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Enterovirus A71 (EV-A71) causes Hand, Foot, and Mouth Disease and has been clinically associated with neurological complications. However, there is a lack of relevant models to elucidate the neuropathology of EV-A71 and its mechanism, as the current models mainly utilize animal models or immortalized cell lines. In this study, we established a human motor neuron model for EV-A71 infection. Single cell transcriptomics of a mixed neuronal population reveal higher viral RNA load in motor neurons, suggesting higher infectivity and replication of EV-A71 in motor neurons. The elevated RNA load in motor neurons correlates with the downregulation of ferritin-encoding genes. Subsequent analysis confirms that neurons infected with EV-A71 undergo ferroptosis, as evidenced by increased levels of labile Fe2+ and peroxidated lipids. Notably, the Fe2+ chelator Deferoxamine improves mitochondrial function and promotes survival of motor neurons by 40% after EV-A71 infection. These findings deepen understanding of the molecular pathogenesis of EV-A71 infection, providing insights which suggest that improving mitochondrial respiration and inhibition of ferroptosis can mitigate the impact of EV-A71 infection in the central nervous system.
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Enterovirus Humano A , Infecciones por Enterovirus , Ferroptosis , Neuronas Motoras , Ferroptosis/efectos de los fármacos , Humanos , Enterovirus Humano A/fisiología , Enterovirus Humano A/genética , Enterovirus Humano A/efectos de los fármacos , Neuronas Motoras/virología , Neuronas Motoras/metabolismo , Infecciones por Enterovirus/virología , Infecciones por Enterovirus/metabolismo , Replicación Viral , Mitocondrias/metabolismo , Deferoxamina/farmacología , Carga Viral , Hierro/metabolismo , Ferritinas/metabolismo , Ferritinas/genéticaRESUMEN
BACKGROUND: Enterovirus 71 (EV-A71) causes Hand, Foot and Mouth Disease (HFMD) in children and has been associated with neurological complications. The molecular mechanisms involved in EV-A71 pathogenesis have remained elusive. METHODS: A siRNA screen in EV-A71 infected-motor neurons was performed targeting 112 genes involved in intracellular membrane trafficking, followed by validation of the top four hits using deconvoluted siRNA. Downstream approaches including viral entry by-pass, intracellular viral genome quantification by qPCR, Western blot analyses, and Luciferase reporter assays allowed determine the stage of the infection cycle the top candidate, RAB11A was involved in. Proximity ligation assay, co-immunoprecipitation and multiplex confocal imaging were employed to study interactions between viral components and RAB11A. Dominant negative and constitutively active RAB11A constructs were used to determine the importance of the protein's GTPase activity during EV-A71 infection. Mass spectrometry and protein interaction analyses were employed for the identification of RAB11A's host interacting partners during infection. RESULTS: Small GTPase RAB11A was identified as a novel pro-viral host factor during EV-A71 infection. RAB11A and RAB11B isoforms were interchangeably exploited by strains from major EV-A71 genogroups and by Coxsackievirus A16, another major causative agent of HFMD. We showed that RAB11A was not involved in viral entry, IRES-mediated protein translation, viral genome replication, and virus exit. RAB11A co-localized with replication organelles where it interacted with structural and non-structural viral components. Over-expression of dominant negative (S25N; GDP-bound) and constitutively active (Q70L; GTP-bound) RAB11A mutants had no effect on EV-A71 infection outcome, ruling out RAB11A's involvement in intracellular trafficking of viral or host components. Instead, decreased ratio of intracellular mature viral particles to viral RNA copies and increased VP0:VP2 ratio in siRAB11-treated cells supported a role in provirion maturation hallmarked by VP0 cleavage into VP2 and VP4. Finally, chaperones, not trafficking and transporter proteins, were found to be RAB11A's top interacting partners during EV-A71 infection. Among which, CCT8 subunit from the chaperone complex TRiC/CCT was further validated and shown to interact with viral structural proteins specifically, representing yet another novel pro-viral host factor during EV-A71 infection. CONCLUSIONS: This study describes a novel, unconventional role for RAB11A during viral infection where it participates in the complex process of virus morphogenesis by recruiting essential chaperone proteins.
