RESUMEN
Repurposing of approved drugs that target host functions also important for virus replication promises to overcome the shortage of antiviral therapeutics. Mostly, virus biology including initial screening of antivirals is studied in conventional monolayer cells. The biology of these cells differs considerably from infected tissues. 3D culture models with characteristics of human tissues may reflect more realistically the in vivo events during infection. We screened first, second, and third generation epidermal growth factor receptor (EGFR)-inhibitors with different modes of action and the EGFR-blocking monoclonal antibody cetuximab in a 3D cell culture infection model with primary human keratinocytes and cowpox virus (CPXV) for antiviral activity. Antiviral activity of erlotinib and osimertinib was nearly unaffected by the cultivation method similar to the virus-directed antivirals tecovirimat and cidofovir. In contrast, the host-directed inhibitors afatinib and cetuximab were approx. 100-fold more efficient against CPXV in the 3D infection model, similar to previous results with gefitinib. In summary, inhibition of EGFR-signaling downregulates virus replication comparable to established virus-directed antivirals. However, in contrast to virus-directed inhibitors, in vitro efficacy of host-directed antivirals might be seriously affected by cell cultivation. Results obtained for afatinib and cetuximab suggest that screening of such drugs in standard monolayer culture might underestimate their potential as antivirals.
Asunto(s)
Antivirales/farmacología , Técnicas de Cultivo de Célula , Descubrimiento de Drogas/métodos , Línea Celular , Supervivencia Celular/efectos de los fármacos , Sinergismo Farmacológico , Receptores ErbB/antagonistas & inhibidores , Receptores ErbB/metabolismo , Ensayos Analíticos de Alto Rendimiento/métodos , Poxviridae/efectos de los fármacos , Poxviridae/fisiología , Cultivo Primario de Células , Inhibidores de Proteínas Quinasas/farmacología , Esferoides Celulares , Replicación ViralRESUMEN
Studies of virus-host interactions in vitro may be hindered by biological characteristics of conventional monolayer cell cultures that differ from in vivo infection. Three-dimensional (3D) cell cultures show more in vivo-like characteristics and may represent a promising alternative for characterisation of infections. In this study, we established easy-to-handle cell culture platforms based on bioprinted 3D matrices for virus detection and characterisation. Different cell types were cultivated on these matrices and characterised for tissue-like growth characteristics regarding cell morphology and polarisation. Cells developed an in vivo-like morphology and long-term cultivation was possible on the matrices. Cell cultures were infected with viruses which differed in host range, tissue tropism, cytopathogenicity, and genomic organisation and virus morphology. Infections were characterised on molecular and imaging level. The transparent matrix substance allowed easy optical monitoring of cells and infection even via live-cell microscopy. In conclusion, we established an enhanced, standardised, easy-to-handle bioprinted 3D-cell culture system. The infection models are suitable for sensitive monitoring and characterisation of virus-host interactions and replication of different viruses under physiologically relevant conditions. Individual cell culture models can further be combined to a multicellular array. This generates a potent diagnostic tool for propagation and characterisation of viruses from diagnostic samples.
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Bioimpresión/métodos , Técnicas de Cultivo de Célula , Interacciones Huésped-Patógeno , Virosis/diagnóstico , Virosis/virología , Animales , Biomarcadores , Supervivencia Celular , Chlorocebus aethiops , Humanos , Imagen Molecular , Esferoides Celulares , Células VeroRESUMEN
The Global Virus Network (GVN) was established in 2011 to strengthen research and responses to emerging viral causes of human disease and to prepare against new viral pandemics. There are now 52 GVN Centers of Excellence and 9 Affiliate laboratories in 32 countries. The 11th International GVN meeting was held from June 9-11, 2019 in Barcelona, Spain and was jointly organized with the Spanish Society of Virology. A common theme throughout the meeting was globalization and climate change. This report highlights the recent accomplishments of GVN researchers in several important areas of medical virology, including severe virus epidemics, anticipation and preparedness for changing disease dynamics, host-pathogen interactions, zoonotic virus infections, ethical preparedness for epidemics and pandemics, one health and antivirals.
