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The human immunodeficiency virus (HIV) epidemic is an ongoing global health problem affecting 38 million people worldwide with nearly 1.6 million new infections every year. Despite the advent of combined antiretroviral therapy (cART), a large percentage of people with HIV (PWH) still develop neurological deficits, grouped into the term of HIV-associated neurocognitive disorders (HAND). Investigating the neuropathology of HIV is important for understanding mechanisms associated with cognitive impairment seen in PWH. The major obstacle for studying neuroHIV is the lack of suitable in vitro human culture models that could shed light into the HIV-CNS interactions. Recent advances in induced pluripotent stem cell (iPSC) culture and 3D brain organoid systems have allowed the generation of 2D and 3D culture methods that possess a potential to serve as a model of neurotropic viral diseases, including HIV. In this study, we first generated and characterized several hiPSC lines from healthy human donor skin fibroblast cells. hiPSCs were then used for the generation of microglia-containing human cerebral organoids (hCOs). Once fully characterized, hCOs were infected with HIV-1 in the presence and absence of cART regimens and viral infection was studied by cellular, molecular/biochemical, and virological assays. Our results revealed that hCOs were productively infected with HIV-1 as evident by viral p24-ELISA in culture media, RT-qPCR and RNAscope analysis of viral RNA, as well as ddPCR analysis of proviral HIV-1 in genomic DNA samples. More interestingly, replication and gene expression of HIV-1 were also greatly suppressed by cART in hCOs as early as 7 days post-infections. Our results suggest that hCOs derived from hiPSCs support HIV-1 replication and gene expression and may serve as a unique platform to better understand neuropathology of HIV infection in the brain.
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Progress in stem cell research has revolutionized the medical field for more than two decades. More recently, the discovery of induced pluripotent stem cells (iPSCs) has allowed for the development of advanced disease modeling and tissue engineering platforms. iPSCs are generated from adult somatic cells by reprogramming them into an embryonic-like state via the expression of transcription factors required for establishing pluripotency. In the context of the central nervous system (CNS), iPSCs have the potential to differentiate into a wide variety of brain cell types including neurons, astrocytes, microglial cells, endothelial cells, and oligodendrocytes. iPSCs can be used to generate brain organoids by using a constructive approach in three-dimensional (3D) culture in vitro. Recent advances in 3D brain organoid modeling have provided access to a better understanding of cell-to-cell interactions in disease progression, particularly with neurotropic viral infections. Neurotropic viral infections have been difficult to study in two-dimensional culture systems in vitro due to the lack of a multicellular composition of CNS cell networks. In recent years, 3D brain organoids have been preferred for modeling neurotropic viral diseases and have provided invaluable information for better understanding the molecular regulation of viral infection and cellular responses. Here we provide a comprehensive review of the literature on recent advances in iPSC-derived 3D brain organoid culturing and their utilization in modeling major neurotropic viral infections including HIV-1, HSV-1, JCV, ZIKV, CMV, and SARS-CoV2.
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COVID-19 , Células Madre Pluripotentes Inducidas , Virosis , Virus , Infección por el Virus Zika , Virus Zika , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Infección por el Virus Zika/genética , Células Endoteliales , ARN Viral/metabolismo , SARS-CoV-2 , Encéfalo , Virosis/metabolismo , Organoides/metabolismoRESUMEN
Zika virus (ZIKV) infection is associated with microcephaly in neonates and Guillain-Barré syndrome in adults. ZIKV produces a class of nonstructural (NS) regulatory proteins that play a critical role in viral transcription and replication, including NS5, which possesses RNA-dependent RNA polymerase (RdRp) activity. Here we demonstrate that rilpivirine (RPV), a non-nucleoside reverse transcriptase inhibitor (NNRTI) used in the treatment of HIV-1 infection, inhibits the enzymatic activity of NS5 and suppresses ZIKV infection and replication in primary human astrocytes. Similarly, other members of the NNRTI family, including etravirine and efavirenz, showed inhibitory effects on viral infection of brain cells. Site-directed mutagenesis identified 14 amino acid residues within the NS5 RdRp domain (AA265-903), which are important for the RPV interaction and the inhibition of NS5 polymerase activity. Administration of RPV to ZIKV-infected interferon-alpha/beta receptor (IFN-A/R) knockout mice improved the clinical outcome and prevented ZIKV-induced mortality. Histopathological examination of the brains from infected animals revealed that RPV reduced ZIKV RNA levels in the hippocampus, frontal cortex, thalamus, and cerebellum. Repurposing of NNRTIs, such as RPV, for the inhibition of ZIKV replication offers a possible therapeutic strategy for the prevention and treatment of ZIKV-associated disease.
