RESUMEN
Viruses cause a wide range of human diseases, ranging from acute self-resolving conditions to acute fatal diseases. Effects that arise long after the primary infection can also increase the propensity for chronic conditions or lead to the development of cancer. Recent advances in the fields of virology and pathology have been fundamental in improving our understanding of viral pathogenesis, in providing improved vaccination strategies and in developing newer, more effective treatments for patients worldwide. The reviews assembled here focus on the interface between virology and pathology and encompass aspects of both the clinical pathology of viral disease and the underlying disease mechanisms. Articles on emerging diseases caused by Ebola virus, Marburg virus, coronaviruses such as SARS and MERS, Nipah virus and noroviruses are followed by reviews of enteroviruses, HIV infection, measles, mumps, human respiratory syncytial virus (RSV), influenza, cytomegalovirus (CMV) and varicella zoster virus (VZV). The issue concludes with a series of articles reviewing the relationship between viruses and cancer, including the role played by Epstein-Barr virus (EBV) in the pathogenesis of lymphoma and carcinoma; how human papillomaviruses (HPVs) are involved in the development of skin cancer; the involvement of hepatitis B virus infection in hepatocellular carcinoma; and the mechanisms by which Kaposi's sarcoma-associated herpesvirus (KSHV) leads to Kaposi's sarcoma. We hope that this collection of articles will be of interest to a wide range of scientists and clinicians at a time when there is a renaissance in the appreciation of the power of pathology as virologists dissect the processes of disease.
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Patología Molecular/métodos , Virología/métodos , Virosis , Virus/patogenicidad , Animales , Conducta Cooperativa , Interacciones Huésped-Patógeno , Humanos , Comunicación Interdisciplinaria , Valor Predictivo de las Pruebas , Pronóstico , Factores de Riesgo , Virulencia , Virosis/patología , Virosis/prevención & control , Virosis/terapia , Virosis/virologíaRESUMEN
The propensity of canine distemper virus (CDV) to spread to the central nervous system is one of the primary features of distemper. Therefore, we developed a reverse genetics system based on the neurovirulent Snyder Hill (SH) strain of CDV (CDV(SH)) and show that this virus rapidly circumvents the blood-brain and blood-cerebrospinal fluid (CSF) barriers to spread into the subarachnoid space to induce dramatic viral meningoencephalitis. The use of recombinant CDV(SH) (rCDV(SH)) expressing enhanced green fluorescent protein (EGFP) or red fluorescent protein (dTomato) facilitated the sensitive pathological assessment of routes of virus spread in vivo. Infection of ferrets with these viruses led to the full spectrum of clinical signs typically associated with distemper in dogs during a rapid, fatal disease course of approximately 2 weeks. Comparison with the ferret-adapted CDV(5804P) and the prototypic wild-type CDV(R252) showed that hematogenous infection of the choroid plexus is not a significant route of virus spread into the CSF. Instead, viral spread into the subarachnoid space in rCDV(SH)-infected animals was triggered by infection of vascular endothelial cells and the hematogenous spread of virus-infected leukocytes from meningeal blood vessels into the subarachnoid space. This resulted in widespread infection of cells of the pia and arachnoid mater of the leptomeninges over large areas of the cerebral hemispheres. The ability to sensitively assess the in vivo spread of a neurovirulent strain of CDV provides a novel model system to study the mechanisms of virus spread into the CSF and the pathogenesis of acute viral meningitis.
