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BACKGROUND: To retain the spread of SARS-CoV-2, fast, sensitive and cost-effective testing is essential, particularly in resource limited settings (RLS). Current standard nucleic acid-based RT-PCR assays, although highly sensitive and specific, require transportation of samples to specialised laboratories, trained staff and expensive reagents. The latter are often not readily available in low- and middle-income countries and this may significantly impact on the successful disease management in these settings. Various studies have suggested a SARS-CoV-2 loop mediated isothermal amplification (LAMP) assay as an alternative method to RT-PCR. METHODS: Four previously published primer pairs were used for detection of SARS-CoV-2 in the LAMP assay. To determine optimal conditions, different temperatures, sample input and incubation times were tested. Ninety-three extracted RNA samples from St. George's Hospital, London, 10 non-extracted nasopharyngeal swab samples from Great Ormond Street Hospital for Children, London, and 92 non-extracted samples from Queen Elisabeth Central Hospital (QECH), Malawi, which have previously been tested for SARS-Cov-2 by quantitative reverse-transcription RealTime PCR (qRT-PCR), were analysed in the LAMP assay. RESULTS: In this study we report the optimisation of an extraction-free colourimetric SARS-CoV-2 LAMP assay and demonstrated that a lower limit of detection (LOD) between 10 and 100 copies/µL of SARS-CoV-2 could be readily detected by a colour change of the reaction within as little as 30 min. We further show that this assay could be quickly established in Malawi, as no expensive equipment is necessary. We tested 92 clinical samples from QECH and showed the sensitivity and specificity of the assay to be 86.7% and 98.4%, respectively. Some viral transport media, used routinely to stabilise RNA in clinical samples during transportation, caused a non-specific colour-change in the LAMP reaction and therefore we suggest collecting samples in phosphate buffered saline (which did not affect the colour) as the assay allows immediate sample analysis on-site. CONCLUSION: SARS-CoV-2 LAMP is a cheap and reliable assay that can be readily employed in RLS to improve disease monitoring and management.
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COVID-19 , SARS-CoV-2 , COVID-19/diagnóstico , Niño , Humanos , Técnicas de Diagnóstico Molecular , Técnicas de Amplificación de Ácido Nucleico/métodos , ARN , SARS-CoV-2/genéticaRESUMEN
The pathogenicity of Campylobacter concisus, increasingly found in the human gastrointestinal (GI) tract, is unclear. Some studies indicate that its role in GI conditions has been underestimated, whereas others suggest that the organism has a commensal-like phenotype. For the enteropathogen C. jejuni, the lipooligosaccharide (LOS) is a main driver of virulence. We investigated the LOS structure of four C. concisus clinical isolates and correlated the inflammatory potential of each isolate with bacterial virulence. Mass spectrometric analyses of lipid A revealed a novel hexa-acylated diglucosamine moiety with two or three phosphoryl substituents. Molecular and fragment ion analysis indicated that the oligosaccharide portion of the LOS had only a single phosphate and lacked phosphoethanolamine and sialic acid substitution, which are hallmarks of the C. jejuni LOS. Consistent with our structural findings, C. concisus LOS and live bacteria induced less TNF-α secretion in human monocytes than did C. jejuni Furthermore, the C. concisus bacteria were less virulent than C. jejuni in a Galleria mellonella infection model. The correlation of the novel lipid A structure, decreased phosphorylation, and lack of sialylation along with reduced inflammatory potential and virulence support the significance of the LOS as a determinant in the relative pathogenicity of C. concisus.
