and, therefore, its activation. Reducing the expression of Slc35b2 to inhibit GAG sulfation in mice impaired responses to vaccinia virus infection. Thus, sGAGs in the Golgi apparatus are necessary and sufficient to drive STING polymerization, providing a mechanistic understanding of the requirement for endoplasmic reticulum (ER)-to-Golgi apparatus translocation for STING activation.
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Glicosaminoglicanos/metabolismo , Aparato de Golgi/metabolismo , Proteínas de la Membrana/metabolismo , Nucleótidos Cíclicos/metabolismo , Animales , Células COS , Línea Celular , Línea Celular Tumoral , Chlorocebus aethiops , Cricetinae , Citosol/metabolismo , Retículo Endoplásmico/metabolismo , Células HeLa , Humanos , Ratones , Polimerizacion , Transducción de Señal/fisiología , Transportadores de Sulfato/metabolismo , Vaccinia/metabolismo , Virus Vaccinia/patogenicidadRESUMEN
Conventional dendritic cells type 1 (cDC1) are critical for inducing protective CD8+ T cell responses to tumour and viral antigens. In many instances, cDC1 access those antigens in the form of material internalised from dying tumour or virally-infected cells. How cDC1 extract dead cell-associated antigens and cross-present them in the form of peptides bound to MHC class I molecules to CD8+ T cells remains unclear. Here we review the biology of dendritic cell natural killer group receptor-1 (DNGR-1; also known as CLEC9A), a C-type lectin receptor highly expressed on cDC1 that plays a key role in this process. We highlight recent advances that support a function for DNGR-1 signalling in promoting inducible rupture of phagocytic or endocytic compartments containing dead cell debris, thereby making dead cell-associated antigens accessible to the endogenous MHC class I processing and presentation machinery of cDC1. We further review how DNGR-1 detects dead cells, as well as the functions of the receptor in anti-viral and anti-tumour immunity. Finally, we highlight how the study of DNGR-1 has opened new perspectives into cross-presentation, some of which may have applications in immunotherapy of cancer and vaccination against viral diseases.
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Reactividad Cruzada , Neoplasias , Humanos , Linfocitos T CD8-positivos , Receptores Inmunológicos , Antígenos/metabolismo , Células Dendríticas , Neoplasias/metabolismoRESUMEN
To define cellular immunity to the intracellular pathogen Toxoplasma gondii, we performed a genome-wide CRISPR loss-of-function screen to identify genes important for (interferon gamma) IFN-γ-dependent growth restriction. We revealed a role for the tumor suppressor NF2/Merlin for maximum induction of Interferon Stimulated Genes (ISG), which are positively regulated by the transcription factor IRF-1. We then performed an ISG-targeted CRISPR screen that identified the host E3 ubiquitin ligase RNF213 as necessary for IFN-γ-mediated control of T. gondii in multiple human cell types. RNF213 was also important for control of bacterial (Mycobacterium tuberculosis) and viral (Vesicular Stomatitis Virus) pathogens in human cells. RNF213-mediated ubiquitination of the parasitophorous vacuole membrane (PVM) led to growth restriction of T. gondii in response to IFN-γ. Moreover, overexpression of RNF213 in naive cells also impaired growth of T. gondii. Surprisingly, growth inhibition did not require the autophagy protein ATG5, indicating that RNF213 initiates restriction independent of a previously described noncanonical autophagy pathway. Mutational analysis revealed that the ATPase domain of RNF213 was required for its recruitment to the PVM, while loss of a critical histidine in the RZ finger domain resulted in partial reduction of recruitment to the PVM and complete loss of ubiquitination. Both RNF213 mutants lost the ability to restrict growth of T. gondii, indicating that both recruitment and ubiquitination are required. Collectively, our findings establish RNF213 as a critical component of cell-autonomous immunity that is both necessary and sufficient for control of intracellular pathogens in human cells.