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Enfermedades Transmisibles Emergentes , Salud Global , Salud Única/tendencias , Virosis , Animales , Antivirales , Arbovirus/efectos de los fármacos , Arbovirus/genética , Arbovirus/metabolismo , Cambio Climático , Enfermedades Transmisibles Emergentes/tratamiento farmacológico , Enfermedades Transmisibles Emergentes/epidemiología , Enfermedades Transmisibles Emergentes/transmisión , Coronavirus/efectos de los fármacos , Coronavirus/genética , Coronavirus/metabolismo , Ebolavirus/efectos de los fármacos , Ebolavirus/genética , Ebolavirus/metabolismo , Fiebre Hemorrágica Ebola/tratamiento farmacológico , Fiebre Hemorrágica Ebola/prevención & control , Hepatitis B/tratamiento farmacológico , Hepatitis B/epidemiología , Hepatitis B/prevención & control , Interacciones Huésped-Patógeno , Humanos , Gripe Humana/tratamiento farmacológico , Gripe Humana/epidemiología , Gripe Humana/prevención & control , Internacionalidad , Pandemias , Vacunas Virales , Virosis/tratamiento farmacológico , Virosis/epidemiología , Virosis/transmisión , Virus/efectos de los fármacos , Virus/genética , Virus/metabolismo , Zoonosis/tratamiento farmacológico , Zoonosis/epidemiología , Zoonosis/transmisiónRESUMEN
An external quality assessment of yellow fever virus (YFV) molecular detection in European laboratories was organised in rapid response to an increase in human cases in Brazil in 2018 with risk of import to Europe. Detection of YFV was assessed among 32 laboratories in 23/31 European Union (EU) and European Economic Area (EEA) countries and two laboratories in one non-EU/EEA country. Adequate capabilities were lacking in 10/23 countries; five did not participate as they lacked implemented assays.
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Laboratorios/normas , Técnicas de Diagnóstico Molecular/normas , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/normas , Pruebas Serológicas/normas , Fiebre Amarilla/diagnóstico , Virus de la Fiebre Amarilla/genética , Virus de la Fiebre Amarilla/aislamiento & purificación , Humanos , Técnicas de Diagnóstico Molecular/métodos , Reacción en Cadena de la Polimerasa , Vigilancia de la Población/métodos , Pruebas Serológicas/métodos , Fiebre Amarilla/inmunología , Fiebre Amarilla/virología , Virus de la Fiebre Amarilla/inmunologíaRESUMEN
Rodent species like Myodes glareolus and Microtus spp. are natural reservoirs for many zoonotic pathogens causing human diseases and are gaining increasing interest in the field of eco-immunology as candidate animal models. Despite their importance the lack of immunological reagents has hampered research in these animal species. Here we report the recombinant production and functional characterization of IFN-γ, a central mediator of host's innate and adaptive immune responses, from the bank vole M. glareolus. Soluble dimeric recMgIFN-γ was purified in high yield from Escherichia coli. Its activity on M. glareolus and Microtus arvalis kidney cell lines was assessed by immunofluorescent detection of nuclear translocation and phosphorylation of the transcription factor STAT1. RecMgIFN-γ also induced expression of an IFN-γ-regulated innate immunity gene. Inhibition of vesicular stomatitis virus replication in vole cells upon recMgIFN-γ treatment provided further evidence of its biological activity. Finally, we established a recMgIFN-γ-responsive bank vole reporter cell line that allows the sensitive titration of the cytokine activity via a bioluminescence reporter assay. Taken together, we report valuable tools for future investigations on the immune response against zoonotic pathogens in their natural animal hosts, which might foster the development of novel animal models.