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Campylobacter/metabolismo , Campylobacter/patogenicidad , Lipopolisacáridos/química , Lipopolisacáridos/metabolismo , Campylobacter/genética , Campylobacter/fisiología , Línea Celular , Genómica , Humanos , Inflamación/microbiología , Lípido A/química , Lipopolisacáridos/farmacología , Transducción de Señal/efectos de los fármacos , Factor de Necrosis Tumoral alfa/metabolismo , VirulenciaRESUMEN
Background: Sudden unexpected death in infancy (SUDI) is the most common cause of post-neonatal death in the developed world. Following an extensive investigation, the cause of ~40% of deaths remains unknown. It is hypothesized that a proportion of deaths are due to an infection that remains undetected due to limitations in routine techniques. This study aimed to apply 16S rRNA gene sequencing to post-mortem (PM) tissues collected from cases of SUDI, as well as those from the childhood equivalent (collectively known as sudden unexpected death in infancy and childhood or SUDIC), to investigate whether this molecular approach could help identify potential infection-causing bacteria to enhance the diagnosis of infection. Methods: In this study, 16S rRNA gene sequencing was applied to de-identified frozen post-mortem (PM) tissues from the diagnostic archive of Great Ormond Street Hospital. The cases were grouped depending on the cause of death: (i) explained non-infectious, (ii) infectious, and (iii) unknown. Results and conclusions: In the cases of known bacterial infection, the likely causative pathogen was identified in 3/5 cases using bacterial culture at PM compared to 5/5 cases using 16S rRNA gene sequencing. Where a bacterial infection was identified at routine investigation, the same organism was identified by 16S rRNA gene sequencing. Using these findings, we defined criteria based on sequencing reads and alpha diversity to identify PM tissues with likely infection. Using these criteria, 4/20 (20%) cases of unexplained SUDIC were identified which may be due to bacterial infection that was previously undetected. This study demonstrates the potential feasibility and effectiveness of 16S rRNA gene sequencing in PM tissue investigation to improve the diagnosis of infection, potentially reducing the number of unexplained deaths and improving the understanding of the mechanisms involved.
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BACKGROUND: Haematopoietic stem cell transplantation is a curative procedure for a variety of conditions. Despite major advances, a plethora of adverse clinical outcomes can develop post-transplantation including graft-versus-host disease and infections, which remain the major causes of morbidity and mortality. There is increasing evidence that the gastrointestinal microbiota is associated with clinical outcomes post-haematopoietic stem cell transplantation. Herein, we investigated the longitudinal dynamics of the gut microbiota and metabolome and potential associations to clinical outcomes in paediatric haematopoietic stem cell transplantation at a single centre. RESULTS: On admission (baseline), the majority of patients presented with a different gut microbial composition in comparison with healthy control children with a significantly lower alpha diversity. A further, marked decrease in alpha diversity was observed immediately post-transplantation and in most microbial diversity, and composition did not return to baseline status whilst hospitalised. Longitudinal trajectories identified continuous fluctuations in microbial composition, with the dominance of a single taxon in a significant proportion of patients. Using pam clustering, three clusters were observed in the dataset. Cluster 1 was common pre-transplantation, characterised by a higher abundance of Clostridium XIVa, Bacteroides and Lachnospiraceae; cluster 2 and cluster 3 were more common post-transplantation with a higher abundance of Streptococcus and Staphylococcus in the former whilst Enterococcus, Enterobacteriaceae and Escherichia predominated in the latter. Cluster 3 was also associated with a higher risk of viraemia. Likewise, further multivariate analysis reveals Enterobacteriaceae, viraemia, use of total parenteral nutrition and various antimicrobials contributing towards cluster 3, Streptococcaceae, Staphylococcaceae, Neisseriaceae, vancomycin and metronidazole contributing towards cluster 2. Lachnospiraceae, Ruminococcaceae, Bifidobacteriaceae and not being on total parenteral nutrition contributed to cluster 1. Untargeted metabolomic analyses revealed changes that paralleled fluctuations in microbiota composition; importantly, low faecal butyrate was associated with a higher risk of viraemia. CONCLUSIONS: These findings highlight the frequent shifts and dominations in the gut microbiota of paediatric patients undergoing haematopoietic stem cell transplantation. The study reveals associations between the faecal microbiota, metabolome and viraemia. To identify and explore the potential of microbial biomarkers that may predict the risk of complications post-HSCT, larger multi-centre studies investigating the longitudinal microbial profiling in paediatric haematopoietic stem cell transplantation are warranted. Video abstract.