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Enterovirus Humano A , Proteínas de Unión al GTP rab , Proteínas de Unión al GTP rab/metabolismo , Proteínas de Unión al GTP rab/genética , Enterovirus Humano A/genética , Enterovirus Humano A/fisiología , Enterovirus Humano A/metabolismo , Humanos , Chaperonas Moleculares/metabolismo , Chaperonas Moleculares/genética , Replicación ViralRESUMEN
BACKGROUND: Due to human error, sample swapping in large cohort studies with heterogeneous data types (e.g., mix of Oxford Nanopore Technologies, Pacific Bioscience, Illumina data, etc.) remains a common issue plaguing large-scale studies. At present, all sample swapping detection methods require costly and unnecessary (e.g., if data are only used for genome assembly) alignment, positional sorting, and indexing of the data in order to compare similarly. As studies include more samples and new sequencing data types, robust quality control tools will become increasingly important. FINDINGS: The similarity between samples can be determined using indexed k-mer sequence variants. To increase statistical power, we use coverage information on variant sites, calculating similarity using a likelihood ratio-based test. Per sample error rate, and coverage bias (i.e., missing sites) can also be estimated with this information, which can be used to determine if a spatially indexed principal component analysis (PCA)-based prescreening method can be used, which can greatly speed up analysis by preventing exhaustive all-to-all comparisons. CONCLUSIONS: Because this tool processes raw data, is faster than alignment, and can be used on very low-coverage data, it can save an immense degree of computational resources in standard quality control (QC) pipelines. It is robust enough to be used on different sequencing data types, important in studies that leverage the strengths of different sequencing technologies. In addition to its primary use case of sample swap detection, this method also provides information useful in QC, such as error rate and coverage bias, as well as population-level PCA ancestry analysis visualization.
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Secuenciación de Nucleótidos de Alto Rendimiento , Análisis de Secuencia de ADN , Humanos , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Análisis de Secuencia de ADN/métodos , Programas Informáticos , Análisis de Componente Principal , Biología Computacional/métodos , AlgoritmosRESUMEN
Dengue virus (DENV) causes approximately 390 million dengue infections worldwide every year. There were 22,777 reported DENV infections in Tainan, Taiwan in 2015. In this study, we sequenced the C-prM-E genes from 45 DENV 2015 strains, and phylogenetic analysis based on C-prM-E genes revealed that all strains were classified as DENV serotype 2 Cosmopolitan genotype. Sequence analysis comparing different DENV-2 genotypes and Cosmopolitan DENV-2 sequences prior to 2015 showed a clade replacement event in the DENV-2 Cosmopolitan genotype. Additionally, a major substitution C-A314G (K73R) was found in the capsid region which may have contributed to the clade replacement event. Reverse genetics virus rgC-A314G (K73R) showed slower replication in BHK-21 and C6/36 cells compared to wildtype virus, as well as a decrease in NS1 production in BHK-21-infected cells. After a series of passaging, the C-A314G (K73R) mutation reverted to wildtype and was thus considered to be unstable. Next generation sequencing (NGS) of three sera collected from a single DENV2-infected patient at 1-, 2-, and 5-days post-admission was employed to examine the genetic diversity over-time and mutations that may work in conjunction with C-A314G (K73R). Results showed that the number of haplotypes decreased with time in the DENV-infected patient. On the fifth day after admission, two new haplotypes emerged, and a single non-synonymous NS4A-L115I mutation was identified. Therefore, we have identified a persistent mutation C-A314G (K73R) in all of the DENV-2 isolates, and during the course of an infection, a single new non-synonymous mutation in the NS4A region appears in the virus population within a single host. The C-A314G (K73R) thus may have played a role in the DENV-2 2015 outbreak while the NS4A-L115I may be advantageous during DENV infection within the host.