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Interferón gamma/farmacología , Animales , Arvicolinae/genética , Arvicolinae/inmunología , Arvicolinae/metabolismo , Línea Celular , Riñón/metabolismo , Ratones , Filogenia , Proteínas Recombinantes/farmacología , Proyectos de Investigación , Enfermedades de los Roedores/virología , Factor de Transcripción STAT1/metabolismo , Homología de Secuencia de Aminoácido , Zoonosis/metabolismoRESUMEN
Gefitinib is a specific inhibitor of the epidermal growth factor receptor (EGFR) and FDA approved for treatment of non-small cell lung cancer. In a previous study we could show the in vitro efficacy of gefitinib for treatment of poxvirus infections in monolayer (2D) cultivated cell lines. Permanent cell lines and 2D cultures, however, are known to be rather unphysiological; therefore it is difficult to predict whether determined effective concentrations or the drug efficacy per se are transferable to the in vivo situation. 3D cell cultures, which meanwhile are widely distributed across all fields of research, are a promising tool for more predictive in vitro investigations of antiviral compounds. In this study the spreading of cowpox virus and the antiviral efficacy of gefitinib were analyzed in primary human keratinocytes (NHEK) grown in a novel 3D extracellular matrix-based cell culture model and compared to the respective monolayer culture. 3D-cultivated NHEK grew in a polarized and thus a more physiological manner with altered morphology and close cell-cell contact. Infected cultures showed a strongly elevated sensitivity towards gefitinib. EGFR phosphorylation, cell proliferation, and virus replication were significantly reduced in 3D cultures at gefitinib concentrations which were at least 100-fold lower than those in monolayer cultures and well below the level of cytotoxicity. Our newly established 3D cell culture model with primary human cells is an easy-to-handle alternative to conventional monolayer cell cultures and previously described more complex 3D cell culture systems. It can easily be adapted to other cell types and a broad spectrum of viruses for antiviral drug screening and many other aspects of virus research under more in vivo-like conditions. In consequence, it may contribute to a more targeted realization of necessary in vivo experiments.
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Antivirales/farmacología , Técnicas de Cultivo de Célula , Descubrimiento de Drogas , Evaluación Preclínica de Medicamentos/métodos , Animales , Línea Celular , Chlorocebus aethiops , Receptores ErbB/metabolismo , Técnica del Anticuerpo Fluorescente , Humanos , Inmunohistoquímica , Queratinocitos/efectos de los fármacos , Queratinocitos/virología , Fosforilación , Células Vero , Virus/efectos de los fármacosRESUMEN
Experimental intranasal infection of marmosets (Callithrix jacchus) with calpox virus results in fatal disease. Route and dose used for viral inoculation of the test animals mimics the natural transmission of smallpox, thus representing a suitable model to study pathogenesis and to evaluate new vaccines against orthopoxvirus infection. However, the pathogenic mechanisms leading to death are still unclear. Therefore, our study aimed at investigating the kinetics of pathological alterations to clarify the pathogenesis in calpox virus infection. Following intranasal inoculation with two different viral doses, common marmosets were sacrificed on days 3, 5, 7, 10 and 12 post inoculation. Collected tissue was screened using histopathology, immunohistochemistry, transmission electron microscopy, and virological assays. Our data suggest that primary replication took place in nasal and bronchial epithelia followed by secondary replication in submandibular lymph nodes and spleen. Parallel to viremia at day 7, virus was detectable in many organs, mainly located in epithelial cells and macrophages, as well as in endothelial cells. Based on the onset of clinical signs, the histological and ultrastructural lesions and the immunohistochemical distribution pattern of the virus, the incubation period was defined to last 11 days, which resembles human smallpox. In conclusion, the data indicate that the calpox model is highly suitable for studying orthopoxvirus-induced disease.