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Virus del Moquillo Canino/patogenicidad , Moquillo/virología , Meningoencefalitis/virología , Animales , Barrera Hematoencefálica/virología , Línea Celular , Líquido Cefalorraquídeo/virología , Chlorocebus aethiops , Plexo Coroideo/virología , Moquillo/patología , Virus del Moquillo Canino/genética , Virus del Moquillo Canino/fisiología , Células Endoteliales/virología , Hurones , Proteínas Fluorescentes Verdes/biosíntesis , Proteínas Fluorescentes Verdes/genética , Leucocitos/virología , Proteínas Luminiscentes/biosíntesis , Proteínas Luminiscentes/genética , Masculino , Meningoencefalitis/patología , Datos de Secuencia Molecular , Genética Inversa , Espacio Subaracnoideo/virología , Células Vero , Proteína Fluorescente RojaRESUMEN
Human respiratory syncytial virus (HRSV) is the leading cause of bronchiolitis in infants. Two subgroups of HRSV (A and B) routinely cocirculate. Most research has been performed with HRSV-A strains because these are easier to culture than HRSV-B strains. In this study, we aimed to compare the replicative fitness and HRSV-induced innate cytokine responses of HRSV-A and HRSV-B strains in disease-relevant cell culture models. We used two recombinant (r) clinical isolate-based HRSV strains (A11 and B05) and one recombinant laboratory-adapted HRSV strain (A2) to infect commercially available nasal, bronchial, and small-airway cultures. Epithelial cells from all anatomical locations were susceptible to HRSV infection despite the induction of a dominant type III interferon response. Subgroup A viruses disseminated and replicated faster than the subgroup B virus. Additionally, we studied HRSV infection and innate responses in airway organoids (AOs) cultured at air-liquid interface (ALI). Results were similar to the commercially obtained bronchial cells. In summary, we show that HRSV replicates well in cells from both the upper and the lower airways, with a slight replicative advantage for subgroup A viruses. Lastly, we showed that AOs cultured at ALI are a valuable model for studying HRSV ex vivo and that they can be used in the future to study factors that influence HRSV disease severity.IMPORTANCE Human respiratory syncytial virus (HRSV) is the major cause of bronchiolitis and pneumonia in young infants and causes almost 200,000 deaths per year. Currently, there is no vaccine or treatment available, only a prophylactic monoclonal antibody (palivizumab). An important question in HRSV pathogenesis research is why only a fraction (1 to 3%) of infants develop severe disease. Model systems comprising disease-relevant HRSV isolates and accurate and reproducible cell culture models are indispensable to study infection, replication, and innate immune responses. Here, we used differentiated AOs cultured at ALI to model the human airways. Subgroup A viruses replicated better than subgroup B viruses, which we speculate fits with epidemiological findings that subgroup A viruses cause more severe disease in infants. By using AOs cultured at ALI, we present a highly relevant, robust, and reproducible model that allows for future studies into what drives severe HRSV disease.
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Bronquios/virología , Nariz/virología , Organoides/microbiología , Virus Sincitial Respiratorio Humano/genética , Virus Sincitial Respiratorio Humano/fisiología , Replicación Viral , Bronquios/citología , Citocinas/análisis , Citocinas/inmunología , Células Epiteliales/virología , Humanos , Inmunidad Innata , Técnicas In Vitro , Cinética , Nariz/citología , Virus Sincitial Respiratorio Humano/clasificación , Virus Sincitial Respiratorio Humano/inmunologíaRESUMEN
This review describes the two interrelated and interdependent processes of transcription and replication for measles virus. First, we concentrate on the ribonucleoprotein (RNP) complex, which contains the negative sense genomic template and in encapsidated in every virion. Second, we examine the viral proteins involved in these processes, placing particular emphasis on their structure, conserved sequence motifs, their interaction partners and the domains which mediate these associations. Transcription is discussed in terms of sequence motifs in the template, editing, co-transcriptional modifications of the mRNAs and the phase of the gene start sites within the genome. Likewise, replication is considered in terms of promoter strength, copy numbers and the remarkable plasticity of the system. The review emphasises what is not known or known only by analogy rather than by direct experimental evidence in the MV replication cycle and hence where additional research, using reverse genetic systems, is needed to complete our understanding of the processes involved.
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Virus del Sarampión/fisiología , Sarampión/virología , Replicación Viral , Animales , Regulación Viral de la Expresión Génica , Genoma Viral , Humanos , Sarampión/metabolismo , Virus del Sarampión/genética , Ribonucleoproteínas/genética , Ribonucleoproteínas/metabolismo , Transcripción Genética , Proteínas Virales/genética , Proteínas Virales/metabolismoRESUMEN
Although the Enders strain of mumps virus (MuV) encodes a functional V protein that acts as an interferon (IFN) antagonist, in multi-cycle growth assays MuV Enders grew poorly in naïve ('IFN-competent' Hep2) cells but grew to high titres in 'IFN-compromised' Hep2 cells. Even so, the growth rate of MuV Enders was significantly slower in 'IFN-compromised' Hep2 cells when compared with its replication rate in Vero cells and with the replication rate of parainfluenza virus type 5 (a closely related paramyxovirus) in both naïve and 'IFN-compromised' Hep2 cells. This suggests that a consequence of slower growth is that the IFN system of naïve Hep2 cells can respond quickly enough to control the growth of MuV Enders. This is supported by the finding that rapidly growing variants of MuV Enders that were selected on 'IFN-compromised' Hep2 cells (i.e. in the absence of any selection pressure exerted by the IFN response) also grew to high titres on naïve Hep2 cells. Sequencing of the complete genome of one of these variants identified a single point mutation that resulted in a substitution of a conserved asparagine by histidine at position 498 of the haemagglutinin-neuraminidase protein, although this mutation was not present in all rapidly growing variants. These results support the concept that there is a race between the ability of a cell to detect and respond to virus infection and the ability of a virus to block the IFN response. Importantly, this emphasizes that factors other than viral IFN antagonists influence the sensitivity of viruses to IFN.