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Toxoplasma , Toxoplasmosis , Humanos , Interferón gamma/metabolismo , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Toxoplasma/metabolismo , Factores de Transcripción , Adenosina Trifosfatasas/metabolismo , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/metabolismoRESUMEN
Human coronavirus (hCoV) OC43 is endemic to global populations and usually causes asymptomatic or mild upper respiratory tract illness. Here, we demonstrate the neutralization efficacy of isolated nanobodies from alpacas immunized with the S1B and S1C domain of the hCoV-OC43 spike glycoprotein. A total of 40 nanobodies bound to recombinant OC43 protein with affinities ranging from 1 to 149 nM. Two nanobodies WNb 293 and WNb 294 neutralized virus at 0.21 and 1.79 nM, respectively. Intranasal and intraperitoneal delivery of WNb 293 fused to an Fc domain significantly reduced nasal viral load in a mouse model of hCoV-OC43 infection. Using X-ray crystallography, we observed that WNb 293 bound to an epitope on the OC43 S1B domain, distal from the sialoglycan-binding site involved in host cell entry. This result suggests that neutralization mechanism of this nanobody does not involve disruption of glycan binding. Our work provides characterization of nanobodies against hCoV-OC43 that blocks virus entry and reduces viral loads in vivo and may contribute to future nanobody-based therapies for hCoV-OC43 infections. IMPORTANCE: The pandemic potential presented by coronaviruses has been demonstrated by the ongoing COVID-19 pandemic and previous epidemics caused by severe acute respiratory syndrome coronavirus and Middle East respiratory syndrome coronavirus. Outside of these major pathogenic coronaviruses, there are four endemic coronaviruses that infect humans: hCoV-OC43, hCoV-229E, hCoV-HKU1, and hCoV-NL63. We identified a collection of nanobodies against human coronavirus OC43 (hCoV-OC43) and found that two high-affinity nanobodies potently neutralized hCoV-OC43 at low nanomolar concentrations. Prophylactic administration of one neutralizing nanobody reduced viral loads in mice infected with hCoV-OC43, showing the potential for nanobody-based therapies for hCoV-OC43 infections.
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Anticuerpos Neutralizantes , Anticuerpos Antivirales , Camélidos del Nuevo Mundo , Infecciones por Coronavirus , Coronavirus Humano OC43 , Anticuerpos de Dominio Único , Glicoproteína de la Espiga del Coronavirus , Carga Viral , Animales , Anticuerpos de Dominio Único/inmunología , Ratones , Anticuerpos Neutralizantes/inmunología , Coronavirus Humano OC43/inmunología , Humanos , Anticuerpos Antivirales/inmunología , Camélidos del Nuevo Mundo/inmunología , Glicoproteína de la Espiga del Coronavirus/inmunología , Infecciones por Coronavirus/inmunología , Infecciones por Coronavirus/prevención & control , Infecciones por Coronavirus/virología , Femenino , Epítopos/inmunología , Cristalografía por Rayos X , Internalización del Virus/efectos de los fármacos , Modelos Animales de Enfermedad , Ratones Endogámicos BALB CRESUMEN
A severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection causes mild-to-severe respiratory symptoms, including acute respiratory distress. Despite remarkable efforts to investigate the virological and pathological impacts of SARS-CoV-2, many of the characteristics of SARS-CoV-2 infection still remain unknown. The interferon-inducible ubiquitin-like protein ISG15 is covalently conjugated to several viral proteins to suppress their functions. It was reported that SARS-CoV-2 utilizes its papain-like protease (PLpro) to impede ISG15 conjugation, ISGylation. However, the role of ISGylation in SARS-CoV-2 infection remains unclear. We aimed to elucidate the role of ISGylation in SARS-CoV-2 replication. We observed that the SARS-CoV-2 nucleocapsid protein is a target protein for the HERC5 E3 ligase-mediated ISGylation in cultured cells. Site-directed mutagenesis reveals that the residue K374 within the C-terminal spacer B-N3 (SB/N3) domain is required for nucleocapsid-ISGylation, alongside conserved lysine residue in MERS-CoV (K372) and SARS-CoV (K375). We also observed that the nucleocapsid-ISGylation results in the disruption of nucleocapsid oligomerization, thereby inhibiting viral replication. Knockdown of ISG15 mRNA enhanced SARS-CoV-2 replication in the SARS-CoV-2 reporter replicon cells, while exogenous expression of ISGylation components partially hampered SARS-CoV-2 replication. Taken together, these results suggest that SARS-CoV-2 PLpro inhibits ISGylation of the nucleocapsid protein to promote viral replication by evading ISGylation-mediated disruption of the nucleocapsid oligomerization.IMPORTANCEISG15 is an interferon-inducible ubiquitin-like protein that is covalently conjugated to the viral protein via specific Lys residues and suppresses viral functions and viral propagation in many viruses. However, the role of ISGylation in SARS-CoV-2 infection remains largely unclear. Here, we demonstrated that the SARS-CoV-2 nucleocapsid protein is a target protein for the HERC5 E3 ligase-mediated ISGylation. We also found that the residue K374 within the C-terminal spacer B-N3 (SB/N3) domain is required for nucleocapsid-ISGylation. We obtained evidence suggesting that nucleocapsid-ISGylation results in the disruption of nucleocapsid-oligomerization, thereby suppressing SARS-CoV-2 replication. We discovered that SARS-CoV-2 papain-like protease inhibits ISG15 conjugation of nucleocapsid protein via its de-conjugating enzyme activity. The present study may contribute to gaining new insight into the roles of ISGylation-mediated anti-viral function in SARS-CoV-2 infection and may lead to the development of more potent and selective inhibitors targeted to SARS-CoV-2 nucleocapsid protein.
