RESUMEN
Although the clinical relevance of GB virus C (GBV-C) is still elusive, this virus has been found with high prevalence in several groups of patients with liver disease. As was shown for hepatitis C virus (HCV), minus-strand RNA is supposed to function as a replicative intermediate. We have established a reliable and sensitive detection system for GBV-C minus-strand RNA based on nested RT-PCR (reverse transcription-polymerase chain reaction) with a tagged primer system. Sensitivity and specificity was extensively tested using in-vitro transcribed GBV-C sequences and genomic viral RNA. Specificity of the amplified fragments was proven by Southern blot hybridization. Using this detection system, we found the presence of GBV-C minus-strand RNA in 6/41 (14.6%) sera of GBV-C infected or GBV-C/HCV coinfected patients. No correlation with virological parameters such as amount of GBV-C plus-strand RNA, genotype or titer of HCV could be detected.
Asunto(s)
Flaviviridae/aislamiento & purificación , Reacción en Cadena de la Polimerasa/métodos , ARN Viral/aislamiento & purificación , Adulto , Southern Blotting , Femenino , Flaviviridae/genética , Genes Virales/genética , Marcadores Genéticos , Hepatitis C/sangre , Hepatitis C/virología , Hepatitis Viral Humana/sangre , Hepatitis Viral Humana/virología , Humanos , Masculino , Persona de Mediana Edad , Sensibilidad y EspecificidadRESUMEN
In 1995, a new human hepatitis virus belonging to the family Flaviviridae was described and designated hepatitis GBV-C. To investigate variations within the genome of GBV-C and to study the relationship of GBV-C to GBV-A/B or hepatitis C virus (HCV), we established a detection system using reverse transcriptase polymerase chain reaction (RT-PCR) of the putative helicase region (NS3). So far, isolates derived from 14 different GBV-C-positive sera were analyzed (GBV-C/S3-36), showing 80.1-89.4% (mean: 85%) identical nucleotides. The deduced amino acid sequences revealed 97.3% homology. Nucleotide sequences of GBV-C/S3-36 revealed about 60% identity to GBV-A as well as to HCV, but only 56% identity to GBV-B. Amino acid sequences revealed 73.4 and 68.6% similarity to GBV-A and GBV-B, respectively, but a slightly higher percentage of 78.5% to HCV sequences. Thus, according to the putative GBV-C helicase sequence, a subtyping of GBV-C into different genotypes may be necessary.
Asunto(s)
Flaviviridae/genética , Flaviviridae/aislamiento & purificación , Hepatitis Viral Humana/virología , Adulto , Secuencia de Aminoácidos , Clonación Molecular , Hepatitis Viral Humana/genética , Humanos , Datos de Secuencia Molecular , Filogenia , Reacción en Cadena de la Polimerasa , Homología de Secuencia de Aminoácido , Homología de Secuencia de Ácido NucleicoRESUMEN
To investigate whether the recently discovered hepatitis G virus (HGV) influences the clinical outcome of heart transplant recipients under immunosuppression, we determined the prevalence of HGV infections correlated with liver function and survival in 51 patients. Presence of HGV RNA and anti-E2, a marker for resolved HGV infection, were serially tested in sera from patients before and after heart transplantation (HTX) by nested RT-PCR and ELISA. Four of 51 (7.8%) patients before transplantation, and 22 of 50 patients (44%) after transplantation showed signs of persistent or resolved HGV infection. HGV infection was not associated with impairment of liver function or with patient survival. In summary, presence of HGV infection does not influence the clinical outcome in heart transplant patients.