Asunto(s)
Callithrix , Modelos Animales de Enfermedad , Orthopoxvirus/patogenicidad , Infecciones por Poxviridae/patología , Administración Intranasal , Animales , Bronquios/virología , Femenino , Inmunohistoquímica , Masculino , Microscopía Electrónica de Transmisión , Mucosa Nasal/virología , Orthopoxvirus/genética , Orthopoxvirus/fisiología , Infecciones por Poxviridae/transmisión , Infecciones por Poxviridae/virología , Viruela/patología , Viruela/transmisión , Viruela/virología , Bazo/patología , Bazo/virología , Virus de la Viruela/genética , Virus de la Viruela/patogenicidad , Virus de la Viruela/fisiología , Carga Viral , Tropismo Viral , Viremia/virología , Replicación ViralRESUMEN
During the recent Ebola outbreak in West Africa several international mobile laboratories were deployed to the mainly affected countries Guinea, Sierra Leone and Liberia to provide ebolavirus diagnostic capacity. Additionally, imported cases and small outbreaks in other countries required global preparedness for Ebola diagnostics. Detection of viral RNA by reverse transcription polymerase chain reaction has proven effective for diagnosis of ebolavirus disease and several assays are available. However, reliability of these assays is largely unknown and requires serious evaluation. Therefore, a proficiency test panel of 11 samples was generated and distributed on a global scale. Panels were analyzed by 83 expert laboratories and 106 data sets were returned. From these 78 results were rated optimal and 3 acceptable, 25 indicated need for improvement. While performance of the laboratories deployed to West Africa was superior to the overall performance there was no significant difference between the different assays applied.
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Fiebre Hemorrágica Ebola/diagnóstico , Laboratorios/normas , Reacción en Cadena de la Polimerasa/métodos , Garantía de la Calidad de Atención de Salud , África Occidental/epidemiología , Ebolavirus , Fiebre Hemorrágica Ebola/epidemiología , Humanos , Reproducibilidad de los ResultadosRESUMEN
The Global Virus Network (GVN) was established in 2011 in order to strengthen research and responses to current viral causes of human disease and to prepare against new viral pandemic threats. There are now 38 GVN Centers of Excellence and 6 Affiliate laboratories in 24 countries. GVN scientists meet annually to learn about each other's current research, address collaborative priorities and plan future programs. The 2016 meeting was held from October 23-25 in Hokkaido, Japan, in partnership with the Japanese Society for Virology, the National Institute of Infectious Diseases of Japan and the Research Center for Zoonosis Control of Hokkaido University. This report highlights the accomplishments of GVN researchers in many priority areas of medical virology, including the current Zika epidemic, infections by human papillomavirus, influenza, Ebola, Lassa, dengue, HIV, hepatitis C, and chikungunya viruses, and the development of improved diagnostics and new vaccines.
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Enfermedades Transmisibles/virología , Cooperación Internacional , Virus/patogenicidad , Animales , Enfermedades Transmisibles/epidemiología , Enfermedades Transmisibles/terapia , Congresos como Asunto , Brotes de Enfermedades , Monitoreo Epidemiológico , Salud Global , Humanos , Japón , Pandemias , Investigación , ZoonosisRESUMEN
This study was undertaken to investigate the susceptibility of rhesus monkeys to the calpox virus, an orthopoxvirus (OPXV) of the Cowpox virus species (CPXV), which is uniformly lethal in common marmosets. Six rhesus monkeys were either intravenously (i.v.) or intranasally (i.n.) exposed to the virus. Monitoring of the macaques after viral exposure included physical examinations, the determination of viral load by real-time PCR and plaque assay, and the analysis of humoral responses. Two i.v. inoculated animals developed numerous classical pox lesions that started after inoculation at days 7 and 10. Both animals became viremic and seroconverted. They exhibited maximal numbers of lesions of approximately 50 and 140 by day 21. One animal completely recovered, while the other one suffered from a phlegmonous inflammation of a leg initially induced by a secondarily infected pox lesion and was euthanized for animal welfare reasons. In contrast to previous pathogenicity studies with the calpox virus in marmosets, none of the four animals inoculated intranasally with doses of the calpox virus exceeding those used in marmosets by orders of magnitude showed typical clinical symptoms. No viral DNA was detectable in the blood of those animals, but three animals seroconverted. In two of these three animals, infectious virus was sporadically isolated from saliva. This indicates that rhesus monkeys are less susceptible to calpox virus infection, which limits their use in further intervention studies with OPXV.