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Microbioma Gastrointestinal , Enfermedad Injerto contra Huésped , Trasplante de Células Madre Hematopoyéticas , Niño , Clostridiales , Enterobacteriaceae , Heces , Trasplante de Células Madre Hematopoyéticas/efectos adversos , Humanos , Metaboloma , Viremia/etiologíaRESUMEN
Sudden unexpected death in infancy (SUDI) is the sudden and unexpected death of an apparently healthy infant occurring within the first year of life where the cause is not immediately obvious. It is believed that a proportion of unexplained infant deaths are due to an infection that remains undiagnosed. The interpretation of post-mortem microbiology results is difficult due to the potential false-positives, a source of which is post-mortem bacterial translocation. Post-mortem bacterial translocation is the spread of viable bacteria from highly colonised sites to extra-intestinal tissues. We hypothesise that although post-mortem bacterial translocation occurs, when carcasses are kept under controlled routine clinical conditions it is not extensive and can be defined using 16S rRNA gene sequencing. With this knowledge, implementation of the 16S rRNA gene sequencing technique into routine clinical diagnostics would allow a more reliable retrospective diagnosis of ante-mortem infection. Therefore, the aim of this study was to establish the extent of post-mortem bacterial translocation in two animal models to establish a baseline sequencing signal for the post-mortem process. To do this we used 16S rRNA gene sequencing in two animal models over a 2 week period to investigate (1) the bacterial community succession in regions of high bacterial colonisation, and (2) the bacterial presence in visceral tissues routinely sampled during autopsy for microbiological investigation. We found no evidence for significant and consistent post-mortem bacterial translocation in the mouse model. Although bacteria were detected in tissues in the piglet model, we did not find significant and consistent evidence for post-mortem bacterial translocation from the gastrointestinal tract or nasal cavity. These data do not support the concept of significant post-mortem translocation as part of the normal post-mortem process.
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BACKGROUND: Evidence suggests an important role for gut-microbiota dysbiosis in the development of rheumatoid arthritis (RA). The link between changes in gut bacteria and the development of joint inflammation is missing. Here, we address whether there are changes to the gut environment and how they contribute to arthritis pathogenesis. METHODS: We analyzed changes in markers of gut permeability, damage, and inflammation in peripheral blood and serum of RA patients. Serum, intestines, and lymphoid organs isolated from K/BxN mice with spontaneous arthritis or from wild-type, genetically modified interleukin (IL)-10R-/-or claudin-8-/-mice with induced arthritis were analyzed by immunofluorescence/histology, ELISA, and flow cytometry. FINDINGS: RA patients display increased levels of serum markers of gut permeability and damage and cellular gut-homing markers, both parameters positively correlating with disease severity. Arthritic mice display increased gut permeability from early stages of disease, as well as bacterial translocation, inflammatory gut damage, increases in interferon γ (IFNγ)+and decreases in IL-10+intestinal-infiltrating leukocyte frequency, and reduced intestinal epithelial IL-10R expression. Mechanistically, both arthritogenic bacteria and leukocytes are required to disrupt gut-barrier integrity. We show that exposing intestinal organoids to IFNγ reduces IL-10R expression by epithelial cells and that mice lacking epithelial IL-10R display increased intestinal permeability and exacerbated arthritis. Claudin-8-/-mice with constitutively increased gut permeability also develop worse joint disease. Treatment of mice with AT-1001, a molecule that prevents development of gut permeability, ameliorates arthritis. CONCLUSIONS: We suggest that breakdown of gut-barrier integrity contributes to arthritis development and propose restoration of gut-barrier homeostasis as a new therapeutic approach for RA. FUNDING: Funded by Versus Arthritis (21140 and 21257) and UKRI/MRC (MR/T000910/1).