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Fármacos Anti-VIH/farmacología , Encéfalo/efectos de los fármacos , Receptor de Interferón alfa y beta/fisiología , Rilpivirina/farmacología , Proteínas no Estructurales Virales/antagonistas & inhibidores , Infección por el Virus Zika/tratamiento farmacológico , Virus Zika/efectos de los fármacos , Animales , Encéfalo/virología , Humanos , Ratones , Ratones Noqueados , Mutagénesis Sitio-Dirigida , Mutación , Unión Proteica , Conformación Proteica , Proteínas no Estructurales Virales/genética , Proteínas no Estructurales Virales/metabolismo , Replicación Viral , Infección por el Virus Zika/patología , Infección por el Virus Zika/virologíaRESUMEN
Cancer is the second leading cause of mortality worldwide. The study of DNA tumor-inducing viruses and their oncoproteins as a causative agent in cancer initiation and tumor progression has greatly enhanced our understanding of cancer cell biology. The initiation of oncogenesis is a complex process. Specific gene mutations cause functional changes in the cell that ultimately result in the inability to regulate cell differentiation and proliferation effectively. The human neurotropic Polyomavirus JC (JCV) belongs to the family Polyomaviridae and it is the causative agent of progressive multifocal leukoencephalopathy (PML), which is a fatal neurodegenerative disease in an immunosuppressed state. Sero-epidemiological studies have indicated JCV infection is prevalent in the population (85%) and that initial infection usually occurs during childhood. The JC virus has small circular, double-stranded DNA that includes coding sequences for viral early and late proteins. Persistence of the virus in the brain and other tissues, as well as its potential to transform cells, has made it a subject of study for its role in brain tumor development. Earlier observation of malignant astrocytes and oligodendrocytes in PML, as well as glioblastoma formation in non-human primates inoculated with JCV, led to the hypothesis that JCV plays a role in central nervous system (CNS) tumorigenesis. Some studies have reported the presence of both JC viral DNA and its proteins in several primary brain tumor specimens. The discovery of new Polyomaviruses such as the Merkel cell Polyomavirus, which is associated with Merkel cell carcinomas in humans, ignited our interest in the role of the JC virus in CNS tumors. The current evidence known about JCV and its effects, which are sufficient to produce tumors in animal models, suggest it can be a causative factor in central nervous system tumorigenesis. However, there is no clear association between JCV presence in CNS and its ability to initiate CNS cancer and tumor formation in humans. In this review, we will discuss the correlation between JCV and tumorigenesis of CNS in animal models, and we will give an overview of the current evidence for the JC virus's role in brain tumor formation.
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Neoplasias del Sistema Nervioso Central/virología , Virus JC/patogenicidad , Leucoencefalopatía Multifocal Progresiva/complicaciones , Animales , Progresión de la Enfermedad , Regulación Viral de la Expresión Génica , Humanos , Virus JC/genética , Leucoencefalopatía Multifocal Progresiva/virología , Mutación , Proteínas Virales/genéticaRESUMEN
BACKGROUND: Infection of glial cells by human neurotropic polyomavirus JC (JCV), the causative agent of the CNS demyelinating disease progressive multifocal leukoencephalopathy (PML), rapidly inflicts damage to cellular DNA. This activates DNA damage response (DDR) signaling including induction of expression of DNA repair factor Rad51. We previously reported that Rad51 co-operates with the transcription factor NF-κB p65 to activate JCV early transcription. Thus Rad51 induction by JCV infection may provide positive feedback for viral activation early in JCV infection. DDR is also known to stimulate NF-κB activity, a phenomenon known as nucleus to cytoplasm or "inside-out" NF-κB signaling, which is initiated by Ataxia telangiectasia mutated (ATM) protein, a serine/threonine kinase recruited and activated by DNA double-strand breaks. Downstream of ATM, there occurs a series of post-translational modifications of NF-κB essential modulator (NEMO), the γ regulatory subunit of inhibitor of NF-κB (IκB) kinase (IKK), resulting in NF-κB activation. METHODS: We analyzed the effects of downstream pathways in the DDR by phosphospecific Western blots and analysis of the subcellular distribution of NEMO by cell fractionation and immunocytochemistry. The role of DDR in JCV infection was analyzed using a small molecule inhibitor of ATM (KU-55933). NEMO sumoylation was investigated by Western and association of ATM and NEMO by immunoprecipitation/Western blots. RESULTS: We show that JCV infection caused phosphorylation and activation of ATM while KU-55933 inhibited JCV replication. JCV infection caused a redistribution of NEMO from cytoplasm to nucleus. Co-expression of JCV large T-antigen and FLAG-tagged NEMO showed the occurrence of sumoylation of NEMO, while co-expression of ATM and FLAG-NEMO demonstrated physical association between ATM and NEMO. CONCLUSIONS: We propose a model where JCV infection induces both overexpression of Rad51 protein and activation of the nucleus to cytoplasm NF-κB signaling pathway, which then act together to enhance JCV gene expression.