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Interferones/antagonistas & inhibidores , Interferones/inmunología , Virus de la Parotiditis/inmunología , Virus de la Parotiditis/fisiología , Replicación Viral , Sustitución de Aminoácidos/genética , Animales , Línea Celular , Chlorocebus aethiops , Análisis Mutacional de ADN , Proteína HN/genética , Humanos , Mutación Missense , Ensayo de Placa ViralRESUMEN
We demonstrate that insertion of the open reading frame of enhanced green fluorescent protein (EGFP) into the coding sequence for the second hinge region of the viral L (large) protein (RNA-dependent RNA polymerase) attenuates a wild-type canine distemper virus. Moreover, we show that single intranasal immunization with this recombinant virus provides significant protection against challenge with the virulent parental virus. Protection against wild-type challenge was gained either after recovery of cellular immunity postimmunization or after development of neutralizing antibodies. Insertion of EGFP seems to result in overattenuation of the virus, while our previous experiments demonstrated that the insertion of an epitope tag into a similar position did not affect L protein function. Thus, a desirable level of attenuation could be reached by manipulating the length of the insert (in the second hinge region of the L protein), providing additional tools for optimization of controlled attenuation. This strategy for controlled attenuation may be useful for a "quick response" in vaccine development against well-known and "new" viral infections and could be combined efficiently with other strategies of vaccine development and delivery systems.
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Virus del Moquillo Canino/inmunología , Moquillo/prevención & control , Proteínas Fluorescentes Verdes/genética , ARN Polimerasa Dependiente del ARN/genética , Recombinación Genética , Vacunas Atenuadas , Vacunas Virales , Animales , Células Cultivadas , Chlorocebus aethiops , Modelos Animales de Enfermedad , Moquillo/virología , Virus del Moquillo Canino/enzimología , Virus del Moquillo Canino/genética , Virus del Moquillo Canino/patogenicidad , Perros , Hurones , Inmunización , Leucocitos Mononucleares , Masculino , Microscopía Confocal , Vacunas Atenuadas/administración & dosificación , Vacunas Atenuadas/genética , Vacunas Atenuadas/inmunología , Células Vero , Vacunas Virales/administración & dosificación , Vacunas Virales/genética , Vacunas Virales/inmunologíaRESUMEN
AIMS: In this study of experimental measles neuropathogenesis, the utility of enhanced green fluorescent protein (EGFP) as a sensitive indicator of measles virus (MV) cell-to-cell spread in the central nervous system (CNS) has been assessed in vibratome-cut brain slices to demonstrate the degree and mechanism of viral spread in the rodent CNS. METHODS: Recombinant MVs expressing EGFP were visualized at different levels in 200-microm vibratome-cut brain sections from infected animals by confocal scanning laser microscopy (CSLM). Comparison was made with 7-microm microtome sections, stained for the N protein of measles by immunocytochemistry (ICC). RESULTS: The recombinant viruses were readily visualized in infected brain tissue, with no loss of neuropathogenicity. No difference was found in the sites of infection when MV infection was detected through EGFP fluorescence or by ICC. MV-infected cells were detected in the cerebral cortex, olfactory bulb and tract, hippocampus, thalamus, hypothalamus, ependyma and subventricular zone. However, the 200-microm vibratome-cut sections and confocal microscopy proved excellent for demonstrating virus distribution in neurites and for in-depth analysis of the extent of tract infection in the white matter of the cerebral hemispheres such as selective infection of the internal capsule and anterior commissure. CONCLUSIONS: The use of self-tracing recombinant MVs, viewed in thick vibratome-cut sections by CSLM, demonstrated that in experimental MV neuropathogenesis the infection is selective and spreads predominately by neurites using defined anatomical pathways.