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Virus del Dengue , Dengue , Brotes de Enfermedades , Genotipo , Epidemiología Molecular , Filogenia , Virus del Dengue/genética , Virus del Dengue/clasificación , Dengue/epidemiología , Dengue/virología , Taiwán/epidemiología , Humanos , Mutación , Análisis Mutacional de ADN , Animales , Línea Celular , Variación GenéticaRESUMEN
Detection of cytosolic nucleic acids by pattern recognition receptors, including STING and RIG-I, leads to the activation of multiple signalling pathways that culminate in the production of type I interferons (IFNs) which are vital for host survival during virus infection. In addition to protective immune modulatory functions, type I IFNs are also associated with autoimmune diseases. Hence, it is important to elucidate the mechanisms that govern their expression. In this study, we identified a critical regulatory function of the DUSP4 phosphatase in innate immune signalling. We found that DUSP4 regulates the activation of TBK1 and ERK1/2 in a signalling complex containing DUSP4, TBK1, ERK1/2 and IRF3 to regulate the production of type I IFNs. Mice deficient in DUSP4 were more resistant to infections by both RNA and DNA viruses but more susceptible to malaria parasites. Therefore, our study establishes DUSP4 as a regulator of nucleic acid sensor signalling and sheds light on an important facet of the type I IFN regulatory system.
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Interferón Tipo I , Proteínas de la Membrana , Proteínas Tirosina Fosfatasas , Receptores de Superficie Celular , Proteínas Roundabout , Virosis , Animales , Ratones , Inmunidad Innata , Interferón Tipo I/metabolismo , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Transducción de Señal , Virosis/inmunología , Virosis/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas Roundabout/metabolismo , Proteínas Tirosina Fosfatasas/metabolismo , Receptores de Superficie Celular/metabolismoRESUMEN
Viruses are a key component of the colon microbiome, but the relationship between virome and colorectal cancer (CRC) remains poorly understood. We seek to identify alterations in the viral community that is characteristic of CRC and examine if they persist after surgery. Forty-nine fecal samples from 25 non-cancer (NC) individuals and 12 CRC patients, before and 6-months after surgery, were collected for metagenomic analysis. The fecal virome of CRC patients demonstrated an increased network connectivity as compared to NC individuals. Co-exclusion of influential viruses to bacterial species associated with healthy gut status was observed in CRC, suggesting an altered virome induced a change in the healthy gut bacteriome. Network analysis revealed lower connectivity within the virome and trans-kingdom interactions in NC. After surgery, the number of strong correlations decreased for trans-kingdom and within the bacteria and virome networks, indicating lower connectivity within the microbiome. Some co-occurrence patterns between dominant viruses and bacteria were also lost after surgery, suggesting a possible return to the healthy state of gut microbiome. Microbial signatures characteristic of CRC include an altered virome besides an altered bacterial composition. Elevated viral correlations and network connectivity were observed in CRC patients relative to healthy individuals, alongside distinct changes in the cross-kingdom correlation network unique to CRC patients. Some patterns of dysbiosis persist after surgery. Future studies should seek to verify if dysbiosis truly persists after surgery in a larger sample size with microbiome data collected at various time points after surgery to explore if there is field-change in the remaining colon, as well as to examine if persistent dysbiosis correlates with patient outcomes.
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Neoplasias Colorrectales , Microbiota , Virus , Humanos , Viroma , Disbiosis/microbiología , Neoplasias Colorrectales/cirugía , Neoplasias Colorrectales/microbiologíaRESUMEN
BACKGROUND: Chikungunya virus (CHIKV) has reemerged as a major public health concern, causing chikungunya fever with increasing cases and neurological complications. METHODS: In the present study, we investigated a low-passage human isolate of the East/ Central/South African (ECSA) lineage of CHIKV strain LK(EH)CH6708, which exhibited a mix of small and large viral plaques. The small and large plaque variants were isolated and designated as CHIKV-SP and CHIKV-BP, respectively. CHIKV-SP and CHIKV-BP were characterized in vitro and in vivo to compare their virus production and virulence. Additionally, whole viral genome analysis and reverse genetics were employed to identify genomic virulence factors. RESULTS: CHIKV-SP demonstrated lower virus production in mammalian cells and attenuated virulence in a murine model. On the other hand, CHIKV-BP induced higher pro-inflammatory cytokine levels, compromised the integrity of the blood-brain barrier, and led to astrocyte infection in mouse brains. Furthermore, the CHIKV-SP variant had limited transmission potential in Aedes albopictus mosquitoes, likely due to restricted dissemination. Whole viral genome analysis revealed multiple genetic mutations in the CHIKV-SP variant, including a Glycine (G) to Arginine (R) mutation at position 55 in the viral E2 glycoprotein. Reverse genetics experiments confirmed that the E2-G55R mutation alone was sufficient to reduce virus production in vitro and virulence in mice. CONCLUSIONS: These findings highlight the attenuating effects of the E2-G55R mutation on CHIKV pathogenicity and neurovirulence and emphasize the importance of monitoring this mutation in natural infections.