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Daño del ADN , Interacciones Huésped-Patógeno , Virus JC/crecimiento & desarrollo , FN-kappa B/metabolismo , Neuroglía/virología , Transducción de Señal , Estrés Fisiológico , Western Blotting , Fraccionamiento Celular , Línea Celular Tumoral , Núcleo Celular/metabolismo , Citoplasma/metabolismo , Regulación Viral de la Expresión Génica , Humanos , Quinasa I-kappa B/análisis , Inmunohistoquímica , Virus JC/genética , Modelos Biológicos , Transporte de Proteínas , Recombinasa Rad51/metabolismo , Transcripción GenéticaRESUMEN
John Cunningham virus (JCPyV) is an ubiquitous human pathogen that causes disease in immunocompromised patients. The JCPyV genome is composed of an early region and a late region, which are physically separated by the non-coding control region (NCCR). The DNA sequence of the NCCR distinguishes two forms of JCPyV, the designated archetype and the prototype, which resulted from a rearrangement of the archetype sequence. To date, the cell culture systems for propagating JCPyV archetype have been very limited in their availability and robustness. Prior to this study, it was demonstrated that JCPyV archetype DNA replicates in COS-7 simian kidney cells expressing SV40 TAg and COS-7 cells expressing HIV-1 Tat. Based on these observations, the present study was conducted to reproduce an in vitro model in COS-7 cells transfected with the JCPyV archetype strain in order to study JCPyV DNA replication and analyze NCCR rearrangements during the viral life cycle. The efficiency of JCPyV replication was evaluated by quantitative PCR (Q-PCR) and by hemagglutination (HA) assay after transfection. In parallel, sequence analysis of JCPyV NCCR was performed. JCPyV efficiently replicated in kidney-derived COS-7 cells, as demonstrated by a progressive increase in viral load and virion particle production after transfection. The archetypal structure of NCCR was maintained during the viral cycle, but two characteristic point mutations were detected 28 days after transfection. This model is a useful tool for analyzing NCCR rearrangements during in vitro replication in cells that are sites of viral persistence, such as tubular epithelial cells of the kidney.
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Adaptación Biológica , Reordenamiento Génico , Virus JC/crecimiento & desarrollo , Virus JC/genética , Animales , Células COS , Chlorocebus aethiops , Pruebas de Hemaglutinación , Humanos , Mutación Puntual , Reacción en Cadena en Tiempo Real de la Polimerasa , Análisis de Secuencia de ADN , Transfección , Cultivo de VirusRESUMEN
The nuclear magnetic resonance (NMR)-based metabolomic approach was used as analytical methodology to study the urine samples of chronic inflammatory rheumatic disease (CIRD) patients. The urine samples of CIRD patients were compared to the ones of both healthy subjects and patients with multiple sclerosis (MS), another immuno-mediated disease. Urine samples collected from 39 CIRD patients, 25 healthy subjects, and 26 MS patients were analyzed using 1H NMR spectroscopy, and the NMR spectra were examined using partial least squares-discriminant analysis (PLS-DA). PLS-DA models were validated by a double cross-validation procedure and randomization tests. Clear discriminations between CIRD patients and healthy controls (average diagnostic accuracy 83.5 ± 1.9%) as well as between CIRD patients and MS patients (diagnostic accuracy 81.1 ± 1.9%) were obtained. Leucine, alanine, 3-hydroxyisobutyric acid, hippuric acid, citric acid, 3-hydroxyisovaleric acid, and creatinine contributed to the discrimination; all of them being in a lower concentration in CIRD patients as compared to controls or to MS patients. The application of NMR metabolomics to study these still poorly understood diseases can be useful to better clarify the pathologic mechanisms; moreover, as a holistic approach, it allowed the detection of, by means of anomalous metabolic traits, the presence of other pathologies or pharmaceutical treatments not directly connected to CIRDs, giving comprehensive information on the general health state of individuals. Graphical abstract NMR-based metabolomic approach as a tool to study urine samples in CIRD patients with respect to MS patients and healthy controls.
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Espectroscopía de Resonancia Magnética/métodos , Metabolómica , Enfermedades Reumáticas/orina , Adulto , Estudios de Casos y Controles , Enfermedad Crónica , Femenino , Humanos , Masculino , Persona de Mediana Edad , Esclerosis Múltiple/orinaRESUMEN
Brd4 is an epigenetic reader protein and a member of the BET (bromodomain and extra terminal domain) family of proteins with two bromodomains that recognize acetylated lysine residues. Brd4 specifically binds to acetylated transcription factor NF-κB p65 and coactivates transcription. Polyomavirus JC (JCV) is regulated by a noncoding control region (NCCR) containing promoter/enhancer elements for viral gene expression including a binding site for NF-κB, which responds to proinflammatory cytokines such as TNF-α, the DNA damage response, calcium signaling and acetylation of the NF-κB p65 subunit on lysine residues K218 and K221. Earlier studies indicated that NF-κB is involved in the reactivation of persistent/latent JCV in glial cells to cause progressive multifocal leukoencephalopathy (PML), a severe demyelinating disease of the brain caused by replication of JCV in glial cells. To investigate the mechanism of action of NF-κB acetylation on JCV transcription, we examined Brd4 and found that JCV early transcription was stimulated by Brd4 via the JCV NF-κB site and that p65 K218 and K221 were involved. Treatment with the Brd4 inhibitor JQ1(+) or mutation of either K218 or K221 to glutamine (K218R or K221) inhibited this stimulation and decreased the proportion of p65 in the nucleus. We conclude that Brd4 is involved in the regulation of the activation status of JCV in glial cells.