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Encéfalo/patología , Proteínas Fluorescentes Verdes/genética , Virus del Sarampión/genética , Sarampión/genética , Panencefalitis Esclerosante Subaguda/patología , Animales , Encéfalo/virología , Chlorocebus aethiops , Genes Reporteros , Genoma Viral , Proteínas Fluorescentes Verdes/análisis , Sarampión/patología , Sarampión/virología , Proteína Cofactora de Membrana/genética , Ratones , Ratones Transgénicos , Proteínas de la Nucleocápside/análisis , Proteínas de la Nucleocápside/genética , Recombinación Genética , Panencefalitis Esclerosante Subaguda/genética , Panencefalitis Esclerosante Subaguda/virología , Células VeroRESUMEN
Products expressed from the second (P/V/C) gene are important in replication and abrogating innate immune responses during acute measles virus (MV) infection. Thirteen clone sets were derived from the P/V/C genes of measles virus (MV) RNA extracted from brains of a unique collection of seven cases of subacute sclerosing panencephalitis (SSPE) caused by persistent MV in the central nervous system (CNS). Whether these functions are fully maintained when MV replicates in the CNS has not been previously determined. Co-transcriptional editing of the P mRNAs by non-template insertion of guanine (G) nucleotides, which generates mRNAs encoding the viral V protein, occurs much less frequently (9%) in the SSPE derived samples than during the acute infection (30-50%). Thus it is likely that less V protein, which is involved in combatting the innate immune response, is produced. The P genes in MV from SSPE cases were not altered by biased hypermutation but exhibited a high degree of variation within each case. Most but not all SSPE derived phospho-(P) proteins were functional in mini genome replication/transcription assays. An eight amino acid truncation of the carboxyl-terminus made the P protein non-functional while the insertion of an additional glycine residue by insertion of G nucleotides at the editing site had no effect on protein function.
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Virus del Sarampión/genética , Fosfoproteínas/genética , Edición de ARN , Panencefalitis Esclerosante Subaguda/virología , Proteínas Virales/genética , Niño , Preescolar , Femenino , Humanos , Lactante , Masculino , Virus del Sarampión/aislamiento & purificación , Virus del Sarampión/metabolismo , Mutación , Fosfoproteínas/metabolismo , Proteínas Virales/metabolismoRESUMEN
BACKGROUND: The association of Chlamydia pneumoniae with atherosclerosis is controversial. We investigated the presence of C. pneumoniae and other Chlamydia spp. in atheromatous carotid artery tissue. METHODS: Forty elective carotid endarterectomy patients were recruited (27 males, mean age 65 and 13 females mean age 68), 4 had bilateral carotid endarterectomies (n= 44 endarterectomy specimens). Control specimens were taken from macroscopically normal carotid artery adjacent to the atheromatous lesions (internal controls), except in 8 cases where normal carotid arteries from post mortem (external controls) were used. Three case-control pairs were excluded when the HLA DRB gene failed to amplify from the DNA. Genus specific primers to the major outer membrane protein (MOMP) gene were used in a nested polymerase chain reaction (nPCR) in 41 atheromatous carotid specimens and paired controls. PCR inhibition was monitored by spiking with target C. trachomatis. Atheroma severity was graded histologically. Plasma samples were tested by microimmunofluorescence (MIF) for antibodies to C. pneumoniae, C. trachomatis and C. psittaci and the corresponding white cells were tested for Chlamydia spp. by nPCR. RESULTS: C. pneumoniae was not detected in any carotid specimen. Twenty-five of 38 (66%) plasma specimens were positive for C. pneumoniae IgG, 2/38 (5%) for C. trachomatis IgG and 1/38 (3%) for C. psittaci IgG. CONCLUSIONS: We were unable to show an association between the presence of Chlamydia spp. and atheroma in carotid arteries in the presence of a high seroprevalence of C. pneumoniae antibodies in Northern Ireland.