RESUMEN
Many viruses, including mammarenaviruses, have evolved mechanisms to counteract different components of the host cell innate immunity, which is required to facilitate robust virus multiplication. The double-stranded RNA (dsRNA) sensor protein kinase receptor (PKR) pathway plays a critical role in the cell anti-viral response. Whether PKR can restrict the multiplication of the Old World mammarenavirus lymphocytic choriomeningitis virus (LCMV) and the mechanisms by which LCMV may counteract the anti-viral functions of PKR have not yet been investigated. Here we present evidence that LCMV infection results in very limited levels of PKR activation, but LCMV multiplication is enhanced in the absence of PKR. In contrast, infection with a recombinant LCMV with a mutation affecting the 3'-5' exonuclease (ExoN) activity of the viral nucleoprotein resulted in robust PKR activation in the absence of detectable levels of dsRNA, which was associated with severely restricted virus multiplication that was alleviated in the absence of PKR. However, pharmacological inhibition of PKR activation resulted in reduced levels of LCMV multiplication. These findings uncovered a complex role of the PKR pathway in LCMV-infected cells involving both pro- and anti-viral activities.IMPORTANCEAs with many other viruses, the prototypic Old World mammarenavirus LCMV can interfere with the host cell innate immune response to infection, which includes the dsRNA sensor PKR pathway. A detailed understanding of LCMV-PKR interactions can provide novel insights about mammarenavirus-host cell interactions and facilitate the development of effective anti-viral strategies against human pathogenic mammarenaviruses. In the present work, we present evidence that LCMV multiplication is enhanced in PKR-deficient cells, but pharmacological inhibition of PKR activation unexpectedly resulted in severely restricted propagation of LCMV. Likewise, we document a robust PKR activation in LCMV-infected cells in the absence of detectable levels of dsRNA. Our findings have revealed a complex role of the PKR pathway during LCMV infection and uncovered the activation of PKR as a druggable target for the development of anti-viral drugs against human pathogenic mammarenaviruses.
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Arenaviridae , Coriomeningitis Linfocítica , Humanos , Arenaviridae/metabolismo , Línea Celular , Proteínas Quinasas/metabolismo , Interacciones Huésped-Patógeno , Virus de la Coriomeningitis Linfocítica/metabolismo , Proteínas Portadoras , Antivirales , eIF-2 Quinasa/genética , eIF-2 Quinasa/metabolismoRESUMEN
T cells rapidly undergo contraction upon viral clearance, but how T cell function and fate are determined during this phase is unclear. During the contraction phase of an acute infection with lymphocytic choriomeningitis virus, we found that virus-specific CD8+ T cells within the splenic red pulp (RP) had higher two-dimensional (2D) effective affinity than those within the white pulp (WP). This increased antigen recognition of RP-derived CD8+ T cells correlated with more efficient target cell killing and improved control of viremia. FoxP3+ regulatory T cells and cytokine TGF-ß limited the 2D-affinity in the WP during the contraction phase. Anatomical location drove gene expression patterns in CD8+ T cells that led to preferential differentiation of memory precursor WP T cells into long-term memory cells. These results highlight that intricate regulation of T cell function and fate is determined by anatomic compartmentalization during the early immune contraction phase.