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Aedes , Virus Chikungunya , Humanos , Ratones , Animales , Virus Chikungunya/genética , Proteínas del Envoltorio Viral/genética , Proteínas del Envoltorio Viral/metabolismo , Aminoácidos , Mutación , MamíferosRESUMEN
The infectious clone has been constructed for years via various mechanisms using reverse genetics of viral RNA into cDNA. The mechanism of construction has evolved to DNA-launch plasmids which simplify infectious clone manipulation and expression in mammalian cells. Infectious clones have enormously allowed manipulation of the enterovirus genome to discover antivirals, viral replication mechanisms, and functions of essential viral proteins. Here we will be discussing methods for the production of DNA-launch human enterovirus infectious clones and viral genome engineered with a fluorescent reporter gene.
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Enterovirus , Humanos , Células Clonales , Clonación Molecular , ADN Complementario/genética , Enterovirus/genética , Plásmidos/genética , Genética Inversa , ARN Viral/genéticaRESUMEN
Satellite DNA are long tandemly repeating sequences in a genome and may be organized as high-order repeats (HORs). They are enriched in centromeres and are challenging to assemble. Existing algorithms for identifying satellite repeats either require the complete assembly of satellites or only work for simple repeat structures without HORs. Here we describe Satellite Repeat Finder (SRF), a new algorithm for reconstructing satellite repeat units and HORs from accurate reads or assemblies without prior knowledge on repeat structures. Applying SRF to real sequence data, we show that SRF could reconstruct known satellites in human and well-studied model organisms. We also find satellite repeats are pervasive in various other species, accounting for up to 12% of their genome contents but are often underrepresented in assemblies. With the rapid progress in genome sequencing, SRF will help the annotation of new genomes and the study of satellite DNA evolution even if such repeats are not fully assembled.
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With approximately 3.8 billion people at risk of infection in tropical and sub-tropical regions, Dengue ranks among the top ten threats worldwide. Despite the potential for severe disease manifestation and the economic burden it places on endemic countries, there is a lack of approved antiviral agents to effectively treat the infection. Flavonoids, including baicalein, have garnered attention for their antimicrobial properties. In this study, we took a rational and iterative approach to develop a series of baicalein derivatives with improved antiviral activity against Dengue virus (DENV). Compound 11064 emerged as a promising lead candidate, exhibiting antiviral activity against the four DENV serotypes and representative strains of Zika virus (ZIKV) in vitro, with attractive selectivity indices. Mechanistic studies revealed that Compound 11064 did not prevent DENV attachment at the cell surface, nor viral RNA synthesis and viral protein translation. Instead, the drug was found to impair the post-receptor binding entry steps (endocytosis and/or uncoating), as well as the late stage of DENV infection cycle, including virus assembly/maturation and/or exocytosis. The inability to raise DENV resistant mutants, combined with significant antiviral activity against an unrelated RNA virus (Enterovirus-A71) suggested that Compound 11064 targets the host rather than a viral protein, further supporting its broad-spectrum antiviral potential. Overall, Compound 11064 represents a promising antiviral candidate for the treatment of Dengue and Zika.