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Anticuerpos Antibacterianos/sangre , Arteriosclerosis/microbiología , Enfermedades de las Arterias Carótidas/microbiología , Chlamydophila pneumoniae/inmunología , Reacción en Cadena de la Polimerasa/métodos , Adulto , Anciano , Anciano de 80 o más Años , Antígenos Bacterianos/inmunología , Arteriosclerosis/sangre , Arteriosclerosis/inmunología , Arteriosclerosis/patología , Enfermedades de las Arterias Carótidas/inmunología , Chlamydophila pneumoniae/genética , Chlamydophila pneumoniae/aislamiento & purificación , Cartilla de ADN , Femenino , Humanos , Masculino , Persona de Mediana Edad , Pruebas SerológicasRESUMEN
Rodent brain-adapted measles virus (MV) strains, such as CAM/RB and recombinant MVs based on the Edmonston strain containing the haemagglutinin (H) of CAM/RB, cause acute encephalitis after intracerebral infection of newborn rodents. We have demonstrated that rodent neurovirulence is modulated by two mutations at amino acid positions 195 and 200 in the H protein, one of these positions (200) being a potential glycosylation site. In order to analyse the effects of specific amino acids at these positions, we introduced a range of individual and combined mutations into the open reading frame of the H gene to generate a number of eukaryotic expression plasmids. The functionality of the mutant H proteins was assessed in transfected cells and by generating recombinant viruses. Interestingly, viruses caused acute encephalitis only if the amino acid Ser at position 200 was coupled with Gly at position 195, whereas viruses with single or combined mutations at these positions, including glycosylation at position 200, were attenuated. Neurovirulence was associated with virus spread and induction of neuronal apoptosis, whereas attenuated viruses failed to infect brain cells. Similar results were obtained by using primary brain-cell cultures. Our findings indicate that a structural alteration in the stem 2 region of the H protein at position 195 or 200 interferes with infectivity of rodent neurons, and suggest that the interaction of the viral attachment protein with cellular receptors on neurons is affected.
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Sistema Nervioso Central/virología , Encefalitis/virología , Virus del Sarampión/genética , Virus del Sarampión/patogenicidad , Proteínas Virales/genética , Proteínas Virales/fisiología , Virulencia/genética , Sustitución de Aminoácidos/genética , Animales , Apoptosis , Línea Celular , Células Cultivadas , Virus del Sarampión/crecimiento & desarrollo , Modelos Moleculares , Mutagénesis Sitio-Dirigida , Neuronas/virología , Ratas , RoedoresRESUMEN
Antibodies to CD9, a member of the tetraspan transmembrane-protein family, selectively inhibit Canine distemper virus (CDV)-induced cell-cell fusion. Neither CDV-induced virus-cell fusion nor cell-cell fusion induced by the closely related morbillivirus Measles virus (MV) is affected by anti-CD9 antibodies. As CDV does not bind CD9, an unknown, indirect mechanism is responsible for the observed inhibition of cell-cell fusion. It was investigated whether this effect was restricted to only one viral glycoprotein, either the haemagglutinin (H) or the fusion (F) protein, which form a fusion complex on the surface of virions and infected cells, or whether it is dependent on both in transient co-transfection assays. The susceptibility to CD9 antibodies segregates with the H protein of CDV. By exchanging portions of the H proteins of CDV and MV, it was determined that the complete extracellular domain, including the predicted stem structure (stem 1, barrel strand 1 and stem 2) and globular head domain, of the CDV-H protein mediates the effect. This suggests that interaction of the CDV-H protein with an unknown cellular receptor(s) is regulated by CD9, rather than F protein-mediated membrane fusion.
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Antígenos CD/inmunología , Fusión Celular , Virus del Moquillo Canino/patogenicidad , Glicoproteínas de Membrana/inmunología , Proteínas Virales/química , Proteínas Virales/metabolismo , Secuencia de Aminoácidos , Animales , Anticuerpos Monoclonales/inmunología , Anticuerpos Antivirales/inmunología , Antígenos CD/metabolismo , Chlorocebus aethiops , Virus del Moquillo Canino/genética , Perros , Células Gigantes/fisiología , Glicoproteínas de Membrana/metabolismo , Ratones , Tetraspanina 29 , Transfección , Células Vero , Proteínas Virales/genéticaRESUMEN
Measles virus (MV) nucleocapsids are present abundantly in brain cells of patients with subacute sclerosing panencephalitis (SSPE). This invariably lethal brain disease develops years after acute measles as result of a persistent MV infection. Various rodent models for MV infection of the central nervous system (CNS) have been described in the past, in which the detection of viral antigens is based on histological staining procedures of paraffin embedded brains. Here, the usage of a recombinant MV (MV-EGFP-CAMH) expressing the haemagglutinin (H) of the rodent-adapted MV-strain CAM/RB and the enhanced green fluorescent protein (EGFP) is described. In newborn rodents the virus infects neurons and causes an acute lethal encephalitis. From 2 weeks on, when the immune system of the genetically unmodified animal is maturating, intracerebral (i.c.) infection is overcome subclinically, however, a focal persistent infection in groups of neurons remains. The complete brain can be analysed in 50 or 100 microm slices, and infected autofluorescent cells are readily detected. Seven and 28 days post-infection (p.i.) 86 and 81% of mice are infected, respectively, and virus persists for more than 50 days p.i. Intraperitoneal immunization with MV 1 week before infection, but not after infection, protects and prevents persistence. The high percentage of persistence demonstrates that this is a reliable and useful model of a persistent CNS infection in fully immunocompetent mice, which allows the investigation of determinants of the immune system.