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Interacciones Huésped-Patógeno , Virus JC/efectos de los fármacos , Proteínas Nucleares/genética , Factor de Transcripción ReIA/genética , Factores de Transcripción/genética , Factor de Necrosis Tumoral alfa/genética , Replicación Viral/efectos de los fármacos , Acetilación , Azepinas/farmacología , Proteínas de Ciclo Celular , Línea Celular Tumoral , Núcleo Celular/efectos de los fármacos , Núcleo Celular/metabolismo , Epigénesis Genética , Genes Reporteros , Humanos , Virus JC/genética , Virus JC/crecimiento & desarrollo , Luciferasas/genética , Luciferasas/metabolismo , Mutación , Neuroglía/efectos de los fármacos , Neuroglía/metabolismo , Neuroglía/patología , Proteínas Nucleares/antagonistas & inhibidores , Proteínas Nucleares/metabolismo , Unión Proteica , Transducción de Señal , Factor de Transcripción ReIA/metabolismo , Factores de Transcripción/antagonistas & inhibidores , Factores de Transcripción/metabolismo , Transcripción Genética/efectos de los fármacos , Triazoles/farmacología , Factor de Necrosis Tumoral alfa/metabolismo , Activación ViralRESUMEN
Progressive multifocal leukoencephalopathy (PML) is a fatal demyelinating disease caused by neurotropic polyomavirus, JC virus (JCV), a virus that causes lytic infection of CNS glial cells. After primary infection, JCV is controlled by the immune system but virus persists asymptomatically. Rarely, when immune function is impaired, it can reemerge to cause PML. The mechanisms of JCV persistence and reactivation are not well understood but our earlier work implicated epigenetic control by protein acetylation since histone deacetylase inhibitors such as trichostatin A (TSA) strongly stimulate JCV transcription. Since both TNF-α and TSA activate JCV transcription via the same unique NF-κB site in the JCV control region, we investigated a role for acetylation of NF-κB in JCV regulation. A site-directed mutagenesis strategy was employed targeting the known lysine acetylation sites of NF-κB p65: K218, K221, and K310. We individually mutated each lysine to arginine, which cannot be acetylated and retains a positive charge like lysine. K218R and K221R impaired transactivation of JCV early promoter transcription either alone or combined with TSA treatment or coexpression of acetyltransferase transcriptional coactivator p300 but K310R was largely without effect. Mutation of lysine to glutamine gives mutants with a negative charge like acetyllysine. However, K218Q and K221Q showed impaired activity and only K310Q showed enhanced transactivation. NF-κB acetylation can regulate several aspects of the process of activation including complex formation with IκB, translocation to the nucleus, and DNA binding and transcriptional activation. Cell fractionation studies revealed that the mutants had no defect in translocation to the nucleus whereas gel shift studies revealed reduced binding to the JCV NF-κB site. Thus, acetylation regulates NF-κB p65 activity toward JCV at the level of p65 binding to the JCV control region and activation of JCV transcription.
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Epigénesis Genética/genética , Virus JC/genética , Factor de Transcripción ReIA/metabolismo , Activación Transcripcional/genética , Activación Viral/genética , Acetilación , Western Blotting , Línea Celular Tumoral , Humanos , Lisina/metabolismo , Mutagénesis Sitio-Dirigida , Oligodendroglía/virología , TransfecciónRESUMEN
The risk of developing progressive multifocal leukoencephalopathy (PML), as a consequence of infection/reactivation with JC virus (JCV), is consistent in natalizumab-treated multiple sclerosis (MS) patients, with 430 cases of PML reported so far. The risk of PML is higher in JCV seropositive patients, and it is recommended that only MS patients without JCV antibodies should be enrolled in the treatment postulating that they do not have JCV infection.We have studied forty-two natalizumab-treated MS patients, and urine and blood were collected monthly for up to 60 months. JCV and BK virus (BKV) DNA presence was verified using quantitative real-time PCR assays, and serum anti-JCV antibodies were measured with the Stratify and/or Stratify DxSelect tests.JCV and BKV DNA were not found in the blood samples, whereas they were found at least once in the urine of 21 of 42 (50 %) and of 25/42 (59.5 %) patients, respectively. JCV DNA urinary shedding increased up to month 24 of natalizumab treatment (45.2 %), and the effect of time was significant for JCV (p = 0.04), but not for BKV (p = 0.39). JCV viruria and seropositivity did not completely correlate, since three patients shedding JCV DNA in the urine were seronegative according to the serological tests.The results indicated that natalizumab therapy may increase the rate of JCV urinary shedding. Additionally, we confirmed that the identification of JCV carriers cannot solely rely on serological tests, but sensitive methods for viral DNA detection should be adopted to more precisely identify the truly JCV uninfected cases.