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Linfocitos T CD8-positivos/inmunología , Diferenciación Celular/inmunología , Memoria Inmunológica/inmunología , Coriomeningitis Linfocítica/inmunología , Receptores de Antígenos de Linfocitos T/inmunología , Bazo/inmunología , Animales , Separación Celular , Citocinas/inmunología , Modelos Animales de Enfermedad , Citometría de Flujo , Perfilación de la Expresión Génica , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , TranscriptomaRESUMEN
RIG-I is our first line of defense against RNA viruses, serving as a pattern recognition receptor that identifies molecular features common among dsRNA and ssRNA viral pathogens. RIG-I is maintained in an inactive conformation as it samples the cellular space for pathogenic RNAs. Upon encounter with the triphosphorylated terminus of blunt-ended viral RNA duplexes, the receptor changes conformation and releases a pair of signaling domains (CARDs) that are selectively modified and interact with an adapter protein (MAVS), thereby triggering a signaling cascade that stimulates transcription of interferons. Here, we describe the structural determinants for specific RIG-I activation by viral RNA, and we describe the strategies by which RIG-I remains inactivated in the presence of host RNAs. From the initial RNA triggering event to the final stages of interferon expression, we describe the experimental evidence underpinning our working knowledge of RIG-I signaling. We draw parallels with behavior of related proteins MDA5 and LGP2, describing evolutionary implications of their collective surveillance of the cell. We conclude by describing the cell biology and immunological investigations that will be needed to accurately describe the role of RIG-I in innate immunity and to provide the necessary foundation for pharmacological manipulation of this important receptor.
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ARN Helicasas DEAD-box , ARN Bicatenario , ARN Helicasas DEAD-box/genética , ARN Helicasas DEAD-box/metabolismo , Inmunidad Innata , Helicasa Inducida por Interferón IFIH1/genética , ARN Viral , Transducción de SeñalRESUMEN
KHNYN protein with a KH-like domain and a NYN endoribonuclease domain interacts with Zinc-finger antiviral protein (ZAP). ZAP isoforms recognize viral or cellular RNAs and recruit KHNYN to form the ZAP: KHNYN complex. Although the structures of several PIN/NYN domains have been determined, the precise substrate RNA binding mode remains poorly understood. This study presents the crystal structure of a complex of the NYN domain of KHNYN and a 7mer RNA from interferon lambda3 (IFNL3). Our structural analysis reveals that NYN domain of human KHNYN shares structural similarities with other NYN domains of ZC3H12Ã C proteins. The RNA is bound in the central groove region of the protein, facilitated by interactions including coordination by two Mg2+ ions, hydrophobic interactions, and hydrogen bonds. In the observed RNA-protein complex, the U5, A6, and U7 bases are stacked on top of one another, while U3 and U4 bases adopt an "open" conformation (as opposed to base-stacked), forming a U-shaped overall structure. Mutagenesis studies underscore the significance of residues involved in RNA binding for RNase activity. Interestingly, NYN domain of human KHNYN forms a head-to-tail dimer in the crystal, a structural feature also observed in other homologous PIN/NYN proteins, with a residue from the symmetry mate contributing to hydrophobic interactions with the bound RNA.
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IMPORTANCE: Porcine epidemic diarrhea caused by porcine coronaviruses remains a major threat to the global swine industry. Fatty acids are extensively involved in the whole life of the virus. In this study, we found that docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) significantly reduced the viral load of porcine epidemic diarrhea virus (PEDV), transmissible gastroenteritis virus (TGEV), and porcine delta coronavirus (PDCoV) and acted on the replication of the viruses rather than attachment and entry. We further confirmed that DHA and EPA inhibited PEDV replication by alleviating the endoplasmic reticulum stress. Meanwhile, DHA and EPA alleviate PEDV-induced inflammation and reactive oxygen species (ROS) levels and enhance the cellular antioxidant capacity. These data indicate that DHA and EPA have antiviral effects on porcine coronaviruses and provide a molecular basis for the development of new fatty acid-based therapies to control porcine coronavirus infection and transmission.