Asunto(s)
Flaviviridae , Trasplante de Corazón , Hepatitis Viral Humana/diagnóstico , Ensayo de Inmunoadsorción Enzimática , Femenino , Flaviviridae/aislamiento & purificación , Rechazo de Injerto , Hepatitis Viral Humana/fisiopatología , Humanos , Pruebas de Función Hepática , Masculino , Persona de Mediana Edad , Reacción en Cadena de la Polimerasa , ARN Viral/análisis , Estudios Retrospectivos , Proteínas del Envoltorio Viral/análisisRESUMEN
The amyloid precursor protein (APP), which is localized on both human chromosome 21 and its murine counterpart, chromosome 16 and which is involved in the formation of deposits in Alzheimer's disease, could be shown to bind effectively to a glytolytic enzyme: rat glyceraldehyde 3-phosphate dehydrogenase (Gapdh). We report here the isolation of a cDNA of murine Gapdh from mouse chromosome 16 (MMU16) originating from microclones of the distal part of MMU16 and the use of homologous genomic DNA sequences to further screen a cDNA phage library. The cDNA was sequenced, confirmed by polymerase chain reaction following reverse transcriptase (RT-PCR) and the open reading frame was expressed in vitro. The possible localization of Gapdh on MMU16--which may provide a mouse model for Down's syndrome and Alzheimer's disease--may lead to new insights into glycolysis and its role in the two syndromes.
Asunto(s)
Enfermedad de Alzheimer/metabolismo , Cromosomas/química , ADN Complementario/aislamiento & purificación , Gliceraldehído-3-Fosfato Deshidrogenasas/metabolismo , Glucólisis , Fragmentos de Péptidos/metabolismo , Animales , Secuencia de Bases , Humanos , Ratones , Sondas Moleculares/genética , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , Transcripción GenéticaAsunto(s)
Infecciones por Flaviviridae/cirugía , Virus GB-C , Trasplante de Corazón/fisiología , Hepatitis Viral Humana/cirugía , Trasplante de Hígado/fisiología , Regiones no Traducidas 3'/genética , Secuencia de Bases , Cardiomiopatía Dilatada/cirugía , Cartilla de ADN , Europa (Continente) , Femenino , Infecciones por Flaviviridae/diagnóstico , Infecciones por Flaviviridae/epidemiología , Virus GB-C/genética , Virus GB-C/aislamiento & purificación , Trasplante de Corazón/patología , Hepatitis Viral Humana/diagnóstico , Hepatitis Viral Humana/epidemiología , Humanos , Pruebas de Función Hepática , Trasplante de Hígado/patología , Masculino , Persona de Mediana Edad , Isquemia Miocárdica/cirugía , Prevalencia , ARN Viral/aislamiento & purificación , Resultado del TratamientoRESUMEN
Programmed cell death is not only known as a mechanism mediating tissue destruction, but also as an organismic tool for body shaping and regulation of morphological events during development. Here we report the tight and vital link of the most prominent form of programmed cell death, apoptosis, to one of the oldest, most basic, and most radical developmental processes, the metamorphosis of the marine hydrozoon Hydractinia echinata. Apoptosis, represented by DNA fragmentation, appears very early during metamorphosis, approximately 20 minutes post induction. It is then executed in a very distinct spatial and temporal pattern, including the removal or phagocytosis of a large number of larval cells prior to the appearance of stolons and tentacles. Our data indicate a developmental program striving to reduce all body parts that are no longer necessary, before reaching a distinct turning point, when the development of adult features is initiated. During these events, morphogenesis of basal and apical structures correlates with recycling of that particular larval region, indicated by the presence of apoptosis. Based on these data, the necessity of apoptosis for normal development of adult patterns is inferred and a fundamental association of apoptosis with developmental processes can be stated.
