RESUMEN
The SARS-associated human coronavirus (SARS-HCoV) is a newly described, emerging virus conclusively established as the etiologic agent of the severe acute respiratory syndrome (SARS). This study presents a single-tube RT-PCR assay that can detect with high analytical sensitivity the SARS-HCoV, as well as several other coronaviruses including other known human respiratory coronaviruses (HCoV-OC43 and HCoV-229E). Species identification is provided by sequencing the amplicon, although a rapid screening test by restriction enzyme analysis has proved to be very useful for the analysis of samples obtained during the SARS outbreak in Toronto, Canada.
Asunto(s)
Infecciones por Coronavirus/diagnóstico , Coronavirus/clasificación , Coronavirus/aislamiento & purificación , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Síndrome Respiratorio Agudo Grave/diagnóstico , Coronavirus Relacionado al Síndrome Respiratorio Agudo Severo/aislamiento & purificación , Secuencia de Bases , Coronavirus/genética , Coronavirus Humano 229E/clasificación , Coronavirus Humano 229E/genética , Coronavirus Humano 229E/aislamiento & purificación , Infecciones por Coronavirus/virología , Coronavirus Humano OC43/clasificación , Coronavirus Humano OC43/genética , Coronavirus Humano OC43/aislamiento & purificación , ADN Complementario , ADN Viral/análisis , ADN Viral/aislamiento & purificación , Desoxirribonucleasas de Localización Especificada Tipo II/metabolismo , Humanos , Datos de Secuencia Molecular , Filogenia , Coronavirus Relacionado al Síndrome Respiratorio Agudo Severo/clasificación , Coronavirus Relacionado al Síndrome Respiratorio Agudo Severo/genética , Sensibilidad y Especificidad , Alineación de Secuencia , Análisis de Secuencia de ADN , Síndrome Respiratorio Agudo Grave/virologíaRESUMEN
Human papillomavirus type 16 (HPV16) E6 and E7 oncoproteins are required for cellular transformation and represent candidate targets for HPV-specific and major histocompatibility complex class I-restricted CD8(+)-T-cell responses in patients with cervical cancer. Recent evidence suggests that cross-reactivity represents the inherent nature of the T-cell repertoire. We identified HLA-A2 binding HPV16 E7 variant peptides from human, bacterial, or viral origin which are able to drive CD8(+)-T-cell responses directed against wild-type HPV16 E7 amino acid 11 to 19/20 (E7(11-19/20)) epitope YMLDLQPET(T) in vitro. CD8(+) T cells reacting to the HLA-A2-presented peptide from HPV16 E7(11-19(20)) recognized also the HLA-A2 binding peptide TMLDIQPED (amino acids 52 to 60) from the human coronavirus OC43 NS2 gene product. Establishment of coronavirus NS2-specific, HLA-A2-restricted CD8(+)-T-cell clones and ex vivo analysis of HPV16 E7 specific T cells obtained by HLA-A2 tetramer-guided sorting from PBL or tumor-infiltrating lymphocytes obtained from patients with cervical cancer showed that cross-reactivity with HPV16 E7(11-19(20)) and coronavirus NS2(52-60) represents a common feature of this antiviral immune response defined by cytokine production. Zero of 10 patients with carcinoma in situ neoplasia and 3 of 18 patients with cervical cancer showed > or =0.1% HPV16 E7-reactive T cells in CD8(+) peripheral blood lymphocytes. In vivo priming with HPV16 was confirmed in patients with cervical cancer or preinvasive HPV16-positive lesions using HLA-A2 tetramer complexes loaded with the E6-derived epitope KLPQLCTEL. In contrast, we could not detect E6-reactive T cells in healthy individuals. These data imply that the measurement of the HPV16 E7(11-19(20)) CD8(+)-T-cell response may reflect cross-reactivity with a common pathogen and that variant peptides may be employed to drive an effective cellular immune response against HPV.