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Infecciones por Coronavirus , Coronavirus , Ácidos Docosahexaenoicos , Ácido Eicosapentaenoico , Enfermedades de los Porcinos , Animales , Coronavirus/fisiología , Infecciones por Coronavirus/tratamiento farmacológico , Infecciones por Coronavirus/veterinaria , Ácidos Docosahexaenoicos/farmacología , Ácido Eicosapentaenoico/farmacología , Virus de la Diarrea Epidémica Porcina/fisiología , Porcinos , Enfermedades de los Porcinos/tratamiento farmacológico , Virus de la Gastroenteritis Transmisible/fisiología , Replicación Viral/efectos de los fármacos , Estrés del Retículo Endoplásmico/efectos de los fármacosRESUMEN
CD8 T cells recognize infected and cancerous cells via their T-cell receptor (TCR), which binds peptide-MHC complexes on the target cell. The affinity of the interaction between the TCR and peptide-MHC contributes to the antigen sensitivity, or functional avidity, of the CD8 T cell. In response to peptide-MHC stimulation, the TCR-CD3 complex and CD8 co-receptor are downmodulated. We quantified CD3 and CD8 downmodulation following stimulation of human CD8 T cells with CMV, EBV, and HIV peptides spanning eight MHC restrictions, observing a strong correlation between the levels of CD3 and CD8 downmodulation and functional avidity, regardless of peptide viral origin. In TCR-transduced T cells targeting a tumor-associated antigen, changes in TCR-peptide affinity were sufficient to modify CD3 and CD8 downmodulation. Correlation analysis and generalized linear modeling indicated that CD3 downmodulation was the stronger correlate of avidity. CD3 downmodulation, simply measured using flow cytometry, can be used to identify high-avidity CD8 T cells in a clinical context.
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Linfocitos T CD8-positivos , Receptores de Antígenos de Linfocitos T , Humanos , Regulación hacia Abajo , Receptores de Antígenos de Linfocitos T/genética , Antígenos CD8/metabolismo , Péptidos/metabolismo , Complejo CD3/metabolismoRESUMEN
Anti-viral surface coatings are under development to prevent viral fomite transmission from high-traffic touch surfaces in public spaces. Copper's anti-viral properties have been widely documented, but the anti-viral mechanism of copper surfaces is not fully understood. We screened a series of metal and metal oxide surfaces for anti-viral activity against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of coronavirus disease (COVID-19). Copper and copper oxide surfaces exhibited superior anti-SARS-CoV-2 activity; however, the level of anti-viral activity was dependent on the composition of the carrier solution used to deliver virus inoculum. We demonstrate that copper ions released into solution from test surfaces can mediate virus inactivation, indicating a copper ion dissolution-dependent anti-viral mechanism. The level of anti-viral activity is, however, not dependent on the amount of copper ions released into solution per se. Instead, our findings suggest that degree of virus inactivation is dependent on copper ion complexation with other biomolecules (e.g., proteins/metabolites) in the virus carrier solution that compete with viral components. Although using tissue culture-derived virus inoculum is experimentally convenient to evaluate the anti-viral activity of copper-derived test surfaces, we propose that the high organic content of tissue culture medium reduces the availability of "uncomplexed" copper ions to interact with the virus, negatively affecting virus inactivation and hence surface anti-viral performance. We propose that laboratory anti-viral surface testing should include virus delivered in a physiologically relevant carrier solution (saliva or nasal secretions when testing respiratory viruses) to accurately predict real-life surface anti-viral performance when deployed in public spaces.IMPORTANCEThe purpose of evaluating the anti-viral activity of test surfaces in the laboratory is to identify surfaces that will perform efficiently in preventing fomite transmission when deployed on high-traffic touch surfaces in public spaces. The conventional method in laboratory testing is to use tissue culture-derived virus inoculum; however, this study demonstrates that anti-viral performance of test copper-containing surfaces is dependent on the composition of the carrier solution in which the virus inoculum is delivered to test surfaces. Therefore, we recommend that laboratory surface testing should include virus delivered in a physiologically relevant carrier solution to accurately predict real-life test surface performance in public spaces. Understanding the mechanism of virus inactivation is key to future rational design of improved anti-viral surfaces. Here, we demonstrate that release of copper ions from copper surfaces into small liquid droplets containing SARS-CoV-2 is a mechanism by which the virus that causes COVID-19 can be inactivated.