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Anticuerpos Antivirales/orina , ADN Viral/orina , Factores Inmunológicos/uso terapéutico , Esclerosis Múltiple Recurrente-Remitente/tratamiento farmacológico , Esclerosis Múltiple Recurrente-Remitente/virología , Natalizumab/uso terapéutico , Adulto , Anticuerpos Antivirales/sangre , ADN Viral/sangre , Ensayo de Inmunoadsorción Enzimática , Femenino , Humanos , Virus JC/aislamiento & purificación , Masculino , Persona de Mediana Edad , Infecciones por Polyomavirus/virología , Reacción en Cadena en Tiempo Real de la Polimerasa , Estudios Seroepidemiológicos , Esparcimiento de Virus/fisiología , Adulto JovenRESUMEN
In the last years, the treatment of multiple sclerosis (MS) patients with natalizumab has been associated with the occurrence of progressive multifocal leukoencephalopathy (PML) caused by human polyomavirus JC (JCV). Here, we have shown a significant correlation between patients with JC viruria and positive JC-specific antibody response and patients without JCV-specific antibodies after 1 year of natalizumab (p = 0.0006). Furthermore, JCV-specific quantitative PCR on urine and plasma samples, collected at the enrollment (t0) and every 4 months (t1, t2, t3) in the first year and at two time points (t4 and t5) in the second year of natalizumab treatment, indicated the prevalence of JC viremia rather than JC viruria only in the second year of treatment (p = 0.04). Moreover, the analysis of JCV non-coding control region (NCCR) sequences in peripheral blood mononuclear cells of patients with JC-specific antibodies after 12 natalizumab infusions (t3) revealed the presence of rearranged sequences, whereas the prevalence of genotypes 1A, 1B, and 4 was detected in these patients by VP1 sequence analysis. In summary, JC viruria evaluation seems to be useful to identify early those patients who do not already develop a humoral immune response against JCV. It may also be interesting to study the JCV NCCR rearrangements since they could give us new insights on the onset of neuro-invasive viral variants.
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ADN Viral/sangre , ADN Viral/orina , Factores Inmunológicos/uso terapéutico , Virus JC/genética , Esclerosis Múltiple Recurrente-Remitente/virología , Natalizumab/uso terapéutico , Adulto , Anticuerpos Antivirales/análisis , Ensayo de Inmunoadsorción Enzimática , Femenino , Humanos , Masculino , Esclerosis Múltiple Recurrente-Remitente/tratamiento farmacológico , Reacción en Cadena en Tiempo Real de la Polimerasa , Carga Viral/efectos de los fármacos , Viremia , Replicación ViralRESUMEN
We report the prevalence of BK virus (BKV) infection before renal transplantation and the dynamics of BKV viremia from pre- to post-transplantation. We assessed 60 kidney transplanted patients from a single cohort in Italy, treated with identical immunosuppressive therapy, for BK viremia at pre-transplantation, 12 h, and three and six months post-transplantation. Polymerase chain reaction showed that the prevalence of plasma BKV replication--considered a marker of infection--was 20% in pre-transplant patients. All pre-transplant-positive patients remained positive post-transplant, whereas the majority of pre-transplant-negative patients remained negative. Viremia dynamics classification revealed three clusters of patients: Cluster A++, pre-transplant-positive patients (20%) who tested positive at least once post-transplant; Cluster B-+, pre-transplant-negative patients (28%) who tested positive at least once post-transplant; and Cluster C- -, pre-transplant-negative patients (52%) who remained negative throughout. These clusters presented significant differences related to the prevalence of substantially positive patients with high plasma viral load (>10(3) copies/mL) in cluster A, but not in donors' or grafts' characteristics. We suggest that pre-transplant viral status should be considered as an additional risk factor for post-transplant BKV replication. Therefore, pre-transplant BKV infection screening in kidney transplant patients should be performed for improving planning of personalized immunosuppressant schemes and specific post-transplant surveillance.
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Virus BK/fisiología , Fallo Renal Crónico/virología , Trasplante de Riñón , Infecciones por Polyomavirus/virología , Infecciones Tumorales por Virus/virología , Replicación Viral , Listas de Espera , Adolescente , Adulto , Anciano , ADN Viral/genética , Femenino , Estudios de Seguimiento , Humanos , Inmunosupresores/uso terapéutico , Italia/epidemiología , Fallo Renal Crónico/cirugía , Masculino , Persona de Mediana Edad , Reacción en Cadena de la Polimerasa , Infecciones por Polyomavirus/cirugía , Prevalencia , Pronóstico , Factores de Riesgo , Tasa de Supervivencia , Infecciones Tumorales por Virus/cirugía , Carga Viral , Viremia/virología , Adulto JovenRESUMEN
Although our understanding of herpes simplex virus type 1 (HSV-1) biology has been considerably enhanced, developing therapeutic strategies to eliminate HSV-1 in latently infected individuals remains a public health concern. Current antiviral drugs used for the treatment of HSV-1 complications are not specific and do not address latent infection. We recently developed a CRISPR-Cas9-based gene editing platform to specifically target the HSV-1 genome. In this study, we further used 2D Vero cell culture and 3D human induced pluripotent stem cell-derived cerebral organoid (CO) models to assess the effectiveness of our editing constructs targeting viral ICP0 or ICP27 genes. We found that targeting the ICP0 or ICP27 genes with AAV2-CRISPR-Cas9 vectors in Vero cells drastically suppressed HSV-1 replication. In addition, we productively infected COs with HSV-1, characterized the viral replication kinetics, and established a viral latency model. Finally, we discovered that ICP0- or ICP27-targeting AAV2-CRISPR-Cas9 vector significantly reduced viral rebound in the COs that were latently infected with HSV-1. In summary, our results suggest that CRISPR-Cas9 gene editing of HSV-1 is an efficient therapeutic approach to eliminate the latent viral reservoir and treat HSV-1-associated complications.