Asunto(s)
Papillomaviridae/inmunología , Secuencia de Aminoácidos , Linfocitos T CD8-positivos/inmunología , Coronavirus/inmunología , Reacciones Cruzadas , Epítopos de Linfocito T/inmunología , Femenino , Antígeno HLA-A2/metabolismo , Humanos , Activación de Linfocitos , Datos de Secuencia Molecular , Proteínas Oncogénicas Virales , Proteínas E7 de Papillomavirus , Infecciones por Papillomavirus/inmunología , Péptidos/química , Péptidos/genética , Péptidos/inmunología , Infecciones Tumorales por Virus/inmunología , Neoplasias del Cuello Uterino/inmunología , Proteínas no Estructurales Virales/inmunologíaRESUMEN
The incidence of nosocomial viral respiratory infections (NVRI) in neonates and children hospitalized in paediatric and neonatal intensive care units (PNICU) is unknown. Human coronaviruses (HCoV) have been implicated in NVRI in hospitalized preterm neonates. The objectives of this study were to determine the incidence of HCoV-related NVRI in neonates and children hospitalized in a PNICU and the prevalence of viral respiratory tract infections in staff. All neonates (age< or =28 days) and children (age>28 days) hospitalized between November 1997 and April 1998 were included. Nasal samples were obtained by cytological brush at admission and weekly thereafter. Nasal samples were taken monthly from staff. Virological studies were performed, using indirect immunofluorescence, for HCoV strains 229E and OC43, respiratory syncytial virus (RSV), influenza virus types A and B, paramyxoviruses types 1, 2 and 3 and adenovirus. A total of 120 patients were enrolled (64 neonates and 56 children). Twenty-two samples from 20 patients were positive (incidence 16.7%). In neonates, seven positive samples, all for HCoV, were detected (incidence 11%). Risk factors for NVRI in neonates were: duration of hospitalization, antibiotic treatment and duration of parenteral nutrition (P<0.01). Monthly prevalence of viral infections in staff was between 0% and 10.5%, mainly with HCoV. In children, 15 samples were positive in 13 children at admission (seven RSV, five influenza and three adenovirus) but no NVRI were observed. In spite of a high rate of community-acquired infection in hospitalized children, the incidence of NVRI with common respiratory viruses appears low in neonates, HCoV being the most important pathogen of NRVI in neonates during this study period. Further research is needed to evaluate the long-term impact on pulmonary function.
Asunto(s)
Infecciones por Coronavirus/epidemiología , Infección Hospitalaria/epidemiología , Infecciones del Sistema Respiratorio/epidemiología , Adolescente , Portador Sano/epidemiología , Niño , Preescolar , Infecciones Comunitarias Adquiridas/epidemiología , Infección Hospitalaria/virología , Femenino , Humanos , Incidencia , Lactante , Recién Nacido , Control de Infecciones , Unidades de Cuidado Intensivo Neonatal , Unidades de Cuidado Intensivo Pediátrico , Masculino , Estudios Prospectivos , Infecciones del Sistema Respiratorio/virologíaRESUMEN
Human coronaviruses, with two known serogroups named 229-E and OC-43, are enveloped positive-stranded RNA viruses. The large RNA is surrounded by a nucleoprotein (protein N). The envelop contains 2 or 3 glycoproteins: spike protein (or protein S), matrix protein (or protein M) and a hemagglutinin (or protein HE). Their pathogen role remains unclear because their isolation is difficult. Reliable and rapid methods as immunofluorescence with monoclonal antibodies and reverse transcription-polymerase chain reaction allow new researches on epidemiology. Human coronaviruses can survive for as long as 6 days in suspension and 3 hours after drying on surfaces, suggesting that they could be a source of hospital-acquired infections. Two prospective studies conducted in a neonatal and paediatric intensive care unit demonstrated a significant association of coronavirus-positive nasopharyngal samples with respiratory illness in hospitalised preterm neonates. Positive samples from staff suggested either a patient-to-staff or a staff-to-patient transmission. No cross-infection were observed from community-acquired respiratory-syncitial virus or influenza-infected children to neonates. Universal precautions with hand washing and surface desinfection could be proposed to prevent coronavirus transmission.