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Virus del Sarampión/genética , Virus del Sarampión/patogenicidad , Sarampión/etiología , Panencefalitis Esclerosante Subaguda/etiología , Factores de Edad , Animales , Animales Recién Nacidos , Encéfalo/inmunología , Encéfalo/patología , Encéfalo/virología , Modelos Animales de Enfermedad , Proteínas Fluorescentes Verdes/genética , Hemaglutininas Virales/genética , Humanos , Inmunización , Inmunocompetencia , Sarampión/inmunología , Sarampión/patología , Sarampión/virología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Neuronas/virología , Proteínas Recombinantes/genética , Recombinación Genética , Panencefalitis Esclerosante Subaguda/inmunología , Panencefalitis Esclerosante Subaguda/patología , Panencefalitis Esclerosante Subaguda/virología , Linfocitos T/patologíaRESUMEN
Chloramphenicol acetyltransferase (CAT)-expressing negative-sense mini-genomic constructs of measles virus (MV) and rinderpest virus (RPV) were rescued by standard technology with helper plasmids expressing the nucleocapsid (N), phospho- (P) and large (L) proteins of MV, canine distemper virus (CDV) or RPV in order to determine whether the proteins of different viruses can function together. Homogeneous sets consisting of N, P and L plasmids derived from one virus were able to generate reporter gene expression from either mini-genomic construct. Heterogeneous sets of proteins from different viruses were not functional, with the exception that a low level of activity was obtained when MV N and P protein were combined with RPV L protein in the rescue of the MV mini-genomic construct, or CDV N was combined with RPV P and L in the rescue of the RPV mini-genome. However, only homogeneous sets of plasmids were able to rescue infectious virus from full-length anti-genome-expressing plasmids.
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Genoma Viral , Virus del Sarampión/genética , Plásmidos , Recombinación Genética , Virus de la Peste Bovina/genética , Proteínas Virales/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Virus del Moquillo Canino/genética , Virus del Moquillo Canino/fisiología , Perros , Genes Reporteros , Células HeLa , Humanos , Virus del Sarampión/fisiología , Datos de Secuencia Molecular , Proteínas de la Nucleocápside/genética , Proteínas de la Nucleocápside/metabolismo , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Virus de la Peste Bovina/fisiología , Proteínas Virales/genéticaRESUMEN
A recombinant measles virus which expresses enhanced green fluorescent protein (MVeGFP) has been used to infect two astrocytoma cell lines (GCCM and U-251) to study the effect of virus infection on the cytoskeleton. Indirect immunocytochemistry was used to demonstrate the cellular localization of the cytoskeletal components. Enhanced green fluorescent protein autofluorescence was used to identify measles virus-infected cells. No alteration of the actin, tubulin, or vimentin components of the cytoskeleton was observed in either cell type, whereas a disruption of the glial-fibrillary-acidic protein filament (GFAP) network was noted in MVeGFP-infected U-251 cells. The relative amounts of GFAP present in infected and uninfected U-251 cells were quantified by image analysis of data sets obtained by confocal microscopy by using vimentin, another intermediate filament on which MVeGFP has no effect, as a control.
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Astrocitoma/virología , Citoesqueleto/metabolismo , Proteína Ácida Fibrilar de la Glía/metabolismo , Virus del Sarampión/fisiología , Citoesqueleto/virología , Proteínas Fluorescentes Verdes , Humanos , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Virus del Sarampión/genética , Virus del Sarampión/metabolismo , Microscopía Confocal , Tubulina (Proteína)/metabolismo , Células Tumorales Cultivadas , Vimentina/metabolismoRESUMEN
Canine distemper virus (CDV) has been rescued from a full-length cDNA clone. Besides Measles virus (MV) and Rinderpest virus, a third morbillivirus is now available for genetic analysis using reverse genetics. A plasmid p(+)CDV was constructed by sequential cloning using the Onderstepoort vaccine strain large-plaque-forming variant. The presence of a T7 promoter allowed transcription of full-length antigenomic RNA by a T7 RNA polymerase, which was provided by a host range mutant of vaccinia virus (MVA-T7). Plasmids expressing the nucleocapsid protein, the phosphoprotein, and the viral RNA-dependent RNA polymerase, also under control of a T7 promoter, have been generated. Infection of HeLa cells with MVA-T7 and subsequent transfection of p(+)CDV plus the helper plasmids led to syncytium formation and release of infectious recombinant (r) CDV. Comparison of the rescued virus with the parental virus revealed no major differences in the progression of infection or in the shape and size of syncytia. A genetic tag, consisting of two nucleotide changes within the coding region of the L protein, has been identified in the rCDV genome. Expression by rCDV of all the major viral structural proteins has been demonstrated by immunofluorescence.