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Artritis Reumatoide , Microbioma Gastrointestinal , Enfermedades Intestinales , Animales , Artritis Reumatoide/metabolismo , Disbiosis/metabolismo , Humanos , Inflamación/metabolismo , Enfermedades Intestinales/metabolismo , Mucosa Intestinal/metabolismo , RatonesRESUMEN
The differentiation of IL-10-producing regulatory B cells (Bregs) in response to gut-microbiota-derived signals supports the maintenance of tolerance. However, whether microbiota-derived metabolites can modulate Breg suppressive function remains unknown. Here, we demonstrate that rheumatoid arthritis (RA) patients and arthritic mice have a reduction in microbial-derived short-chain fatty acids (SCFAs) compared to healthy controls and that in mice, supplementation with the SCFA butyrate reduces arthritis severity. Butyrate supplementation suppresses arthritis in a Breg-dependent manner by increasing the level of the serotonin-derived metabolite 5-Hydroxyindole-3-acetic acid (5-HIAA), which activates the aryl-hydrocarbon receptor (AhR), a newly discovered transcriptional marker for Breg function. Thus, butyrate supplementation via AhR activation controls a molecular program that supports Breg function while inhibiting germinal center (GC) B cell and plasmablast differentiation. Our study demonstrates that butyrate supplementation may serve as a viable therapy for the amelioration of systemic autoimmune disorders.
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Artritis Reumatoide/metabolismo , Linfocitos B Reguladores/metabolismo , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico , Butiratos/farmacología , Ácidos Grasos Volátiles/metabolismo , Receptores de Hidrocarburo de Aril , Animales , Linfocitos B Reguladores/citología , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Células Cultivadas , Femenino , Microbioma Gastrointestinal , Humanos , Ácido Hidroxiindolacético/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Persona de Mediana Edad , Receptores de Hidrocarburo de Aril/metabolismoRESUMEN
Respiratory syncytial virus (RSV) is the cause of one-fifth of all lower respiratory tract infections worldwide and is increasingly being recognized as representing a serious threat to patient groups with poorly functioning or immature immune systems. Racemic 1,4-benzodiazepines show potent anti-RSV activity in vitro. Anti-RSV evaluation of 3-position R- and S-benzodiazepine enantiomers and subsequent optimization of this series resulted in selection of a clinical candidate. Antiviral activity was found to reside mainly in the S-enantiomer, and the R-enantiomers were consistently less active against RSV. Analogues of 1,4-(S)-benzodiazepine were synthesized as part of the lead optimization program at Arrow and tested in the XTT assay. From this exercise, (S)-1-(2-fluorophenyl)-3-(2-oxo-5-phenyl-2,3-dihydro-1H-benzo[e][1,4]-diazepin-3-yl)-urea, 17b (RSV-604) was identified as a clinical candidate, exhibiting potent anti-RSV activity in the XTT assay, which was confirmed in secondary assays. Compound 17b also possessed a good pharmacokinetic profile and has now progressed into the clinic.