Asunto(s)
Infecciones por Coronavirus , Coronavirus/aislamiento & purificación , Infección Hospitalaria , Adulto , Factores de Edad , Coronavirus/genética , Coronavirus Humano 229E/aislamiento & purificación , Infecciones por Coronavirus/diagnóstico , Infecciones por Coronavirus/virología , Coronavirus Humano OC43/aislamiento & purificación , Infección Hospitalaria/diagnóstico , Infección Hospitalaria/prevención & control , Infección Hospitalaria/virología , ADN Viral/análisis , Técnica del Anticuerpo Fluorescente , Humanos , Lactante , Recién Nacido , Unidades de Cuidado Intensivo Neonatal , Estudios Prospectivos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factores de Riesgo , Estaciones del Año , Factores de TiempoAsunto(s)
Enfermedades Autoinmunes/virología , Enfermedades Virales del Sistema Nervioso Central/inmunología , Sistema Nervioso Central/inmunología , Animales , Axones/patología , Encéfalo/patología , Movimiento Celular , Sistema Nervioso Central/virología , Enfermedades Virales del Sistema Nervioso Central/virología , Coronavirus/patogenicidad , Humanos , Imagen por Resonancia Magnética , Microglía/inmunología , Imitación Molecular , Esclerosis Múltiple/patología , Esclerosis Múltiple/virología , Virus de la Hepatitis Murina/patogenicidad , Vaina de Mielina/inmunología , Linfocitos T/inmunologíaRESUMEN
Acute infection of fibroblastic cell lines by the Indiana strain of vesicular stomatitis virus (VSV) usually induces dramatic cytopathic effects and shutoff of cellular gene expression. We have compared a series of independent mutants with differences in shutoff induction and found that M was mutated either in the N-terminus (M(51)R) or C-terminus (V(221)F and S(226)R). Furthermore, only double mutants (M mutation and a ts mutation related or not to M) were able to persist on fibroblast cell lines at 39 degrees C. A more detailed investigation of the infection was performed for the mutants T1026, TP3 and G31, differing in their host shutoff effects related to M protein. Viral activity in persistently infected mouse L-929 and monkey Vero cell lines was followed by viral proteins detection, RNA synthesis throughout infection and finally detection of infectious particles. All three mutants cause extensive CPE followed by emergence of persistently infected cells on Vero cells. The same thing is seen on L-929 cells except for T1026 which causes little CPE. Taken together, the results form a basis of further studies to clarify how various viral and cellular factors interact in the establishment of a persistent infection by VSV mutants.
Asunto(s)
Regulación hacia Abajo , Fibroblastos/metabolismo , Fibroblastos/virología , Virus de la Estomatitis Vesicular Indiana/fisiología , Proteínas de la Matriz Viral/metabolismo , Replicación Viral , Animales , Tamaño de la Célula , Supervivencia Celular , Chlorocebus aethiops , Fibroblastos/citología , Células L , Ratones , Mutación , Biosíntesis de Proteínas , Proteínas/análisis , ARN Mensajero/análisis , ARN Mensajero/biosíntesis , ARN Viral/análisis , Células Vero , Virus de la Estomatitis Vesicular Indiana/genética , Virus de la Estomatitis Vesicular Indiana/patogenicidad , Proteínas de la Matriz Viral/química , Proteínas de la Matriz Viral/genética , Proteínas Virales/análisisAsunto(s)
Enfermedades Virales del Sistema Nervioso Central/virología , Infecciones por Coronavirus/virología , Coronavirus Humano OC43/patogenicidad , Modelos Animales de Enfermedad , Animales , Astrocitos/virología , Encéfalo/virología , Células Cultivadas , Enfermedades Virales del Sistema Nervioso Central/fisiopatología , Infecciones por Coronavirus/fisiopatología , Coronavirus Humano OC43/genética , Coronavirus Humano OC43/aislamiento & purificación , Humanos , Macrófagos/virología , Ratones , Ratones Endogámicos BALB C , Microglía/virología , ARN Viral/análisis , Médula Espinal/virologíaRESUMEN
Strains OC43 and 229E of human coronaviruses (HCoV) cause one-third of common colds and hospital-acquired upper respiratory tract HCoV infections have been reported in premature newborns. To evaluate possible sources of infection, virus survival was studied in aqueous suspensions and on absorptive and non-absorptive surfaces representative of a hospital environment. Virus susceptibility to chemical disinfection with standard products was also characterized. Virus survived in saline solution for as long as six days but less in culture medium, with or without added cells. After drying, HCoV-229E infectivity was still detectable after 3h on various surfaces (aluminum, sterile latex surgical gloves, sterile sponges) but HCoV-OC43 survived 1h or less. Of the various chemical disinfectants tested, Proviodine reduced the virus infectious titre by at least 50%. This study suggests that surfaces and suspensions can be considered as possible sources of contamination that may lead to hospital-acquired infections with HCoV and should be appropriately disinfected.