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Virus del Dengue , Dengue , Flavivirus , Infección por el Virus Zika , Virus Zika , Humanos , Infección por el Virus Zika/tratamiento farmacológico , Antivirales/uso terapéutico , Dengue/tratamiento farmacológicoRESUMEN
The fight against hand, foot, and mouth disease (HFMD) remains an arduous challenge without existing point-of-care (POC) diagnostic platforms for accurate diagnosis and prompt case quarantine. Hence, the purpose of this salivary biomarker discovery study is to set the fundamentals for the realization of POC diagnostics for HFMD. Whole salivary proteome profiling was performed on the saliva obtained from children with HFMD and healthy children, using a reductive dimethylation chemical labeling method coupled with high-resolution mass spectrometry-based quantitative proteomics technology. We identified 19 upregulated (fold change = 1.5-5.8) and 51 downregulated proteins (fold change = 0.1-0.6) in the saliva samples of HFMD patients in comparison to that of healthy volunteers. Four upregulated protein candidates were selected for dot blot-based validation assay, based on novelty as biomarkers and exclusions in oral diseases and cancers. Salivary legumain was validated in the Singapore (n = 43 healthy, 28 HFMD cases) and Taiwan (n = 60 healthy, 47 HFMD cases) cohorts with an area under the receiver operating characteristic curve of 0.7583 and 0.8028, respectively. This study demonstrates the feasibility of a broad-spectrum HFMD POC diagnostic test based on legumain, a virus-specific host systemic signature, in saliva.
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Enfermedad de Boca, Mano y Pie , Niño , Humanos , Enfermedad de Boca, Mano y Pie/diagnóstico , Biomarcadores/metabolismo , Cisteína Endopeptidasas/genética , Curva ROCRESUMEN
To reduce the error induced by overfitting or underfitting in predicting non-invasive fasting blood glucose (NIBG) levels using photoplethysmography (PPG) data alone, we previously demonstrated that incorporating HbA1c led to a notable 10% improvement in NIBG prediction accuracy (the ratio in zone A of Clarke's error grid). However, this enhancement came at the cost of requiring an additional HbA1c measurement, thus being unfriendly to users. In this study, the enhanced HbA1c NIBG deep learning model (blood glucose level predicted from PPG and HbA1c) was trained with 1494 measurements, and we replaced the HbA1c measurement (explicit HbA1c) with "implicit HbA1c" which is reversely derived from pretested PPG and finger-pricked blood glucose levels. The implicit HbA1c is then evaluated across intervals up to 90 days since the pretest, achieving an impressive 87% accuracy, while the remaining 13% falls near the CEG zone A boundary. The implicit HbA1c approach exhibits a remarkable 16% improvement over the explicit HbA1c method by covering personal correction items automatically. This improvement not only refines the accuracy of the model but also enhances the practicality of the previously proposed model that relied on an HbA1c input. The nonparametric Wilcoxon paired test conducted on the percentage error of implicit and explicit HbA1c prediction results reveals a substantial difference, with a p-value of 2.75 × 10-7.
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The COVID-19 pandemic has resulted in a large number of fatalities and, at present, lacks a readily available curative treatment for patients. Here, we demonstrate that unmodified red blood cell-derived extracellular vesicles (RBCEVs) can inhibit SARS-CoV-2 infection in a phosphatidylserine (PS) dependent manner. Using T cell immunoglobulin mucin domain-1 (TIM-1) as an example, we demonstrate that PS receptors on cells can significantly increase the adsorption and infection of authentic and pseudotyped SARS-CoV-2 viruses. RBCEVs competitively inhibit this interaction and block TIM-1-mediated viral entry into cells. We further extend the therapeutic efficacy of this antiviral treatment by loading antisense oligonucleotides (ASOs) designed to target conserved regions of key SARS-CoV-2 genes into RBCEVs. We establish that ASO-loaded RBCEVs are efficiently taken up by cells in vitro and in vivo to suppress SARS-CoV-2 replication. Our findings indicate that this RBCEV-based SARS-CoV-2 therapeutic displays promise as a potential treatment capable of inhibiting SARS-CoV-2 entry and replication.