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Antivirales/síntesis química , Benzodiazepinas/síntesis química , Benzodiazepinonas/síntesis química , Compuestos de Fenilurea/síntesis química , Virus Sincitiales Respiratorios/efectos de los fármacos , Animales , Antivirales/farmacocinética , Antivirales/farmacología , Benzodiazepinas/farmacocinética , Benzodiazepinas/farmacología , Benzodiazepinonas/farmacocinética , Benzodiazepinonas/farmacología , Línea Celular Tumoral , Cristalografía por Rayos X , Perros , Ensayo de Inmunoadsorción Enzimática , Humanos , Técnicas In Vitro , Microsomas/metabolismo , Estructura Molecular , Compuestos de Fenilurea/farmacocinética , Compuestos de Fenilurea/farmacología , Ratas , Estereoisomerismo , Relación Estructura-Actividad , Ensayo de Placa ViralRESUMEN
Respiratory syncytial virus (RSV) is the cause of one-fifth of all lower respiratory tract infections worldwide and is increasingly being recognized as a serious threat to patient groups with poorly functioning immune systems. Our approach to finding a novel inhibitor of this virus was to screen a 20 000-member diverse library in a whole cell XTT assay. Parallel assays were carried out in the absence of virus in order to quantify any associated cell toxicity. This identified 100 compounds with IC(50)'s less than 50 muM. A-33903 (18), a 1,4-benzodiazepine analogue, was chosen as the starting point for lead optimization. This molecule was moderately active and demonstrated good pharmacokinetic properties. The most potent compounds identified from this work were A-58568 (47), A-58569 (44), and A-62066 (46), where modifications to the aromatic substitution enhanced potency, and A-58175 (42), where the amide linker was modified.
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Antivirales/síntesis química , Benzodiazepinas/síntesis química , Virus Sincitiales Respiratorios/efectos de los fármacos , Animales , Antivirales/farmacocinética , Antivirales/farmacología , Benzodiazepinas/química , Benzodiazepinas/farmacología , Línea Celular Tumoral , Técnicas Químicas Combinatorias , Ensayo de Inmunoadsorción Enzimática , Ratas , Ratas Sprague-Dawley , Relación Estructura-Actividad , Ensayo de Placa ViralRESUMEN
Viral and bacterial infectious agents have been implicated in the etiology of atherosclerosis. We have previously shown that a gamma-herpesvirus can accelerate atherosclerosis in the apolipoprotein E-deficient (apoE-/-) mouse. To address whether a virally induced systemic immune response is sufficient to trigger enhanced atheroma formation, we infected apoE-/- mice with murine gamma-herpesvirus-68 (MHV-68) or herpes simplex virus-1 (HSV-1). In this study, we show that both viruses were able to induce a cell-mediated and humoral immune response in the apoE-/- mouse, which was sustained over a period of 24 weeks. Although intranasal or intraperitoneal infection with MHV-68 induced similar levels of virus-specific IgG1 and IgG2a antibodies in the serum of apoE-/- mice, those infected with HSV-1 showed higher anti-HSV-1 IgG2a compared with IgG1 antibody levels. In addition, viral message was not detected in the aortas of HSV-1-infected animals, whereas we have shown previously that MHV-68 mRNA can be detected in the aortas of infected mice as early as 5 days after infection. Compared with control mice, apoE-/- mice infected with MHV-68 showed accelerated atherosclerosis, whereas mice infected with HSV-1 did not. These data indicate that a systemic immune response to any particular infectious agent is insufficient to induce enhanced atherosclerosis in the apoE-/- mouse and point to specific infections or immune mechanisms that might be essential for virally enhanced atherogenesis.