Asunto(s)
Coronavirus Humano 229E , Infecciones por Coronavirus/prevención & control , Coronavirus Humano OC43 , Coronavirus/fisiología , Infección Hospitalaria/prevención & control , Desinfección , Equipos y Suministros de Hospitales/microbiología , Infecciones del Sistema Respiratorio/prevención & control , Coronavirus/clasificación , Coronavirus/efectos de los fármacos , Coronavirus/aislamiento & purificación , Desinfectantes/farmacología , Desinfección/métodos , Monitoreo del Ambiente/métodos , Contaminación de Equipos , Francia , Humanos , Recién Nacido , Recien Nacido Prematuro , SuspensionesRESUMEN
Human coronaviruses (HCoV) cause common colds but can also infect neural cell cultures. To provide definitive experimental evidence for the neurotropism and neuroinvasion of HCoV and its possible association with multiple sclerosis (MS), we have performed an extensive search and characterization of HCoV RNA in a large panel of human brain autopsy samples. Very stringent reverse transcription-PCR with two primer pairs for both viral strains (229E and OC43), combined with Southern hybridization, was performed on samples from 90 coded donors with various neurological diseases (39 with MS and 26 with other neurological diseases) or normal controls (25 patients). We report that 44% (40 of 90) of donors were positive for 229E and that 23% (21 of 90) were positive for OC43. A statistically significant higher prevalence of OC43 in MS patients (35.9%; 14 of 39) than in controls (13.7%; 7 of 51) was observed. Sequencing of nucleocapsid protein (N) gene amplicons revealed point mutations in OC43, some consistently found in three MS patient brains and one normal control but never observed in laboratory viruses. In situ hybridization confirmed the presence of viral RNA in brain parenchyma, outside blood vessels. The presence of HCoV in human brains is consistent with neuroinvasion by these respiratory pathogens. Further studies are needed to distinguish between opportunistic and disease-associated viral presence in human brains.
Asunto(s)
Coronavirus Humano 229E , Infecciones por Coronavirus/virología , Coronavirus Humano OC43 , Coronavirus/aislamiento & purificación , Esclerosis Múltiple/virología , Neuronas/virología , Encéfalo/patología , Encéfalo/virología , Infecciones por Coronavirus/patología , Humanos , Hibridación in Situ , Esclerosis Múltiple/patología , Replicación ViralRESUMEN
Multiple sclerosis (MS) is an immune-mediated demyelinating disease that could be triggered by a viral infection. Coronaviruses induce an MS-like disease in rodents, are neuroinvasive in humans and can infect primary cultures of human astrocytes and microglia. Infection of the human astrocytic cell line U-373MG by the OC43 strain of human coronavirus caused an upregulation of IL-6, TNF-alpha, and MCP-1 mRNA expression. This virus also modulated the activity of matrix metalloproteinases-2 and -9 and augmented nitric oxide production in both U-373MG cells and the human microglial cell line CHME-5. Thus, a coronaviral infection of glial cells could lead to the production of inflammatory molecules that have been associated with central nervous system pathologies such as MS.