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COVID-19 , SARS-CoV-2 , Humanos , Cobre/farmacología , Antivirales , Óxidos , IonesRESUMEN
BACKGROUND: Despite increasing evidence that Epstein-Barr virus (EBV) plays a causal role in MS, no treatments have been shown to reduce EBV turnover. We studied the effect of famciclovir on salivary EBV shedding in people with MS (NCT05283551) in a pilot, proof-of-concept study. METHODS: People with MS receiving natalizumab provided weekly saliva samples for 12 weeks before starting famciclovir 500 mg twice daily for 12 weeks. Twelve saliva samples were provided on treatment and 12 following treatment. A real-time qPCR Taqman assay was used to detect EBV DNA in saliva. The proportion of saliva samples containing EBV DNA was compared using the Friedman test. RESULTS: Of 30 participants (19 F; mean age 41 years; median EDSS 3.5), 29 received famciclovir, and 24 completed the 12-week course. Twenty-one participants provided at least one usable saliva sample in all epochs. Ten of the 21 had shedding in at least one sample pre-drug; 7/21 when taking famciclovir (not significant). No difference in EBV DNA copy number was seen. There were no drug-related serious adverse events. CONCLUSION: No significant effect of famciclovir on EBV shedding was seen in this small pilot study. Given the low numbers, a small effect of famciclovir cannot be excluded. Salivary EBV shedding in this natalizumab-treated cohort was lower than in previous studies, which requires replication.
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Infecciones por Virus de Epstein-Barr , Esclerosis Múltiple , Humanos , Adulto , Herpesvirus Humano 4 , Esclerosis Múltiple/tratamiento farmacológico , Infecciones por Virus de Epstein-Barr/complicaciones , Infecciones por Virus de Epstein-Barr/tratamiento farmacológico , Famciclovir , Saliva , Natalizumab , Proyectos Piloto , ADN , ADN Viral/análisisRESUMEN
BACKGROUND & AIM: To evaluate the risk of early hepatocellular carcinoma (HCC) in chronic hepatitis C patients treated with direct-acting antivirals (DAAs) in Hong Kong, as it has not been studied before in this locality. METHODS: Three hundred thirty-three consecutive chronic hepatitis C patients treated with DAAs from two hospitals over the past 6 years were identified. Kaplan-Meier method was used to calculate cumulative HCC incidence. Cox regression was used to identify factors associated with HCC development. RESULTS: During a median follow-up of 23.4 months after DAA started, 15 (5.4%, 95% CI 3.3-8.7%) out of 279 total included patients developed HCC. The overall sustained virological response (SVR) rate was 98.9%. The 1-year cumulative incidence for de-novo HCC and HCC recurrence were 0.8 and 30.9%, respectively (log-rank test p < 0.001). The 1-year cumulative HCC incidence for patients without and with cirrhosis were 0.7 and 5.1%, respectively (log-rank test p = 0.036). Univariate analysis showed that significant factors associated with HCC after DAA were: history of treated HCC, cirrhosis, evidence of portal hypertension, higher AFP at the start or end of DAA therapy, higher bilirubin, lower platelets, lower albumin, and older age. From receiver operating characteristic curve analysis, the optimal cut-off level of AFP for predicting HCC was 10.5 ng/mL at the start and 5.6 ng/mL at the end of DAA therapy. CONCLUSIONS: The risk of early HCC recurrence remains high despite achieving SVR following DAA therapy, whereas the risk of early de-novo HCC occurence is low. AFP levels, both at the start and end of DAA therapy, can be useful in stratifying risks of HCC development.
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Carcinoma Hepatocelular , Hepatitis C Crónica , Neoplasias Hepáticas , Humanos , Carcinoma Hepatocelular/epidemiología , Carcinoma Hepatocelular/etiología , Carcinoma Hepatocelular/patología , Antivirales/uso terapéutico , Hepatitis C Crónica/complicaciones , Hepatitis C Crónica/tratamiento farmacológico , Hepatitis C Crónica/epidemiología , Neoplasias Hepáticas/epidemiología , Neoplasias Hepáticas/etiología , Neoplasias Hepáticas/patología , alfa-Fetoproteínas/análisis , Hong Kong/epidemiología , Cirrosis Hepática/complicaciones , Fibrosis , Respuesta Virológica SostenidaRESUMEN
Japanese encephalitis (JE) is a vector-borne neurotropic disease caused by Japanese encephalitis virus (JEV) associated with high mortality rate distributed from Eastern and Southern Asia to Northern Queensland (Australia). The challenges in early detection and lack of point-of-care biomarkers make it the most important Flavivirus causing encephalitis. There is no specific treatment for the disease, although vaccines are licenced. In this review, we focussed on point-of-care biomarkers as early detection tools and developing the effective therapeutic agents that could halt JE. We have also provided molecular details of JEV, disease progression, and its pathogenesis with recent findings which might bring insights to overcome the disease burden.