Asunto(s)
Apoptosis , Cnidarios/citología , Cnidarios/crecimiento & desarrollo , Animales , Tipificación del Cuerpo , Cnidarios/metabolismo , Fragmentación del ADN , Larva/citología , Larva/crecimiento & desarrollo , Larva/metabolismo , Metamorfosis Biológica , MorfogénesisRESUMEN
BACKGROUND: Haemodialysis patients run a high risk of acquiring viral hepatitis B (HBV) or C (HCV) infection. Recently a new parenterally transmittable RNA virus, designated GBV-C, was isolated. METHODS: We therefore screened 266 patients on maintenance dialysis and 358 blood donors as a control group for GBV-C by nested PCR and correlated the data with AST, ALT, duration of dialysis, transfusions, renal transplants and coinfections with HBV and HCV. RESULTS: The prevalence of GBV-C among haemodialysis patients was 7.9%, and 3.6% among blood donors. Neither duration of dialysis nor number of blood transfusions were associated with GBV-C infection, whereas GBV-C-positive patients were significantly more often transplanted than GBV-C-negative individuals. Transaminases of GBV-C-positive individuals remained within normal limits in all haemodialysis patients and normal in all infected blood donors. Coinfections of GBV-C with HBV and HCV were only present in 0.7% and 1% respectively. CONCLUSIONS: We conclude that GBV-C virus infection is frequent among haemodialysis patients. Transaminases cannot serve as surrogate markers, and parenteral as well as community-acquired infection seems to be possible.
Asunto(s)
Flaviviridae , Hepatitis Viral Humana/epidemiología , Diálisis Renal/efectos adversos , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Alanina Transaminasa/sangre , Secuencia de Aminoácidos , Femenino , Humanos , Masculino , Persona de Mediana Edad , Datos de Secuencia Molecular , Prevalencia , ARN Viral/análisisRESUMEN
A novel RNA virus of the Flaviviridae family has been discovered recently and designated hepatitis GB-C virus (GBV-C). Previous studies have reported that GBV-C is associated with posttransfusion hepatitis, chronic viral hepatitis, and cryptogenic hepatitis. However, the clinical significance of GBV-C infection has been questioned increasingly in patients not undergoing transplantation. To investigate whether GBV-C infection under immunosuppression affects the clinical or the histological outcome in liver transplant recipients, we determined the prevalence and incidence of GBV-C infections and the clinical and histological signs in patients after orthotopic liver transplantation (OLT). The presence of GBV-C was tested in sera from patients before and in regular intervals up to 6 years after OLT by nested reverse transcription-polymerase chain reaction using primers derived from the NS3 region. A total of 72 patients were studied. Before OLT, 8 of 72 (11.1%) patients were positive for GBV-C. After OLT, 7 of 8 (87.5%) remained positive. Of 64 patients who were negative for GBV-C before OLT, 23 became positive after OLT, resulting in a de novo rate of GBV-C infection of 35.9%. We could not detect a higher rate of histologically proven hepatitis in GBV-C-positive patients (29.1%) than in GBV-C-negative patients (14.6%, P > 0.057). Comparing GBV-C-positive with GBV-C-negative liver transplant patients, we could not find any differences in age, gender, liver function tests, number of blood transfusions, histological degree of hepatitis, or number of rejection episodes. Survival was not negatively influenced by GBV-C positivity. In conclusion, the presence of GBV-C did not influence the clinical or histological outcome in liver transplant patients.