Asunto(s)
Coronavirus Humano OC43 , Coronavirus/fisiología , Regulación de la Expresión Génica , Neuroglía/metabolismo , Neuroglía/virología , Astrocitos/enzimología , Astrocitos/inmunología , Astrocitos/metabolismo , Astrocitos/virología , Línea Celular , Quimiocina CCL2/genética , Humanos , Interleucina-6/genética , Metaloproteinasa 2 de la Matriz/metabolismo , Metaloproteinasa 9 de la Matriz/metabolismo , Microglía/enzimología , Microglía/inmunología , Microglía/metabolismo , Microglía/virología , Esclerosis Múltiple/metabolismo , Esclerosis Múltiple/virología , Neuroglía/enzimología , Neuroglía/inmunología , Óxido Nítrico/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Factor de Necrosis Tumoral alfa/genética , Regulación hacia ArribaRESUMEN
Phage-displayed peptide libraries were used to map immunologically relevant epitopes on the surface (S) glycoprotein of a neurotropic murine coronavirus (MHV-A59). Three in vitro virus-neutralizing and in vivo protective mAbs against either continuous or discontinuous epitopes on the S glycoprotein were used to screen 12 different peptide libraries expressed on the pVIII major coat protein of the fd filamentous bacteriophage. Consensus sequences that matched short sequences within the S glycoprotein were identified. The sequence of a tight-binding, mAb-selected peptide suggested the location of a discontinuous epitope within the N-terminal S1 subunit. Several tightly binding phage were amplified and used directly as immunogens in BALB/c and C57BL/6 mice. Partial protection of C57BL/6 mice against a lethal acute virus infection was achieved with a phage preparation that displayed a linear epitope. Protection correlated with the presence of sufficient levels of specific antiviral antibodies recognizing the same immunodominant domain and 13-mer peptide, located within the C-terminal S2 subunit, as the selecting mAb. Thus, the direct use of phage-displayed peptides to evaluate protective antiviral immune responses complements their use to characterize antibody-binding epitopes. This is the first evaluation of protective immunization induced by mAb-selected phage-displayed peptides.
Asunto(s)
Anticuerpos Antivirales/inmunología , Infecciones por Coronavirus/prevención & control , Coronavirus/inmunología , Mapeo Epitopo , Biblioteca de Péptidos , Secuencia de Aminoácidos , Animales , Anticuerpos Monoclonales/genética , Anticuerpos Monoclonales/inmunología , Anticuerpos Antivirales/biosíntesis , Clonación Molecular , Infecciones por Coronavirus/inmunología , Reacciones Cruzadas , Ensayo de Inmunoadsorción Enzimática , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Datos de Secuencia Molecular , Pruebas de Neutralización , VacunaciónRESUMEN
Previously, we have reported protective vaccination of mice against a coronavirus infection using rabbit polyclonal noninternal image Ab2gamma anti-idiotypic (anti-Id) antibody specific for a virus-neutralizing and protective monoclonal antibody (mAb) 7-10A against the viral surface S glycoprotein. To characterize further the mechanisms involved in the induction of protective immunity by this noninternal image anti-Id, plasma and splenocytes from Ab2gamma-immunized BALB/c mice were passively transferred to naive BALB/c mice, followed by viral challenge. A reproducible significant delay in mortality observed in mice to which plasma was passively transferred, together with the presence of specific in vitro neutralizing antiviral Ab3 identified the humoral immune response as the major element responsible for protection. The activation of specific and cross-reactive T lymphocytes by both virus and anti-Id in immunized mice and the absence of adoptive transfer of protection by splenocytes suggested the participation of T helper activity in the induction of protective virus-neutralizing Ab3. To obtain more defined monoclonal reagents for a better understanding of anti-Id-induced protection, mAb2 were generated against the same mAb1 7-10A and characterized. We report the successful generation of mAb2 of the gamma type. However, unlike the polyclonal Ab2gamma, they were not capable of inducing a protective immune response.