RESUMEN
BACKGROUND: A unit of the European Mobile Laboratory (EMLab) consortium was deployed to the Ebola virus disease (EVD) treatment unit in Guéckédou, Guinea, from March 2014 through March 2015. METHODS: The unit diagnosed EVD and malaria, using the RealStar Filovirus Screen reverse transcription-polymerase chain reaction (RT-PCR) kit and a malaria rapid diagnostic test, respectively. RESULTS: The cleaned EMLab database comprised 4719 samples from 2741 cases of suspected EVD from Guinea. EVD was diagnosed in 1231 of 2178 hospitalized patients (57%) and in 281 of 563 who died in the community (50%). Children aged <15 years had the highest proportion of Ebola virus-malaria parasite coinfections. The case-fatality ratio was high in patients aged <5 years (80%) and those aged >74 years (90%) and low in patients aged 10-19 years (40%). On admission, RT-PCR analysis of blood specimens from patients who died in the hospital yielded a lower median cycle threshold (Ct) than analysis of blood specimens from survivors (18.1 vs 23.2). Individuals who died in the community had a median Ct of 21.5 for throat swabs. Multivariate logistic regression on 1047 data sets revealed that low Ct values, ages of <5 and ≥45 years, and, among children aged 5-14 years, malaria parasite coinfection were independent determinants of a poor EVD outcome. CONCLUSIONS: Virus load, age, and malaria parasite coinfection play a role in the outcome of EVD.
Asunto(s)
Ebolavirus/aislamiento & purificación , Epidemias , Infecciones por Filoviridae/diagnóstico , Fiebre Hemorrágica Ebola/diagnóstico , Malaria/complicaciones , Unidades Móviles de Salud , Adolescente , Adulto , Anciano , Niño , Preescolar , Servicios de Laboratorio Clínico , Ebolavirus/genética , Femenino , Filoviridae , Infecciones por Filoviridae/complicaciones , Infecciones por Filoviridae/virología , Guinea , Fiebre Hemorrágica Ebola/complicaciones , Fiebre Hemorrágica Ebola/virología , Humanos , Lactante , Malaria/parasitología , Masculino , Persona de Mediana Edad , ARN Viral/sangre , Carga Viral , Adulto JovenRESUMEN
The Hepatitis C virus (HCV) infection exhibits a high global prevalence frequently associated with hepatocellular carcinoma, taking years to develop. Despite the standardization of highly sensitive HCV quantitative RT-PCR (qRT-PCR) detection methods, false-negative diagnoses may be generated with current methods, mainly due to the presence of PCR inhibitors and/or low viral loads in the patient's sample. These false-negative diagnoses impact both public health systems, in developing countries, and an in lesser extent, in developed countries, including both the risk of virus transmission during organ transplantation and/or blood transfusion and the quality of the antiviral treatment monitoring. To adopt an appropriate therapeutic strategy to improve the patient's prognosis, it is urgent to increase the HCV detection sensitivity. Based upon previous studies on HBV, we worked on the capacity of the scavenger acute phase protein, Apolipoprotein H (ApoH) to interact with HCV. Using different approaches, including immunoassays, antibody-inhibition, oxidation, ultracentrifugation, electron microscopy and RT-PCR analyses, we demonstrated specific interactions between HCV particles and ApoH. Moreover, when using a two-step HCV detection process, including capture of HCV by ApoH-coated nanomagnetic beads and a home-made real-time HCV-RT-PCR, we confirmed the presence of HCV for all samples from a clinical collection of HCV-seropositive patients exhibiting an RT-PCR COBAS® TaqMan® HCV Test, v2.0 (COBAS)-positive result. In contrast, for HCV-seropositive patients with either low HCV-load as determined with COBAS or exhibiting HCV-negative COBAS results, the addition of the two-step ApoH-HCV-capture and HCV-detection process was able to increase the sensitivity of HCV detection or more interestingly, detect in a genotype sequence-independent manner, a high-proportion (44%) of HCV/RNA-positive among the COBAS HCV-negative patients. Thus, the immune interaction between ApoH and HCV could be used as a sample preparation tool to enrich and/or cleanse HCV patient's samples to enhance the detection sensitivity of HCV and therefore significantly reduce the numbers of false-negative HCV diagnosis results.