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COVID-19 , Vesículas Extracelulares , Humanos , Antivirales/farmacología , Oligonucleótidos , Pandemias , SARS-CoV-2 , EritrocitosRESUMEN
Virus genome recoding is an attenuation method that confers genetically stable attenuation by rewriting a virus genome with numerous silent mutations. Prior flavivirus genome recoding attempts utilised codon deoptimisation approaches. However, these codon deoptimisation approaches act in a species dependent manner and were unable to confer flavivirus attenuation in mosquito cells or in mosquito animal models. To overcome these limitations, we performed flavivirus genome recoding using the contrary approach of codon optimisation. The genomes of flaviviruses such as dengue virus type 2 (DENV2) and Zika virus (ZIKV) contain functional RNA elements that regulate viral replication. We hypothesised that flavivirus genome recoding by codon optimisation would introduce silent mutations that disrupt these RNA elements, leading to decreased replication efficiency and attenuation. We chose DENV2 and ZIKV as representative flaviviruses and recoded them by codon optimising their genomes for human expression. Our study confirms that this recoding approach of codon optimisation does translate into reduced replication efficiency in mammalian, human, and mosquito cells as well as in vivo attenuation in both mice and mosquitoes. In silico modelling and RNA SHAPE analysis confirmed that DENV2 recoding resulted in the extensive disruption of genomic structural elements. Serial passaging of recoded DENV2 resulted in the emergence of rescue or adaptation mutations, but no reversion mutations. These rescue mutations were unable to rescue the delayed replication kinetics and in vivo attenuation of recoded DENV2, demonstrating that recoding confers genetically stable attenuation. Therefore, our recoding approach is a reliable attenuation method with potential applications for developing flavivirus vaccines.
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Culicidae , Flavivirus , Infección por el Virus Zika , Virus Zika , Humanos , Animales , Ratones , Flavivirus/genética , Virus Zika/genética , Replicación Viral/genética , Codón , MamíferosRESUMEN
Zika virus (ZIKV) is a mosquito-borne virus that has re-emerged as a significant threat to global health in the recent decade. Whilst infections are primarily asymptomatic, the virus has been associated with the manifestation of severe neurological complications. At present, there is still a lack of approved antivirals for ZIKV infections. In this study, chelerythrine chloride, a benzophenanthridine alkaloid, was identified from a mid-throughput screen conducted on a 502-compound natural products library to be a novel and potent inhibitor of ZIKV infection in both in-vitro and in-vivo assays. Subsequent downstream studies demonstrated that the compound inhibits a post-entry step of the viral replication cycle and is capable of disrupting viral RNA synthesis and protein expression. The successful generation and sequencing of a ZIKV resistant mutant revealed that a single S61T mutation on the viral NS4B allowed ZIKV to overcome chelerythrine chloride inhibition. Further investigation revealed that chelerythrine chloride could directly inhibit ZIKV protein synthesis, and that the NS4B-S61T mutation confers resistance to this inhibition. This study has established chelerythrine chloride as a potential candidate for further development as a therapeutic agent against ZIKV infection.
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Infección por el Virus Zika , Virus Zika , Animales , Chlorocebus aethiops , Infección por el Virus Zika/tratamiento farmacológico , Benzofenantridinas/farmacología , Benzofenantridinas/metabolismo , Benzofenantridinas/uso terapéutico , Células Vero , Proteínas Virales/metabolismo , Replicación Viral , Antivirales/uso terapéuticoRESUMEN
Over the years, the hand, foot and mouth disease (HFMD) has sparked epidemics across many countries which mainly affected young children. While symptoms are usually mild, severe complications may arise, and some even lead to death. Such concerns, coupled with the lack of approved vaccines and antivirals to date, create an urgency in the identification of safe therapeutics against HFMD. The disease is mainly transmitted by enteroviruses like enterovirus A71 (EV-A71). Essential for enterovirus replication is the host protein, PI4KB. In this study, we investigate the antiviral efficacy of a novel PI4KB inhibitor, CUR-N399. We found that CUR-N399 displayed broad-spectrum antiviral activity against picornaviruses in cell culture models. Using a suckling mouse model of lethal EV-A71 infection, CUR-N399 was found to be well-tolerated, promote survival and reduce viral titre in mice organs. Together, these support the discovery of CUR-N399 as an antiviral against EV-A71 and potentially other closely related viruses.