Asunto(s)
Anticuerpos Antiidiotipos/inmunología , Infecciones por Coronavirus/prevención & control , Virus de la Hepatitis Murina/inmunología , Vacunación , Animales , Anticuerpos Antiidiotipos/administración & dosificación , Anticuerpos Monoclonales/inmunología , Citotoxicidad Celular Dependiente de Anticuerpos , Proteínas del Sistema Complemento/inmunología , Infecciones por Coronavirus/inmunología , Citotoxicidad Inmunológica , Ensayo de Inmunoadsorción Enzimática , Femenino , Inmunización Pasiva , Activación de Linfocitos , Ratones , Ratones Endogámicos BALB C , Pruebas de Neutralización , Conejos , Ratas , Linfocitos T Colaboradores-Inductores/inmunologíaRESUMEN
Human coronaviruses (HuCV) cause common colds. Previous reports suggest that these infectious agents may be neurotropic in humans, as they are for some mammals. With the long-term aim of providing experimental evidence for the neurotropism of HuCV and the establishment of persistent infections in the nervous system, we have evaluated the susceptibility of various human neural cell lines to acute and persistent infection by HuCV-229E. Viral antigen, infectious virus progeny and viral RNA were monitored during both acute and persistent infections. The astrocytoma cell lines U-87 MG, U-373 MG, and GL-15, as well as neuroblastoma SK-N-SH, neuroglioma H4, and oligodendrocytic MO3.13 cell lines, were all susceptible to an acute infection by HuCV-229E. The CHME-5 immortalized fetal microglial cell line was not susceptible to infection by this virus. The MO3.13 and H4 cell lines also sustained a persistent viral infection, as monitored by detection of viral antigen and infectious virus progeny. Sequencing of the S1 gene from viral RNA after approximately 130 days of infection showed two point mutations, suggesting amino acid changes during persistent infection of MO3.13 cells but none for H4 cells. Thus, persistent in vitro infection did not generate important changes in the S1 portion of the viral spike protein, which was shown for murine coronaviruses to bear hypervariable domains and to interact with cellular receptor. These results are consistent with the potential persistence of HuCV-229E in cells of the human nervous system, such as oligodendrocytes and possibly neurons, and the virus's apparent genomic stability.
Asunto(s)
Astrocitoma/virología , Coronavirus Humano 229E , Infecciones por Coronavirus , Coronavirus , Glioma/virología , Neuroblastoma/virología , Oligodendroglía/virología , Susceptibilidad a Enfermedades , Humanos , Especificidad de Órganos , Células Tumorales CultivadasRESUMEN
Human coronaviruses (HuCV) are recognized respiratory pathogens. Data accumulated by different laboratories suggest their neurotropic potential. For example, primary cultures of human astrocytes and microglia were shown to be susceptible to an infection by the OC43 strain of HuCV (A. Bonavia, N. Arbour, V. W. Yong, and P. J. Talbot, J. Virol. 71:800-806, 1997). We speculate that the neurotropism of HuCV will lead to persistence within the central nervous system, as was observed for murine coronaviruses. As a first step in the verification of our hypothesis, we have characterized the susceptibility of various human neural cell lines to infection by HuCV-OC43. Viral antigen, infectious virus progeny, and viral RNA were monitored during both acute and persistent infections. The astrocytoma cell lines U-87 MG, U-373 MG, and GL-15, as well as neuroblastoma SK-N-SH, neuroglioma H4, oligodendrocytic MO3.13, and the CHME-5 immortalized fetal microglial cell lines, were all susceptible to an acute infection by HuCV-OC43. Viral antigen and RNA and release of infectious virions were observed during persistent HuCV-OC43 infections ( approximately 130 days of culture) of U-87 MG, U-373 MG, MO3.13, and H4 cell lines. Nucleotide sequences of RNA encoding the putatively hypervariable viral S1 gene fragment obtained after 130 days of culture were compared to that of initial virus input. Point mutations leading to amino acid changes were observed in all persistently infected cell lines. Moreover, an in-frame deletion was also observed in persistently infected H4 cells. Some point mutations were observed in some molecular clones but not all, suggesting evolution of the viral population and the emergence of viral quasispecies during persistent infection of H4, U-87 MG, and MO3.13 cell lines. These results are consistent with the potential persistence of HuCV-OC43 in cells of the human nervous system, accompanied by the production of infectious virions and molecular variation of viral genomic RNA.
