RESUMEN
The objective of this study was to determine the initial and subsequent phenotypes of HIV-1 isolated from the blood, duodenal, and colonic biopsies of 51 HIV-1 positive patients followed prospectively over 2 years. Blood and tissues were cocultured with stimulated peripheral blood monocytes, and HIV was analyzed for phenotypic expression of syncytia-induction (SI). A total of 45/51 patients had HIV-1 isolated from the blood and 35/51 had HIV isolated from gastrointestinal tract. In 12/45 patients SI-HIV-1 was isolated from the blood. In 6/12 patients the blood phenotype reverted to the NSI phenotype. SI phenotypes were also isolated from the colon and duodenum of 8/35 patients and reversion from SI to NSI virus phenotype was again observed in gut tissue of 3/8 patients. These results show that gastrointestinal tissues can harbor SI HIV phenotype. Discordant phenotypes can be found in tissue and blood of late-stage patients. Reversion of phenotypic SI expression to NSI may occur in patients receiving monotherapy as antiretroviral treatment. These results suggest that gastrointestinal tissues may act as a separate and distinct site involved in HIV replication and its associated pathogenesis.
Asunto(s)
Sistema Digestivo/virología , Infecciones por VIH/sangre , VIH/genética , VIH/aislamiento & purificación , Biopsia , Didanosina/uso terapéutico , Femenino , Células Gigantes/virología , VIH/química , Enteropatía por VIH/genética , Enteropatía por VIH/virología , Infecciones por VIH/genética , Infecciones por VIH/virología , Humanos , Masculino , Mutación/genética , Mutación/fisiología , Fenotipo , Estudios Prospectivos , Estavudina/uso terapéutico , Carga Viral , Zidovudina/uso terapéuticoRESUMEN
Ohio HeLa cells in multichamber slides were inoculated with nasal samples from patients presenting with common cold symptoms and incubated at 33 degrees C with gentle shaking for 48 hours. The cultures were fixed with cold acetone, and viral antigens were detected by immunofluorescence using an antirhinovirus type 2 (HRV-2) polyclonal serum. Of 158 samples, 58 (36.7%) and 57 (36%) were positive for HRV by virus isolation (confirmed by acid lability test) and by culture-amplified immunofluorescent (CAIF) test, respectively. The correlation between the two tests was highly significant (P = 0.0001). Nasal washings or nasal/throat swabs were equally suitable for detecting virus by isolation but not by CAIF. On the other hand, nasal washings were better than nasal/throat swabs for detecting HRV by CAIF. In an ELISA system, the polyclonal anti-HRV-2 serum recognized a rhinovirus antigen expressed in situ within 48 hr postinfection by all the 11 HRV serotypes investigated. However, 60 hr postinfection, the anti-HRV-2 serum recognized only homologous and closely related HRV antigens. These results suggest that a rhinovirus "common" antigen may be expressed some 48 hr after infection of Ohio HeLa cells with rhinoviruses. The CAIF test provides a sensitive, rapid and reliable procedure to detect wild-type rhinovirus infection as well as a clear alternative to detection by isolation.
Asunto(s)
Resfriado Común/microbiología , Epítopos , Técnica del Anticuerpo Fluorescente , Rhinovirus/aislamiento & purificación , Antígenos Virales/aislamiento & purificación , Resfriado Común/epidemiología , Resfriado Común/inmunología , Ensayo de Inmunoadsorción Enzimática , Células HeLa , Humanos , Kuwait/epidemiología , Nariz/microbiología , Rhinovirus/crecimiento & desarrollo , Rhinovirus/inmunología , Manejo de EspecímenesRESUMEN
A polymerase chain reaction (PCR) assay was used to detect and differentiate picornaviruses (PVs), using primers homologous to the 5' non-coding and VP2 regions of the PV genome. The PCR resulted in a 530 bp PCR product for human rhinoviruses (HRVs) and a 650 bp product for polioviruses, coxsackieviruses (CV) or echoviruses. The PCR assay could detect as little as 1 p.f.u. of virus in either cerebrospinal fluid (CSF) or stool, using ethidium bromide-stained gels. Standard strains of poliovirus, CV, echovirus and HRV were detected, with the exception of echovirus type 22. In contrast, heterologous viruses, such as herpes simplex virus, human cytomegalovirus, adenovirus, influenza virus and rotavirus, as well as human and monkey cell DNA, were not amplified. In nasal swabs taken from patients with respiratory infections, the PCR detected 27 of 28 HRV isolation-positive specimens. All specimens from which viruses other than HRVs were isolated were negative by PCR. The PCR definitively identified poliovirus and CVs from the CSF or stool of patients with aseptic meningitis, as well as CV in the pericardial fluid of a patient who had suffered a myocardial infarction. Specimens taken from patients with similar pathologies, and from which heterologous viruses were isolated, were uniformly negative by PCR.