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Virus del Moquillo Canino/genética , Virus del Moquillo Canino/fisiología , Recombinación Genética , Proteínas Virales/metabolismo , Animales , Chlorocebus aethiops , Clonación Molecular , Efecto Citopatogénico Viral , ADN Complementario/genética , Virus del Moquillo Canino/crecimiento & desarrollo , Perros , Técnica del Anticuerpo Fluorescente , Marcadores Genéticos , Genoma Viral , Células HeLa/virología , Humanos , Datos de Secuencia Molecular , Plásmidos/genética , Reacción en Cadena de la Polimerasa , Células Vero , Proteínas Virales/genéticaRESUMEN
Certain members of the morbillivirus genus, canine distemper virus, phocine distemper virus, and the cetacean viruses of dolphins and porpoises exhibit high levels of central nervous system (CNS) infection in their natural hosts. CNS complications are rare for measles virus (MV) and are not associated with rinderpest virus (RPV) and peste des petits ruminants virus (PPRV) infection. However, both RPV and PPRV are neurovirulent in permissive murine strains. Human postmortem tissue, neural cell cultures, and animal models have been used to answer major questions concerning morbillivirus neurovirulence. Studies of the MV CNS complication subacute sclerosing panencephalitis (SSPE) indicate that virus could enter the CNS either by direct infection of endothelial cells or in infected leucocytes, followed by infection of predominately neurones and oligodendrocytes. It has been established that MV neurovirulence in mice is partially determined by the virus-receptor specificity. The two known MV receptors, CD46 and SLAM, have been examined in normal and SSPE brain tissue and the findings suggest that further receptors may be necessary to explain infection of the CNS with wild-type strains of MV. In both humans and mice (and in vitro), once infection of neurones has been established, virus spreads transneuronally. It is possible that all morbilliviruses transiently infect the CNS in their natural hosts, but development of disease is dependent on the efficiency of the immune response. Alternatively, for RPV and PPRV, virus entry may be restricted due either to absence of viral receptors or failure of virus to replicate or spread in the CNS.
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Infecciones por Morbillivirus/virología , Morbillivirus/patogenicidad , Animales , Humanos , Infecciones por Morbillivirus/patología , VirulenciaRESUMEN
A recombinant measles virus (MV) which expresses enhanced green fluorescent protein (EGFP) has been rescued. This virus, MVeGFP, expresses the reporter gene from an additional transcription unit which is located prior to the gene encoding the measles virus nucleocapsid protein. The recombinant virus was used to infect human astrocytoma cells (GCCM). Immunocytochemistry (ICC) together with EGFP autofluorescence showed that EGFP is both an early and very sensitive indicator of cell infection. Cells that were EGFP-positive and ICC-negative were frequently observed. Confocal microscopy was used to indirectly visualize MV infection of GCCM cells and to subsequently follow cell-to-cell spread in real time. These astrocytoma cells have extended processes, which in many cases are intimately associated. The processes appear to have an important role in cell-to-cell spread, and MVeGFP was observed to utilize them in the infection of surrounding cells. Heterogeneity was seen in cell-to-cell spread in what was expected to be a homogeneous monolayer. In tissue culture, physical constraints govern the integrity of the syncytia which are formed upon extensive cell fusion. When around 50 cells were fused, the syncytia rapidly disintegrated and many of the infected cells detached. Residual adherent EGFP-positive cells were seen to either continue to be involved in the infection of surrounding cells or to remain EGFP positive but no longer participate in the transmission of MV infection to neighboring cells.