Asunto(s)
Infecciones por Coronavirus , Coronavirus Humano OC43 , Coronavirus , Tejido Nervioso/virología , Línea Celular , Infecciones por Coronavirus/genética , Infecciones por Coronavirus/inmunología , Infecciones por Coronavirus/fisiopatología , Susceptibilidad a Enfermedades , Variación Genética , Genoma Viral , Humanos , Mutación PuntualRESUMEN
Erythrocytes offer the exciting opportunity of being used as carriers of therapeutic agents. Encapsulation within erythrocytes will give the therapeutic agent a clearance equivalent to the normal life of the erythrocyte therefore maintaining therapeutic blood levels over prolonged periods and also giving a sustained delivery to the monocyte-macrophage system (reticulo-endothelial system). Both the dose and frequency of therapeutic interventions could thus be reduced. Ensuring a near-physiological survival time of carrier erythrocytes is essential to their successful use as a sustained drug delivery system, and this has not been demonstrated in man. In this study we assessed the survival in vivo of autologous unloaded energy-replete carrier erythrocytes in nine volunteers, using a standard 51Cr erythrocyte-labelling technique. Within 144 h after infusion there was a 3 to 49% fall in circulating labelled cells, followed thereafter by an almost complete return to initial circulating levels; surface counting demonstrated an initial sequestration of erythrocytes by the spleen and subsequent release. Mean cell life and cell half-life of the carrier erythrocytes were within the normal range of 89 to 131 days and 19 to 29 days respectively. These results demonstrate the viability of carrier erythrocytes as a sustained drug delivery system.
Asunto(s)
Portadores de Fármacos , Envejecimiento Eritrocítico , Adulto , Radioisótopos de Cromo , Diálisis , Recuento de Eritrocitos , Femenino , Corazón , Humanos , Pulmón , Masculino , Bazo , Factores de TiempoRESUMEN
Human coronaviruses (HCV) have been associated mainly with infections of the respiratory tract. Accumulating evidence from in vitro and in vivo observations is consistent with the neurotropism of these viruses in humans. To verify the possibility of a persistent infection within the central nervous system (CNS), various human cell lines of neural origin were tested for their ability to maintain chronic infection by both known strains of HCV, OC43 and 229E. Production of infectious progeny virions was monitored by an immunoperoxydase assay on a susceptible cell line and viral RNA was observed after RT-PCR. Astrocytic cell lines U-373 MG and U-87 MG did not sustain a persistent HCV-229E infection, even though they were susceptible to an acute infection by this virus. On the other hand, these two cell lines could maintain a persistent infection by HCV-OC43 for as many as 25 cell passages (about 130 days of culture). Relatively stable titers of infectious viral particles, as well as apparently constant amounts of viral RNA were detected throughout the persistent infection of U-87 MG cells. However, persistent infection of U-373 MG cells was accompanied by the detection of infectious viral particles from passage 0 to passage 13 and then from passage 20 to the end of the experiment. This gap in the production of infectious virions was correlated by a drop in the apparent amount of viral RNA detected at passages 15 and 20. These results confirm the ability of HCV-OC43 to persistently infect cells of an astrocytic lineage and, together with our previous observations of HCV infection of primary cultures of human astrocytes and the detection of HCV RNA in human brains, are consistent with the possibility that this human coronavirus could persist in the human CNS by targeting astrocytes.