Asunto(s)
Flaviviridae/aislamiento & purificación , Hepatitis Viral Humana/diagnóstico , Trasplante de Hígado , Adulto , Femenino , Hepatitis Viral Humana/mortalidad , Hepatitis Viral Humana/patología , Humanos , Trasplante de Hígado/mortalidad , Masculino , Persona de Mediana Edad , Datos de Secuencia Molecular , Tasa de SupervivenciaRESUMEN
BACKGROUND/AIMS: Recently, the hepatitis GB-C virus (GBV-C) has been identified as another virus potentially causing chronic hepatitis. Although high rates of coinfection are emerging in drug addicts with chronic hepatitis C virus infection, no detailed data on clinical presentation are available. Therefore, co-infection was sought in hepatitis C virus patients to determine the impact of GB-C virus on clinical presentation. METHODS: GBV-C was determined by nested reverse transcriptase-polymerase chain reaction in serum of 70 HIV negative intravenous drug abusers with chronic hepatitis C. Biochemical, histological and virological parameters were compared between patients with or without GBV-C coinfection. RESULTS: Hepatitis C virus and GBV-C coinfection was found in 18 of 70 (25.7%) patients. Cases with coinfection were younger and had shorter duration of disease (31.4+/-6.2 vs. 35.3+/-7.3 (p=0.09) and 9.9+/-6.8 vs. 12.9+/-7.7 (p=0.17) years) than those without coinfection. Neither hepatitis C virus genotype distribution and HCV RNA levels nor serum liver function tests, titers of immunoglobulins or autoantibodies differed between the two groups. Histologically, chronic active hepatitis (16.7 vs. 46.4%, p=0.07), fibrosis (8.3% vs. 21.4%, p=0.3), and cirrhosis (0% vs. 8.2%, p=0.31) were less prevalent in coinfected patients. After interferon treatment, 5/6 coinfected and 11/19 patients with hepatitis C virus infection alone had cleared HCV RNA and 4/6 lost GBV-C RNA from serum. The two patients with GBV-C/HCV infection who persistently cleared hepatitis C virus but not GBV-C from serum had normal transaminases during follow-up despite persistence of GBV-C. CONCLUSIONS: Coinfection of chronic hepatitis C patients with GBV-C does not lead to a significant change in clinical presentation, severity of liver disease, hepatitis C viremia, or response to interferon treatment.
Asunto(s)
Flaviviridae/genética , Hepacivirus/genética , Hepatitis C/complicaciones , Hepatitis Viral Humana/complicaciones , ARN Viral/análisis , Abuso de Sustancias por Vía Intravenosa/complicaciones , Adulto , Alanina Transaminasa/sangre , Anticuerpos Antinucleares/análisis , Antivirales/uso terapéutico , Enfermedad Crónica , Cartilla de ADN/química , Ensayo de Inmunoadsorción Enzimática , Femenino , Flaviviridae/inmunología , Estudios de Seguimiento , Genotipo , Hepacivirus/inmunología , Hepatitis C/patología , Hepatitis C/terapia , Anticuerpos contra la Hepatitis C/análisis , Hepatitis Viral Humana/patología , Hepatitis Viral Humana/terapia , Humanos , Interferón-alfa/uso terapéutico , Masculino , Reacción en Cadena de la Polimerasa , Estudios Retrospectivos , Abuso de Sustancias por Vía Intravenosa/sangre , Abuso de Sustancias por Vía Intravenosa/virologíaRESUMEN
Hepatitis C virus (HCV) is a major cause of chronic viral hepatitis. Development of anti-viral strategies has been hampered by the lack of efficient cell systems to propagate HCV in vitro. To establish a long-term culture system, we tested human hepatoma (HuH7, HepG2) and porcine non-hepatoma (PK15, STE) cell lines, as well as several culture and infection conditions. As a marker for virus replication, minus-strand HCV RNA in infected cells was detected by an enhanced detection system using nested RT-PCR followed by hybridization analysis. Short-term efficiency of HCV infection (10 days) was slightly increased by addition of polyethylene glycol (PEG) and/or dimethyl sulfoxide (DMSO) to culture media during inoculation of HuH7, PK15 and STE cells, but no augmentation in long-term culture was achieved, suggesting enhanced attachment of HCV to cells rather than more efficient infection. A stabilizing effect on HCV propagation was observed for 50 days in a serum-free medium with stimulation of the low-density lipoprotein (LDL) receptor expression by lovastatin. Using partially serum-free culture conditions, long-term persistence of HCV in cells and release of virions into supernatant was achieved for up to 130 days. Infectivity of released virions in supernatants after long-term culturing (day 30-80) was shown by successful infection of fresh cells. In conclusion, supplementation with PEG, DMSO and lovastatin during inoculation did not enhance virus replication substantially, but continued stimulation of LDL-receptor expression resulted in infections which persisted for over 4 months. These data support the hypothesis of an LDL-receptor mediated uptake of HCV into cells in vitro.