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John Cunningham virus (JCV), the etiological agent of progressive multifocal leukoencephalopathy (PML), contains a hyper-variable non-coding control region usually detected in urine of healthy individuals as archetype form and in the brain and cerebrospinal fluid (CSF) of PML patients as rearranged form. We report a case of HIV-related PML with clinical, immunological and virological data longitudinally collected. On admission (t0), after 8-week treatment with a rescue highly active antiretroviral therapy (HAART), the patient showed a CSF-JCV load of 16,732 gEq/ml, undetectable HIV-RNA and an increase of CD4+ cell count. Brain magnetic resonance imaging (MRI) showed PML-compatible lesions without contrast enhancement. We considered PML-immune reconstitution inflammatory syndrome as plausible because of the sudden onset of neurological symptoms after the effective HAART. An experimental JCV treatment with mefloquine and mirtazapine was added to steroid boli. Two weeks later (t1), motor function worsened and MRI showed expanded lesions with cytotoxic oedema. CSF JCV-DNA increased (26,263 gEq/ml) and JCV viremia was detected. After 4 weeks (t2), JCV was detected only in CSF (37,719 gEq/ml), and 8 weeks after admission (t3), JC viral load decreased in CSF and JCV viremia reappeared. The patient showed high level of immune activation both in peripheral blood and CSF. He died 4 weeks later. Considering disease progression, combined therapy failure and immune hyper-activation, we finally classified the case as classical PML. The archetype variant found in CSF at t0/t3 and a rearranged sequence detected at t1/t2 suggest that PML can develop from an archetype virus and that the appearance of rearranged genotypes contribute to faster disease progression.
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Reordenamiento Génico , Infecciones por VIH/virología , Inmunidad Innata , Virus JC/genética , Leucoencefalopatía Multifocal Progresiva/virología , Corticoesteroides/uso terapéutico , Terapia Antirretroviral Altamente Activa , Antivirales/uso terapéutico , Coinfección , Resultado Fatal , VIH/aislamiento & purificación , Infecciones por VIH/líquido cefalorraquídeo , Infecciones por VIH/tratamiento farmacológico , Infecciones por VIH/inmunología , Humanos , Virus JC/aislamiento & purificación , Virus JC/patogenicidad , Leucoencefalopatía Multifocal Progresiva/líquido cefalorraquídeo , Leucoencefalopatía Multifocal Progresiva/tratamiento farmacológico , Leucoencefalopatía Multifocal Progresiva/inmunología , Masculino , Mefloquina/uso terapéutico , Mianserina/análogos & derivados , Mianserina/uso terapéutico , Persona de Mediana Edad , MirtazapinaRESUMEN
Acute renal dysfunction (ARD) is a common complication in renal transplant recipients. Multiple factors contribute to ARD development, including acute rejection and microbial infections. Many viral infections after kidney transplantation result from reactivation of "latent" viruses in the host or from the graft, such as the human Polyomavirus BK (BKV). We report the case of a 39 year-old recipient of a 2nd kidney graft who experienced BKV reactivation after a second episode of acute humoral rejection. A 10-day treatment with the quinolone antibiotic ciprofloxacin was administered with an increase of immunosuppressive therapy despite the active BKV replication. Real Time PCR analysis performed after treatment with ciprofloxacin, unexpectedly showed clearance of BK viremia and regression of BK viruria. During the follow-up, BK viremia persisted undetectable while viruria decreased further and disappeared after 3 months.BKV non-coding control region sequence analysis from all positive samples always showed the presence of archetypal sequences, with two single-nucleotide substitutions and one nucleotide deletion that, interestingly, were all representative of the subtype/subgroup I/b-1 we identified by the viral protein 1 sequencing analysis.We report the potential effect of the quinolone antibiotic ciprofloxacin in the decrease of the BKV load in both blood and urine.