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Virus de la Encefalitis Japonesa (Especie) , Encefalitis Japonesa , Animales , Humanos , Encefalitis Japonesa/diagnóstico , Encefalitis Japonesa/epidemiología , Encefalitis Japonesa/prevención & control , Virus de la Encefalitis Japonesa (Especie)/genética , Zoonosis/diagnóstico , Zoonosis/epidemiología , Sur de AsiaRESUMEN
AIM: The high incidence of virus-related infections and the large diffusion of drug-resistant pathogens stimulate the search and identification of new antiviral agents with a broad spectrum of action. Antivirals can be designed to act on a single target by interfering with a specific step in the viral lifecycle. On the contrary, antiviral peptides (AVPs) are known for acting on a wide range of viruses, with a diversified mechanism of action targeting virus and/or host cell. In the present study, we evaluated the antiviral potential of the peptide Hylin-a1 secreted by the frog Hypsiobas albopunctatus against members of the Herpesviridae family. METHODS AND RESULTS: The inhibitory capacity of the peptide was evaluated in vitro by plaque assays in order to understand the possible mechanism of action. The results were also confirmed by real-time PCR and Western blot evaluating the expression of viral genes. Hylin-a1 acts to block the herpetic infection interfering at the early stages of both herpes simplex virus type 1 (HSV-1) and type 2 infection. Its mechanism is mainly directed on the membrane, probably by damaging the viral envelope. The same effect was also observed against HSV-1 strains resistant to acyclovir. CONCLUSIONS: The data presented in this study, such as the increased activity of the peptide when combined to acyclovir, a weak hemolytic profile, an anti-inflammatory effect, and a tolerable half-life in serum, indicates Hylin-a1 as a novel antiherpetic molecule with promising potential in the clinical setting.
Asunto(s)
Antiinflamatorios , Antivirales , Anuros , Animales , Antivirales/farmacología , Antiinflamatorios/farmacología , Herpesvirus Humano 1/efectos de los fármacos , Proteínas Anfibias/farmacología , Péptidos/farmacología , Células Vero , Chlorocebus aethiopsRESUMEN
In hemato-oncological patients, COVID-19 can present as a persistent infection with ongoing symptoms and viral replication over a prolonged period of time. Data are scarce on the preferred treatment options for these patients. We describe our experience with a five-day course of dual anti-viral treatment with remdesivir and nirmatrelvir/ritonavir for hemato-oncological immunocompromised patients with persistent COVID-19. Fifteen patients with a history of lymphoma, CLL, and MM were included. Eight were male, median age was 74. All patients had an immediate clinical and virological response. In 73 % of patients, PCR for SARS-CoV-2 became negative at the end of treatment and the rest had an increase in PCR cycle threshold (CT) values, with a median increase of 6 cycles. After a follow-up of three months, 60 % of patients remained in full clinical and virological remission. None required invasive mechanical ventilation or died. The side effects we observed, neutropenia, lactatemia and elevated transaminases, were mild and almost all transient in nature. We conclude that dual anti-viral treatment appears to be a valid treatment option for persistent COVID-19.
Asunto(s)
COVID-19 , Humanos , Masculino , Anciano , Femenino , COVID-19/complicaciones , SARS-CoV-2 , Pronóstico , Factores de Tiempo , Antivirales/efectos adversosRESUMEN
Influenza A virus (IAV) can cause infection and illness in a wide range of animals, including humans, poultry, and swine, and cause annual epidemics, resulting in thousands of deaths and millions of hospitalizations all over the world. Thus, there is an urgent need to develop novel anti-IAV drugs with high efficiency and low toxicity. In this study, the anti-IAV activity of a marine-derived compound mycophenolic acid methyl ester (MAE) was intensively investigated both in vitro and in vivo. The results showed that MAE inhibited the replication of different influenza A virus strains in vitro with low cytotoxicity. MAE can mainly block some steps of IAV infection post adsorption. MAE may also inhibit viral replication through activating the cellular Akt-mTOR-S6K pathway. Importantly, oral treatment of MAE can significantly ameliorate pneumonia symptoms and reduce pulmonary viral titers, as well as improving the survival rate of mice, and this was superior to the effect of oseltamivir. In summary, the marine compound MAE possesses anti-IAV effects both in vitro and in vivo, which merits further studies for its development into a novel anti-IAV drug in the future.