Asunto(s)
Coronavirus Humano 229E , Coronavirus Humano OC43 , Coronavirus/fisiología , Neuronas/virología , Línea Celular , Coronavirus/crecimiento & desarrollo , Humanos , Neuroglía/virología , ARN Viral/análisis , Virión , Latencia del VirusRESUMEN
Human coronaviruses, with two known serogroups named 229E and OC43, cause up to one third of common colds and may be associated with serious diseases such as nosocomial respiratory infections, enterocolitis, pericarditis and neurological disorders. Reliable methods of detection in clinical samples are needed for a better understanding of their role in pathology. As a first step in the design of such diagnostic procedures, the sensitivities and specificities of two viral diagnostic assays were compared in an experimental cell culture model: an indirect immuno-fluorescence assay using monoclonal antibodies and reverse transcriptase-polymerase chain reaction amplification of viral RNA from infected cells. Immunofluorescence detected human coronaviruses in cells infected at a MOI as low as 10(-2) (log TCID50/ml = 4.25 for HCV-229E and 2.0 for HCV-OC43; log PFU/ml = 4.83 for HCV-229E and 1.84 for HCV-OC43) versus 10(-3) (HCV-OC43) or 10(-4) (HCV-229E) for reverse transcriptase-polymerase chain reaction amplification (log TCID50/ml = 1.75 for HCV-229E and 1.5 for HCV-OC43; log PFU/ml = 2.3 for HCV-229E and 1.34 for HCV-OC43). There were no false positive signals with other human respiratory pathogens: influenza virus, respiratory syncytial virus and adenovirus. Moreover, each assay was coronavirus serogroup-specific. These results demonstrate the potential usefulness of immunofluorescence with monoclonal antibodies and reverse transcriptase-polymerase chain reaction RNA amplification for the rapid detection of human coronaviruses in infected cell cultures. Both methods could be applied to clinical specimens for the diagnosis of human infections.
Asunto(s)
Coronavirus Humano 229E , Coronavirus Humano OC43 , Coronavirus/aislamiento & purificación , Técnica del Anticuerpo Fluorescente Indirecta , Reacción en Cadena de la Polimerasa/métodos , Anticuerpos Monoclonales , Línea Celular/virología , Coronavirus/clasificación , Humanos , Sensibilidad y EspecificidadRESUMEN
Attachment to a cell surface receptor can be a major determinant of virus tropism. Previous studies have shown that human respiratory coronavirus HCV-229E uses human aminopeptidase N (hAPN [CD13]) as its cellular receptor for infection of lung fibroblasts. Although human coronaviruses are recognized respiratory pathogens, occasional reports have suggested their possible neurotropism. We have previously shown that human neural cells, including glial cells in primary cultures, are susceptible to human coronavirus infection in vitro (A. Bonavia, N. Arbour, V. W. Yong, and P. J. Talbot, J. Virol. 71:800-806, 1997). However, the only reported expression of hAPN in the nervous system is at the level of nerve synapses. Therefore, we asked whether hAPN is utilized as a cellular receptor for infection of these human neural cell lines. Using flow cytometry, we were able to show the expression of hAPN on the surfaces of various human neuronal and glial cell lines that are susceptible to HCV-229E infection. An hAPN-specific monoclonal antibody (WM15), but not control antibody, inhibited the attachment of radiolabeled HCV-229E to astrocytic, neuronal, and oligodendrocytic cell lines. A correlation between the apparent amount of cell surface hAPN and the level of virus attachment was observed. Furthermore, the presence of WM15 inhibited virus infection of these cell lines, as detected by indirect immunofluorescence. These results indicate that hAPN (CD13) is expressed on neuronal and glial cell lines in vitro and serves as the receptor for infection by HCV-229E. This further strengthens the neurotropic potential of this human respiratory virus.