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Proteínas de Fase Aguda/metabolismo , Hepacivirus/metabolismo , Hepatitis C/diagnóstico , beta 2 Glicoproteína I/metabolismo , Proteínas de Fase Aguda/inmunología , Centrifugación por Gradiente de Densidad/métodos , Reacciones Falso Negativas , Hepacivirus/inmunología , Humanos , Técnicas para Inmunoenzimas/métodos , Microscopía Electrónica , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Sensibilidad y Especificidad , beta 2 Glicoproteína I/inmunologíaRESUMEN
West Africa is currently witnessing the most extensive Ebola virus (EBOV) outbreak so far recorded. Until now, there have been 27,013 reported cases and 11,134 deaths. The origin of the virus is thought to have been a zoonotic transmission from a bat to a two-year-old boy in December 2013 (ref. 2). From this index case the virus was spread by human-to-human contact throughout Guinea, Sierra Leone and Liberia. However, the origin of the particular virus in each country and time of transmission is not known and currently relies on epidemiological analysis, which may be unreliable owing to the difficulties of obtaining patient information. Here we trace the genetic evolution of EBOV in the current outbreak that has resulted in multiple lineages. Deep sequencing of 179 patient samples processed by the European Mobile Laboratory, the first diagnostics unit to be deployed to the epicentre of the outbreak in Guinea, reveals an epidemiological and evolutionary history of the epidemic from March 2014 to January 2015. Analysis of EBOV genome evolution has also benefited from a similar sequencing effort of patient samples from Sierra Leone. Our results confirm that the EBOV from Guinea moved into Sierra Leone, most likely in April or early May. The viruses of the Guinea/Sierra Leone lineage mixed around June/July 2014. Viral sequences covering August, September and October 2014 indicate that this lineage evolved independently within Guinea. These data can be used in conjunction with epidemiological information to test retrospectively the effectiveness of control measures, and provides an unprecedented window into the evolution of an ongoing viral haemorrhagic fever outbreak.
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Brotes de Enfermedades/estadística & datos numéricos , Ebolavirus/genética , Evolución Molecular , Fiebre Hemorrágica Ebola/epidemiología , Fiebre Hemorrágica Ebola/virología , Filogenia , Análisis Espacio-Temporal , Sustitución de Aminoácidos/genética , Ebolavirus/aislamiento & purificación , Femenino , Guinea/epidemiología , Fiebre Hemorrágica Ebola/transmisión , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Liberia/epidemiología , Masculino , Malí/epidemiología , Datos de Secuencia Molecular , Sierra Leona/epidemiologíaRESUMEN
BACKGROUND: Reliable reverse transcription polymerase chain reaction (RT-PCR)-based diagnosis of Ebola virus infection currently requires a blood sample obtained by intravenous puncture. During the current Ebola outbreak in Guinea, we evaluated the usability of capillary blood samples collected from fingersticks of patients suspected of having Ebola virus disease (EVD) for field diagnostics during an outbreak emergency. METHODS: A total of 120 venous and capillary blood samples were collected from 53 patients admitted to the Ebola Treatment Centre in Guéckédou, Guinea, between July and August 2014. All sample specimens were analyzed by RT-PCR using the RealStar Filovirus Screen RT-PCR Kit 1.0 from altona Diagnostics (Germany). We compared samples obtained by venipuncture and those obtained by capillary blood sampling absorbed onto swab devices. RESULTS: The resulting sensitivity and specificity of tests performed with capillary blood samples were 86.8% (95% confidence interval [CI], 71.9%-95.6%; 33/38 patients) and 100% (95% CI, 84.6%-100%; 22/22 patients), respectively. CONCLUSIONS: Our data suggest that capillary blood samples could serve as an alternative to venous blood samples for the diagnosis of EVD in resource-limited settings during a crisis. This can be of particular advantage in cases when venipuncture is difficult to perform-for example, with newborns and infants or when adult patients reject venipuncture for cultural or religious reasons.