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Apolipoproteínas E/deficiencia , Arteriosclerosis/virología , Herpes Simple/inmunología , Infecciones por Herpesviridae/inmunología , Herpesvirus Humano 1/patogenicidad , Rhadinovirus/patogenicidad , Animales , Anticuerpos Antivirales/biosíntesis , Aorta/virología , Arteriosclerosis/inmunología , Chlorocebus aethiops , Modelos Animales de Enfermedad , Herpesvirus Humano 1/crecimiento & desarrollo , Herpesvirus Humano 1/aislamiento & purificación , Inmunidad Celular , Inmunoglobulina G/biosíntesis , Isotipos de Inmunoglobulinas/biosíntesis , Pulmón/virología , Ratones , Ratones Endogámicos C57BL , ARN Viral/aislamiento & purificación , Rhadinovirus/crecimiento & desarrollo , Rhadinovirus/aislamiento & purificación , Bazo/virología , Infecciones Tumorales por Virus/inmunología , Células Vero/virología , Cultivo de Virus/métodosRESUMEN
Chronic rhinosinusitis (CRS) is a common and potentially debilitating disease characterized by inflammation of the sinus mucosa for longer than 12 weeks. Bacterial colonization of the sinuses and its role in the pathogenesis of this disease is an ongoing area of research. Recent advances in culture-independent molecular techniques for bacterial identification have the potential to provide a more accurate and complete assessment of the sinus microbiome, however there is little concordance in results between studies, possibly due to differences in the sampling location and techniques. This study aimed to determine whether the microbial communities from one sinus could be considered representative of all sinuses, and examine differences between two commonly used methods for sample collection, swabs, and tissue biopsies. High-throughput DNA sequencing of the bacterial 16S rRNA gene was applied to both swab and tissue samples from multiple sinuses of 19 patients undergoing surgery for treatment of CRS. Results from swabs and tissue biopsies showed a high degree of similarity, indicating that swabbing is sufficient to recover the microbial community from the sinuses. Microbial communities from different sinuses within individual patients differed to varying degrees, demonstrating that it is possible for distinct microbiomes to exist simultaneously in different sinuses of the same patient. The sequencing results correlated well with culture-based pathogen identification conducted in parallel, although the culturing missed many species detected by sequencing. This finding has implications for future research into the sinus microbiome, which should take this heterogeneity into account by sampling patients from more than one sinus.
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The flavonoid components of New Zealand manuka (Leptospermum scoparium) honey have been quantified in a series of 31 honeys of varying non-peroxide antibacterial activity to clarify discrepancies between previous studies reported in the literature. Total flavonoid content was 1.16 mg/100 g honey. The principal flavonoids present were pinobanksin, pinocembrin, luteolin and chrysin and together these represented 61% of the total flavonoid content. 1, 2-formyl-5-(2-methoxyphenyl)-pyrrole, which was weakly correlated with the non-peroxide antibacterial activity, was isolated from the flavonoid fraction and separately synthesised. 1 did not display inhibitory activity against Staphylococcus aureus in vitro and thus the origin of the correlation, which is still unknown, is not a direct contribution.
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Flavonoides/química , Miel/análisis , Leptospermum/química , Pirroles/química , Antibacterianos/síntesis química , Antibacterianos/química , Antibacterianos/farmacología , Flavonoides/aislamiento & purificación , Flavonoides/farmacología , Pruebas de Sensibilidad Microbiana , Pirroles/síntesis química , Pirroles/aislamiento & purificación , Pirroles/farmacología , Staphylococcus aureus/efectos de los fármacosRESUMEN
Skin and chronic wound infections caused by highly antibiotic resistant bacteria such as methicillin-resistant Staphylococcus aureus (MRSA) are an increasing and urgent health problem worldwide, particularly with sharp increases in obesity and diabetes. New Zealand manuka honey has potent broad-spectrum antimicrobial activity, has been shown to inhibit the growth of MRSA strains, and bacteria resistant to this honey have not been obtainable in the laboratory. Combinational treatment of chronic wounds with manuka honey and common antibiotics may offer a wide range of advantages including synergistic enhancement of the antibacterial activity, reduction of the effective dose of the antibiotic, and reduction of the risk of antibiotic resistance. The aim of this study was to investigate the effect of Medihoney in combination with the widely used antibiotic rifampicin on S. aureus. Using checkerboard microdilution assays, time-kill curve experiments and agar diffusion assays, we show a synergism between Medihoney and rifampicin against MRSA and clinical isolates of S. aureus. Furthermore, the Medihoney/rifampicin combination stopped the appearance of rifampicin-resistant S. aureus in vitro. Methylglyoxal (MGO), believed to be the major antibacterial compound in manuka honey, did not act synergistically with rifampicin and is therefore not the sole factor responsible for the synergistic effect of manuka honey with rifampicin. Our findings support the idea that a combination of honey and antibiotics may be an effective new antimicrobial therapy for chronic wound infections.