Asunto(s)
Hepacivirus/crecimiento & desarrollo , Hepatitis C/virología , ARN Viral/análisis , Anciano , Animales , Células Cultivadas , Femenino , Hepacivirus/genética , Humanos , Reacción en Cadena de la Polimerasa/métodos , ARN Viral/biosíntesis , Porcinos , Factores de TiempoRESUMEN
BACKGROUND/AIMS: Recently, GB virus C (GBV-C) has been identified as another virus potentially causing viral hepatitis. However, its hepatotropism and pattern of infection in humans is still unknown. To elucidate the presence and replication of GBV-C in the human liver, we investigated tissue samples of six explanted livers from five GBV-C mono- or GBV-C/HCV co-infected patients for GBV-C RNA plus- and minus-strand RNA. METHODS: These tissues were examined using nested RT-PCR followed by Southern blot hybridization as well as fluorescence in situ hybridization on liver cryosections. To further substantiate susceptibility of liver cells for GBV-C, in vitro infection of human hepatoma cells (HuH7, HepG2) with GBV-C mono-infected serum was performed. RESULTS: By reverse transcription followed by nested PCR (RT-PCR), 5 of 6 liver specimens (4/5 patients) were positive for GBV-C plus-strand RNA, and viral minus-strand RNA could be detected in 4 of 6 liver specimens (4/5 patients). One liver sample was negative for GBV-C RNA. In two specimens we could identify GBV-C infection by in situ hybridization. Virus infection appeared to be restricted to hepatocytes and detection of minus-strand RNA showed viral replication in a few highly infected liver cells. In vitro infection of HepG2 or HuH7 cells confirmed these findings by a release of virions into supernatant. CONCLUSION: In conclusion, our results establish GBV-C as a hepatotropic virus infecting human cells of hepatic origin in vivo and in vitro.
Asunto(s)
Carcinoma Hepatocelular/virología , Flaviviridae/fisiología , Hepatitis Viral Humana/virología , Neoplasias Hepáticas/virología , Hígado/virología , Replicación Viral , Línea Celular , Clonación Molecular , Flaviviridae/aislamiento & purificación , Hepatitis Viral Humana/sangre , Hepatitis Viral Humana/patología , Humanos , Hibridación Fluorescente in Situ , Hígado/patología , ARN Viral/genética , ARN Viral/aislamiento & purificación , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transcripción Genética , Células Tumorales CultivadasRESUMEN
BACKGROUND/AIMS: The aim of the study was to assess the impact factor of HCV and HGV in fulminant hepatic failure. METHODS: The 5'-untranslated regions of HCV RNA and HGV RNA and a segment of the core antigen sequence of HBV were amplified after extracting the nucleic acids from snap-frozen tissue aliquots from explanted livers of 26 consecutive patients undergoing orthotopic liver transplantation for fulminant hepatic failure preoperatively diagnosed as either autoimmune (n=2), HAV/HBV (n=8), toxic (n=4) or aetiologically unknown (n=12). RESULTS: HCV RNA was detected in five of 26 (19.2%) livers with fulminant hepatic failure. All five HCV RNA-positive livers belonged to the group of non-toxic, non-autoimmune liver failure (n=20), three of them were found in the group of liver failure with unknown aetiology (n=12) and two in the group of HBV-associated liver failure (n=7), making an HCV incidence of 25%, 25% and 28.6%, in the different groups, respectively. HGV RNA was detected in 10 of 17 (58.8%) explants and in all four groups of fulminant hepatic failure as defined preoperatively. HBV DNA was identified in six livers of 26 patients (23.1%) with fulminant hepatic failure. Neither HCV RNA nor HBV DNA was detected in the livers of patients with toxic or autoimmune fulminant hepatic failure. CONCLUSIONS: These results indicate that HBV and HCV, but not HGV, play an aetiologic role in fulminant hepatic failure. HCV-positive cases were concentrated either in the group of otherwise unexplained fulminant hepatic failure or in the group of HBV fulminant hepatic failure. HGV-positive cases, on the other hand, were found within all four preoperatively defined groups, indicating a role as cofactor rather than as single aetiologic agent.