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Antibacterianos/uso terapéutico , Virus BK/aislamiento & purificación , Ciprofloxacina/uso terapéutico , ADN Intergénico , ADN Viral/genética , Infecciones por Polyomavirus/virología , Secuencias Reguladoras de Ácidos Nucleicos , Adulto , Antibacterianos/farmacología , Virus BK/clasificación , Virus BK/efectos de los fármacos , Virus BK/genética , Ciprofloxacina/farmacología , ADN Viral/sangre , ADN Viral/orina , Humanos , Trasplante de Riñón , Masculino , Mutación Puntual , Infecciones por Polyomavirus/diagnóstico , Reacción en Cadena en Tiempo Real de la Polimerasa , Eliminación de Secuencia , Resultado del Tratamiento , Viremia/diagnóstico , Viremia/virologíaRESUMEN
BACKGROUND: Progressive multifocal leukoencephalopathy (PML) onset, caused by Polyomavirus JC (JCPyV) in patients affected by immune-mediated diseases during biological treatment, raised concerns about the safety profile of these agents. Therefore, the aims of this study were the JCPyV reactivation monitoring and the noncoding control region (NCCR) and viral protein 1 (VP1) analysis in patients affected by different immune-mediated diseases and treated with biologics. METHODS: We performed JCPyV-specific quantitative PCR of biological samples collected at moment of recruitment (t0) and every 4 months (t1, t2, t3, t4). Subsequently, rearrangements' analysis of NCCR and VP1 was carried out. Data were analyzed using χ2 test. RESULTS: Results showed that at t0 patients with chronic inflammatory rheumatic diseases presented a JCPyV load in the urine significantly higher (p≤0.05) than in patients with multiple sclerosis (MS) and Crohn's disease (CD). It can also be observed a significant association between JC viruria and JCPyV antibodies after 1 year of natalizumab (p=0.04) in MS patients. Finally, NCCR analysis showed the presence of an archetype-like sequence in all urine samples, whereas a rearranged NCCR Type IR was found in colon-rectal biopsies collected from 2 CD patients after 16 months of infliximab. Furthermore, sequences isolated from peripheral blood mononuclear cells (PBMCs) of 2 MS patients with JCPyV antibody at t0 and t3, showed a NCCR Type IIR with a duplication of a 98 bp unit and a 66 bp insert, resulting in a boxB deletion and 37 T to G transversion into the Spi-B binding site. In all patients, a prevalence of genotypes 1A and 1B, the predominant JCPyV genotypes in Europe, was observed. CONCLUSIONS: It has been important to understand whether the specific inflammatory scenario in different immune-mediated diseases could affect JCPyV reactivation from latency, in particular from kidneys. Moreover, for a more accurate PML risk stratification, testing JC viruria seems to be useful to identify patients who harbor JCPyV but with an undetectable JCPyV-specific humoral immune response. In these patients, it may also be important to study the JCPyV NCCR rearrangement: in particular, Spi-B expression in PBMCs could play a crucial role in JCPyV replication and NCCR rearrangement.
Asunto(s)
Productos Biológicos/uso terapéutico , ADN Intergénico , Enfermedades del Sistema Inmune/tratamiento farmacológico , Enfermedades del Sistema Inmune/virología , Virus JC/genética , Virus JC/fisiología , Activación Viral , Adulto , Colon/virología , Femenino , Genotipo , Humanos , Leucocitos Mononucleares/virología , Masculino , Estudios Prospectivos , Reacción en Cadena en Tiempo Real de la Polimerasa , Recombinación Genética , Orina/virología , Carga Viral , Proteínas Estructurales Virales/genéticaRESUMEN
John Cunningham virus (JCV) is a member of the Polyomaviridae family. It was first isolated from the brain of a patient with Hodgkin disease in 1971, and since then the etiological agent of the progressive multifocal leukoencephalopathy (PML) was considered. Until the human immunodeficiency virus (HIV) pandemic, PML was rare: in fact HIV-induced immunodeficiency is the most common predisposing factor accounting for 85% of all instances of PML. This data led to intense research on JCV infection and resulted in better understanding of epidemiology and clinic-pathologic spectrum. Recently, cases of PML have been observed after the introduction of monoclonal antibodies, such as natalizumab, rituximab, efalizumab, and infliximab, in the treatment of autoimmune disease, underlining the important role of host immunity in PML pathogenesis. In this review current understanding of the JCV infection and the new findings relating to the pathogenesis of PML has been comprehensively revised, focusing our attention on the interaction between the cellular and viral molecular pathways implicated in the JCV infection and the modulating role of host immune surveillance in the viral reactivation from a latent state.
Asunto(s)
Encéfalo/patología , Regulación Viral de la Expresión Génica/inmunología , Huésped Inmunocomprometido , Virus JC/inmunología , Leucoencefalopatía Multifocal Progresiva/patología , Activación Viral/inmunología , Anticuerpos Monoclonales/farmacología , Enfermedades Autoinmunes/inmunología , Enfermedades Autoinmunes/patología , Enfermedades Autoinmunes/virología , Encéfalo/inmunología , Encéfalo/virología , VIH/inmunología , Infecciones por VIH/inmunología , Infecciones por VIH/patología , Infecciones por VIH/virología , Interacciones Huésped-Patógeno , Humanos , Vigilancia Inmunológica , Leucoencefalopatía Multifocal Progresiva/inmunología , Leucoencefalopatía Multifocal Progresiva/virología , Activación Viral/efectos de los fármacosRESUMEN
We previously reported the discovery and characterization of two novel proteins (ORF1 and ORF2) generated by the alternative splicing of the JC virus (JCV) late coding region. Here, we report the discovery and partial characterization of three additional novel ORFs from the same coding region, ORF3, ORF4 and ORF5, which potentially encode 70, 173 and 265 amino acid long proteins respectively. While ORF3 protein exhibits a uniform distribution pattern throughout the cells, we were unable to detect ORF5 expression. Surprisingly, ORF4 protein was determined to be the only JCV protein specifically targeting the promyelocytic leukemia nuclear bodies (PML-NBs) and inducing their reorganization in nucleus. Although ORF4 protein has a modest effect on JCV replication, it is implicated to play major roles during the JCV life cycle, perhaps by regulating the antiviral response of PML-NBs against JCV infections and thus facilitating the progression of the JCV-induced disease in infected individuals.