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Recolección de Muestras de Sangre/métodos , Brotes de Enfermedades , Fiebre Hemorrágica Ebola/diagnóstico , ARN Viral/sangre , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Recolección de Muestras de Sangre/normas , Niño , Preescolar , Urgencias Médicas , Estudios de Factibilidad , Femenino , Guinea , Humanos , Lactante , Masculino , Persona de Mediana Edad , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/normas , Sensibilidad y Especificidad , Adulto JovenAsunto(s)
Infecciones por Parvoviridae/veterinaria , Parvovirinae/clasificación , Parvovirinae/genética , Enfermedades de los Porcinos/epidemiología , Animales , Camerún/epidemiología , Genes Virales , Datos de Secuencia Molecular , Filogenia , Prevalencia , Vigilancia en Salud Pública , Sus scrofa , PorcinosRESUMEN
Simian T-lymphotropic virus type 1 (STLV-1) strains occasionally infect humans. However, the frequency of such infections is unknown. We show that direct transmission of STLV-1 from nonhuman primates to humans may be responsible for a substantial proportion of human T-lymphotropic virus type 1 infections in rural Côte d'Ivoire, where primate hunting is common.
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Infecciones por HTLV-I/transmisión , Virus Linfotrópico T Tipo 1 Humano/genética , Animales , Côte d'Ivoire , Genes env , Virus Linfotrópico T Tipo 1 Humano/inmunología , Humanos , Filogenia , Primates , Virus Linfotrópico T Tipo 1 de los Simios/genética , Virus Linfotrópico T Tipo 1 de los Simios/inmunología , Secuencias Repetidas TerminalesRESUMEN
During 2010-2011, we investigated interspecies transmission of partetraviruses between predators (humans and chimpanzees) and their prey (colobus monkeys) in Côte d'Ivoire. Despite widespread infection in all species investigated, no interspecies transmission could be detected by PCR and genome analysis. All sequences identified formed species- or subspecies (chimpanzee)-specific clusters, which supports a co-evolution hypothesis.
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Variación Genética , Infecciones por Parvoviridae/transmisión , Parvovirus/genética , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Animales , Niño , Colobus/virología , Côte d'Ivoire , ADN Viral/química , Humanos , Persona de Mediana Edad , Datos de Secuencia Molecular , Pan troglodytes/virología , Infecciones por Parvoviridae/virología , Parvovirus/clasificación , Filogenia , Análisis de Secuencia de ADN , Proteínas Virales/genética , Adulto JovenRESUMEN
Tropical rainforests show the highest level of terrestrial biodiversity and may be an important contributor to microbial diversity. Exploitation of these ecosystems may foster the emergence of novel pathogens. We report the discovery of the first insect-associated nidovirus, tentatively named Cavally virus (CAVV). CAVV was found with a prevalence of 9.3% during a survey of mosquito-associated viruses along an anthropogenic disturbance gradient in Côte d'Ivoire. Analysis of habitat-specific virus diversity and ancestral state reconstruction demonstrated an origin of CAVV in a pristine rainforest with subsequent spread into agriculture and human settlements. Virus extension from the forest was associated with a decrease in virus diversity (P<0.01) and an increase in virus prevalence (P<0.00001). CAVV is an enveloped virus with large surface projections. The RNA genome comprises 20,108 nucleotides with seven major open reading frames (ORFs). ORF1a and -1b encode two large proteins that share essential features with phylogenetically higher representatives of the order Nidovirales, including the families Coronavirinae and Torovirinae, but also with families in a basal phylogenetic relationship, including the families Roniviridae and Arteriviridae. Genetic markers uniquely conserved in nidoviruses, such as an endoribonuclease- and helicase-associated zinc-binding domain, are conserved in CAVV. ORF2a and -2b are predicted to code for structural proteins S and N, respectively, while ORF3a and -3b encode proteins with membrane-spanning regions. CAVV produces three subgenomic mRNAs with 5' leader sequences (of different lengths) derived from the 5' end of the genome. This novel cluster of mosquito-associated nidoviruses is likely to represent a novel family within the order Nidovirales.