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Antibacterianos/farmacología , Leptospermum/química , Staphylococcus aureus Resistente a Meticilina/efectos de los fármacos , Extractos Vegetales/farmacología , Rifampin/farmacología , Antibacterianos/uso terapéutico , Farmacorresistencia Bacteriana/efectos de los fármacos , Sinergismo Farmacológico , Staphylococcus aureus Resistente a Meticilina/fisiología , Extractos Vegetales/química , Extractos Vegetales/uso terapéutico , Rifampin/uso terapéutico , Riesgo , Infecciones Cutáneas Estafilocócicas/tratamiento farmacológicoRESUMEN
Respiratory syncytial virus (RSV) is the most common cause of lower respiratory tract infections worldwide, yet no effective vaccine or antiviral treatment is available. Here we report the discovery and initial development of RSV604, a novel benzodiazepine with submicromolar anti-RSV activity. It proved to be equipotent against all clinical isolates tested of both the A and B subtypes of the virus. The compound has a low rate of in vitro resistance development. Sequencing revealed that the resistant virus had mutations within the nucleocapsid protein. This is a novel mechanism of action for anti-RSV compounds. In a three-dimensional human airway epithelial cell model, RSV604 was able to pass from the basolateral side of the epithelium effectively to inhibit virus replication after mucosal inoculation. RSV604, which is currently in phase II clinical trials, represents the first in a new class of RSV inhibitors and may have significant potential for the effective treatment of RSV disease.
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Antivirales/farmacología , Benzodiazepinonas/farmacología , Compuestos de Fenilurea/farmacología , Virus Sincitiales Respiratorios/efectos de los fármacos , Secuencia de Aminoácidos , Antivirales/síntesis química , Benzodiazepinonas/síntesis química , Línea Celular , Fenómenos Químicos , Química Física , Efecto Citopatogénico Viral , Relación Dosis-Respuesta a Droga , Farmacorresistencia Viral/genética , Células Epiteliales/efectos de los fármacos , Células Epiteliales/virología , Humanos , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Mutación , Proteínas de la Nucleocápside/efectos de los fármacos , Compuestos de Fenilurea/síntesis química , Virus Sincitiales Respiratorios/genética , Sales de Tetrazolio , Replicación Viral/efectos de los fármacosRESUMEN
Gammaherpesviruses are associated with a number of diseases including lymphomas and other malignancies. Murine gammaherpesvirus 68 (MHV-68) constitutes the most amenable animal model for this family of pathogens. However experimental characterization of gammaherpesvirus gene expression, at either the protein or RNA level, lags behind that of other, better-studied alpha- and beta-herpesviruses. We have developed a cDNA array to globally characterize MHV-68 gene expression profiles, thus providing an experimental supplement to a genome that is chiefly annotated by homology. Viral genes started to be transcribed as early as 3 h postinfection (p.i.), and this was followed by a rapid escalation of gene expression that could be seen at 5 h p.i. Individual genes showed their own transcription profiles, and most genes were still being expressed at 18 h p.i. Open reading frames (ORFs) M3 (chemokine-binding protein), 52, and M9 (capsid protein) were particularly noticeable due to their very high levels of expression. Hierarchical cluster analysis of transcription profiles revealed four main groups of genes and allowed functional predictions to be made by comparing expression profiles of uncharacterized genes to those of genes of known function. Each gene was also categorized according to kinetic class by blocking de novo protein synthesis and viral DNA replication in vitro. One gene, ORF 73, was found to be expressed with alpha-kinetics, 30 genes were found to be expressed with beta-kinetics, and 42 genes were found to be expressed with gamma-kinetics. This fundamental characterization furthers the development of this model and provides an experimental basis for continued investigation of gammaherpesvirus pathology.