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Dengue virus (DENV) is the cause of dengue fever, infecting 390 million people worldwide per year. It is transmitted to humans through the bites of mosquitoes and could potentially develop severe symptoms. In spite of the rising social and economic impact inflicted by the disease on the global population, a conspicuous lack of efficacious therapeutics against DENV still persists. In this study, catechin, a natural polyphenol compound, was evaluated as a DENV infection inhibitor in vitro. Through time-course studies, catechin was shown to inhibit a post-entry stage of the DENV replication cycle. Further investigation revealed its role in affecting viral protein translation. Catechin inhibited the replication of all four DENV serotypes and chikungunya virus (CHIKV). Together, these results demonstrate the ability of catechin to inhibit DENV replication, hinting at its potential to be used as a starting scaffold for further development of antivirals against DENV infection.
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Catequina , Virus del Dengue , Dengue , Animales , Humanos , Antivirales/farmacología , Antivirales/uso terapéutico , Catequina/farmacología , Catequina/uso terapéutico , Replicación ViralRESUMEN
BACKGROUND: RNA viruses account for many human diseases and pandemic events but are often not targetable by traditional therapeutics modalities. Here, we demonstrate that adeno-associated virus (AAV) -delivered CRISPR-Cas13 directly targets and eliminates the positive-strand EV-A71 RNA virus in cells and infected mice. METHODS: We developed a Cas13gRNAtor bioinformatics pipeline to design CRISPR guide RNAs (gRNAs) that cleave conserved viral sequences across the virus phylogeny and developed an AAV-CRISPR-Cas13 therapeutics using in vitro viral plaque assay and in vivo EV-A71 lethally-infected mouse model. FINDINGS: We show that treatment with a pool of AAV-CRISPR-Cas13-gRNAs designed using the bioinformatics pipeline effectively blocks viral replication and reduces viral titers in cells by >99.99%. We further demonstrate that AAV-CRISPR-Cas13-gRNAs prophylactically and therapeutically inhibited viral replication in infected mouse tissues and prevented death in a lethally challenged EV-A71-infected mouse model. INTERPRETATION: Our results show that the bioinformatics pipeline designs efficient CRISPR-Cas13 gRNAs for direct viral RNA targeting to reduce viral loads. Additionally, this new antiviral AAV-CRISPR-Cas13 modality represents an effective direct-acting prophylactic and therapeutic agent against lethal RNA viral infections. FUNDING: Agency for Science, Technology and Research (A∗STAR) Assured Research Budget, A∗STAR Central Research Fund UIBR SC18/21-1089UI, A∗STAR Industrial Alignment Fund Pre-Positioning (IAF-PP) grant H17/01/a0/012, MOE Tier 2 2017 (MOE2017-T2-1-078; MOE-T2EP30221-0005), and NUHSRO/2020/050/RO5+5/NUHS-COVID/4.
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COVID-19 , Enterovirus Humano A , Enterovirus , Humanos , Ratones , Animales , Sistemas CRISPR-Cas , Dependovirus/genética , COVID-19/genética , Enterovirus/genética , Enterovirus Humano A/genéticaRESUMEN
Positive-sense RNA viruses modify intracellular calcium stores, endoplasmic reticulum and Golgi apparatus (Golgi) to generate membranous replication organelles known as viral factories. Viral factories provide a conducive and substantial enclave for essential virus replication via concentrating necessary cellular factors and viral proteins in proximity. Here, we identified the vital role of a broad-spectrum antiviral, peruvoside in limiting the formation of viral factories. Mechanistically, we revealed the pleiotropic cellular effect of Src and PLC kinase signaling via cyclin-dependent kinase 1 signaling leads to Golgi-specific brefeldin A-resistance guanine nucleotide exchange factor 1 (GBF1) phosphorylation and Golgi vesiculation by peruvoside treatment. The ramification of GBF1 phosphorylation fosters GBF1 deprivation consequentially activating downstream antiviral signaling by dampening viral factories formation. Further investigation showed signaling of ERK1/2 pathway via cyclin-dependent kinase 1 activation leading to GBF1 phosphorylation at Threonine 1337 (T1337). We also showed 100% of protection in peruvoside-treated mouse model with a significant reduction in viral titre and without measurable cytotoxicity in serum. These findings highlight the importance of dissecting the broad-spectrum antiviral therapeutics mechanism and pave the way for consideration of peruvoside, host-directed antivirals for positive-sense RNA virus-mediated disease, in the interim where no vaccine is available.