Asunto(s)
Astrocitos/virología , Virus del Sarampión/genética , Virus del Sarampión/fisiología , Animales , Astrocitoma , Línea Celular , Técnica del Anticuerpo Fluorescente , Genes Reporteros , Proteínas Fluorescentes Verdes , Humanos , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Microscopía Confocal , Proteínas Recombinantes/metabolismo , Células Tumorales CultivadasRESUMEN
This study focused on the in vitro infection of mouse and human neuroblastoma cells and the in vivo infection of the murine central nervous system with a recombinant measles virus. An undifferentiated mouse neuroblastoma cell line (TMN) was infected with the vaccine strain of measles virus (MVeGFP), which expresses enhanced green fluorescent protein (EGFP). MVeGFP infected the cells, and cell-to-cell spread was studied by virtue of the resulting EGFP autofluorescence, using real-time confocal microscopy. Cells were differentiated to a neuronal phenotype, and extended processes, which interconnected the cells, were observed. It was also possible to infect the differentiated neuroblastoma cells (dTMN) with MVeGFP. Single autofluorescent EGFP-positive cells were selected at the earliest possible point in the infection, and the spread of EGFP autofluorescence was monitored. In this instance the virus used the interconnecting processes to spread from cell to cell. Human neuroblastoma cells (SH-SY-5Y) were also infected with MVeGFP. The virus infected these cells, and existing processes were used to initiate new foci of infection at distinct regions of the monolayer. Transgenic animals expressing CD46, a measles virus receptor, and lacking interferon type 1 receptor gene were infected intracerebrally with MVeGFP. A productive infection ensued, and the mice exhibited clinical signs of infection, such as ataxia and an awkward gait, identical to those previously observed for the parental virus (Edtag). Mice were sacrificed, and brain sections were examined for EGFP autofluorescence by confocal scanning laser microscopy over a period of 6 h. EGFP was detected in discrete focal regions of the brain and in processes, which extended deep into the parenchyma. Collectively, these results indicate (i) that MVeGFP can be used to monitor virus replication sensitively, in real time, in animal tissues, (ii) that infection of ependymal cells and neuroblasts provides a route by which measles virus can enter the central nervous system in mouse models of encephalitis, and (iii) that upon infection, the virus spreads transneuronally.
Asunto(s)
Encéfalo/virología , Virus del Sarampión/crecimiento & desarrollo , Neuronas/virología , Animales , Animales Lactantes , Antígenos CD/metabolismo , Encéfalo/patología , Chlorocebus aethiops , Encefalitis Viral/patología , Técnica del Anticuerpo Fluorescente , Proteínas Fluorescentes Verdes , Humanos , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Virus del Sarampión/genética , Virus del Sarampión/metabolismo , Proteína Cofactora de Membrana , Glicoproteínas de Membrana/metabolismo , Ratones , Ratones Endogámicos , Ratones Transgénicos , Microscopía Confocal , Neuroblastoma , Neuronas/patología , Receptores Virales/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Células Tumorales Cultivadas , Células VeroRESUMEN
Molecular determinants of neuropathogenesis have been shown to be present in the hemagglutinin (H) protein of measles virus (MV). An H gene insertion vector has been generated from the Edmonston B vaccine full-length infectious clone of MV. Using this vector, it is possible to insert complete H open reading frames into the parental (Edtag) background. The H gene from a rodent brain-adapted MV strain (CAM/RB) was inserted into this vector, and a recombinant virus (EdtagCAMH) was rescued by using a modified vaccinia virus which expresses T7 RNA polymerase (MVA-T7). The recombinant virus grew at an equivalent rate and to similar titers as the CAM/RB and Edtag parental viruses. Neurovirulence was assayed in a mouse model for MV encephalitis. Viruses were injected intracerebrally into the right cortex of C57/BL/6 suckling mice. After infection mice inoculated with the CAM/RB strain developed hind limb paralysis and ataxia. Clinical symptoms were never observed with an equivalent dose of Edtag virus or in sham infections. Immunohistochemistry (IHC) was used to detect viral antigen in formalin-fixed brain sections. Measles antigen was observed in neurons and neuronal processes of the hippocampus, frontal, temporal, and olfactory cortices and neostriatum on both sides of symmetrical structures. Viral antigen was not detected in mice infected with Edtag virus. Mice infected with the recombinant virus, EdtagCAMH, became clinically ill, and virus was detected by IHC in regions of the brain similar to those in which it was detected in animals infected with CAM/RB. The EdtagCAMH infection had, however, progressed much less than the CAM/RB virus at 4 days postinfection. It therefore appears that additional determinants are encoded in other regions of the MV genome which are required for full neurovirulence equivalent to CAM/RB. Nevertheless, replacement of the H gene alone is sufficient to cause neuropathology.