Asunto(s)
Genes Virales , Infecciones por Picornaviridae/diagnóstico , Picornaviridae/aislamiento & purificación , Secuencia de Bases , Reacciones Cruzadas , Replicación del ADN , ADN Viral/genética , ADN Viral/aislamiento & purificación , Enterovirus Humano B/genética , Humanos , Datos de Secuencia Molecular , Picornaviridae/clasificación , Picornaviridae/genética , Poliovirus/genética , Reacción en Cadena de la Polimerasa/métodosRESUMEN
The stimulatory effects of the 13S adenovirus E1A gene product on the human cytomegalovirus (HCMV) major immediate early (IE) enhancer were examined. Chimeric plasmids containing cloned portions of the HCMV major IE enhancer-promoter positioned upstream of the chloramphenicol acetyltransferase gene (cat) were cotransfected into HeLa cells with the plasmid p13S-wt which contained a cDNA encoding the adenovirus 13S E1A gene product. CAT expression from chimeric plasmids containing at least one copy of the HCMV 19 base pair (bp) repetitive motif was stimulated 10-fold in the presence of p13S-wt. The 19-bp motif contains a potential binding site for the cellular transcription factor ATF/CREB. Deletion analysis indicated that the ATF/CREB site was crucial for E1A-mediated stimulation. Insertion of a synthetic oligonucleotide homologous to a 19-bp motif and containing an ATF/CREB binding site into an HCMV chimera lacking ATF/CREB motifs conferred E1A responsivity on HCMV promoter-mediated CAT expression whereas insertion of a similar oligonucleotide containing a change of two bases in the sequence of the ATF/CREB site did not. Measurement of CAT-specific RNA verified the results of the CAT enzyme experiments. The ATF/CREB motif may be a target for stimulation of HCMV gene expression through either viral or cellular transcription factors.
Asunto(s)
Antígenos Virales/genética , Elementos de Facilitación Genéticos , Regulación Viral de la Expresión Génica , Proteínas Inmediatas-Precoces , Proteínas Oncogénicas Virales/genética , Regiones Promotoras Genéticas , Factores de Transcripción Activadores , Proteínas Precoces de Adenovirus , Secuencia de Bases , Proteínas Sanguíneas/metabolismo , Cloranfenicol O-Acetiltransferasa/genética , Deleción Cromosómica , Proteína de Unión a Elemento de Respuesta al AMP Cíclico , ADN Viral/metabolismo , Proteínas de Unión al ADN/metabolismo , Genes Virales , Células HeLa , Humanos , Oligonucleótidos/biosíntesis , Oligonucleótidos/genética , Plásmidos , ARN Viral/genética , Factores de Transcripción/metabolismo , TransfecciónRESUMEN
A hybridization assay using a biotinylated DNA probe was compared to both ELISA and direct isolation methods for detecting human cytomegalovirus (HCMV). The biotin labeled HCMV AD 169 HindIII-O-DNA fragment was used in a dot-blot assay to screen for the presence of HCMV in 186 urine specimens obtained from kidney transplant patients. The biotinylated HCMV HindIII-O probe could detect 3 log10 TCID50 units of HCMV. Urine specimens were also examined for the presence of HCMV by either ELISA or direct isolation of virus in tissue culture. The HindIII-O fragment detected 12 of 20 culture positive samples (sensitivity, 60%). There were 5 samples which were probe positive and cell culture negative (specificity, 97%). The ELISA assay also detected 12 of 20 culture positive samples (sensitivity, 60%). Eight samples were ELISA positive, cell culture negative (specificity, 95%). Seven specimens were positive by all three criteria. Five specimens which were both ELISA positive and probe positive were cell culture negative. The ELISA positive, probe positive, culture negative specimens originated from patients who gave a culture positive specimen within 10 days of the original sample. The combination of probe and ELISA assays detected 16 of the 20 culture positive specimens (sensitivity, 80%). The combined use of biotinylated DNA probes and ELISA allows the detection of HCMV in urine specimens with good sensitivity and specificity.