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Culicidae/virología , Nidovirales/clasificación , Nidovirales/aislamiento & purificación , Animales , Análisis por Conglomerados , Secuencia Conservada , Côte d'Ivoire , Femenino , Datos de Secuencia Molecular , Nidovirales/genética , Sistemas de Lectura Abierta , Filogenia , ARN Viral/genética , Análisis de Secuencia de ADN , Árboles , Clima Tropical , Proteínas Virales/genéticaRESUMEN
Increasing infections with Monkeypox and Cowpox viruses pose a continuous and growing threat to human health. The standard method for detecting poxvirus neutralizing antibodies is the plaque-reduction neutralization test that is specific but also time-consuming and laborious. Therefore, a rapid and reliable method was developed to determine neutralizing antibody titers within twelve hours. The new assay measures viral mRNA transcription as a marker for actively replicating virus after incomplete neutralization using real-time PCR.
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Anticuerpos Neutralizantes/análisis , Anticuerpos Antivirales/análisis , Reacción en Cadena de la Polimerasa/métodos , Virus Vaccinia/inmunología , Vaccinia/diagnóstico , Anticuerpos Neutralizantes/inmunología , Anticuerpos Antivirales/inmunología , Línea Celular , Humanos , Pruebas de Neutralización , Vaccinia/inmunología , Virus Vaccinia/genética , Virus Vaccinia/aislamiento & purificación , Virus Vaccinia/fisiología , Replicación ViralRESUMEN
BACKGROUND: The principle of a capture ELISA is binding of specific capture antibodies (polyclonal or monoclonal) to the surface of a suitable 96 well plate. These immobilized antibodies are capable of specifically binding a virus present in a clinical sample. Subsequently, the captured virus is detected using a specific detection antibody. The drawback of this method is that a capture ELISA can only function for a single virus captured by the primary antibody. Human Apolipoprotein H (ApoH) or ß2-glycoprotein 1 is able to poly-specifically bind viral pathogens. Replacing specific capture antibodies by ApoH should allow poly-specific capture of different viruses that subsequently could be revealed using specific detection antibodies. Thus, using a single capture ELISA format different viruses could be analysed depending on the detection antibody that is applied. In order to demonstrate that this is a valid approach we show detection of group A rotaviruses from stool samples as a proof of principle for a new method of capture ELISA that should also be applicable to other viruses. RESULTS: Stool samples of different circulating common human and potentially zoonotic group A rotavirus strains, which were pretested in commercial EIAs and genotyped by PCR, were tested in parallel in an ApoH-ELISA set-up and by quantitative real-time PCR (qPCR). Several control samples were included in the analysis. The ApoH-ELISA was suitable for the capture of rotavirus-particles and the detection down to 1,000 infectious units (TCID(50/ml)). Subsets of diagnostic samples of different G- and P-types were tested positive in the ApoH-ELISA in different dilutions. Compared to the qPCR results, the analysis showed high sensitivity, specificity and low cross-reactivity for the ApoH-ELISA, which was confirmed in receiver operating characteristics (ROC) analysis. CONCLUSIONS: In this study the development of a highly sensitive and specific capture ELISA was demonstrated by combining a poly-specific ApoH capture step with specific detection antibodies using group A rotaviruses as an example.