Asunto(s)
Virus JC , Leucoencefalopatía Multifocal Progresiva , Poliomavirus , Humanos , Virus JC/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Sistemas de Lectura Abierta , Cuerpos Nucleares de la Leucemia PromielocíticaRESUMEN
Psoriasis vulgaris (PsV) and psoriatic arthritis (PSA) are inter-related heritable inflammatory skin diseases. Psoriatic lesions develop as a result of abnormal immune responses, hyperproliferation and altered differentiation of keratinocytes, and a notable subset of psoriatic patients develops PsA, characterized by joints inflammation. Recently, biological drugs were introduced to treat these diseases. However, this therapy has already been associated with the development of serious life-threatening infections, such as the reactivation of human polyomavirus JC (JCV), responsible for the progressive multifocal leukoencephalopathy (PML), a lethal demyelinating disease caused by oligodendrocytes lytic infection. Therefore, the aims of our study were the investigation of the possible JCV reactivation in PsV and PsA patients treated with adalimumab, etanercept, and methotrexate, performing quantitative real-time PCR in sera and skin biopsies at the time of recruitment (T0) and after 3 (T3) and 6 (T6) months of treatment, and the sequencing analysis of the JCV non-coding control region (NCCR). We found JCV DNA in 5/15 PsV patients and in 2/15 PsA patients and JCV NCCR sequence analysis always showed a structure similar to non-pathogenic CY archetype, with random occurrence of a few irrelevant point mutations. Nevertheless the poor number of patients analyzed, our preliminary data can pave the way for taking into account that the follow-up of JCV DNA detection and the JCV NCCR sequence analysis in psoriatic patients may be important to evaluate the risk of PML onset, considering that patients affected by autoimmune diseases and treated with biologics continue to rise.
Asunto(s)
Inmunosupresores/uso terapéutico , Virus JC/fisiología , Infecciones por Polyomavirus/virología , Psoriasis/tratamiento farmacológico , Infecciones Tumorales por Virus/virología , Activación Viral/inmunología , Adalimumab , Adulto , Anticuerpos Monoclonales Humanizados/farmacología , Anticuerpos Monoclonales Humanizados/uso terapéutico , Antígenos Virales/análisis , Artritis Psoriásica/complicaciones , Artritis Psoriásica/tratamiento farmacológico , Artritis Psoriásica/inmunología , Secuencia de Bases , ADN Viral/análisis , ADN Viral/química , Etanercept , Femenino , Humanos , Inmunoglobulina G/farmacología , Inmunoglobulina G/uso terapéutico , Inmunosupresores/farmacología , Masculino , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , Infecciones por Polyomavirus/patología , Psoriasis/complicaciones , Psoriasis/inmunología , Receptores del Factor de Necrosis Tumoral/uso terapéutico , Infecciones Tumorales por Virus/patología , Activación Viral/efectos de los fármacosRESUMEN
The recent introduction of monoclonal antibodies in Crohn's disease (CD) management has been associated with the development of serious complications, such as the progressive multifocal leukoencephalopathy (PML), caused by JC polyomavirus (JCV) reactivation. Therefore, the aims of our study have been the investigation of the possible JCV reactivation in pediatric CD patients treated or not with infliximab, performing quantitative PCR in urine, plasma, and intestinal biopsies at the time of recruitment (t0) and every 4 months in 1 year of follow-up (t1, t2, and t3), and the analysis of the JCV noncoding control region (NCCR) to detect cellular transcription factors binding site mutations. Results obtained showed that, in urine and ileal specimens, JCV load significantly increased in infliximab-treated patients after 1 year of treatment (t3), while viremia was significantly higher at t1. JCV NCCR sequence analysis showed a structure similar to CY archetype in 65/80 analyzed sequences, but the remaining 15/80, obtained exclusively from plasma and biopsies, evidenced a CY NCCR organization with two recurrent nucleotide changes, the 37-T to G transversion in box A Spi-B binding site and the 217-G to A transition in box F, and a box D deletion. These rearrangements were always found at t3 within seven infliximab-treated CD patients, who presented a very severe disease at t0. We can conclude that our rearranged NCCR sequences could be considered a marker of JCV virulence during mAb treatment, although none of our examined patients developed PML, and further studies on a larger cohort of patients should be performed.