RESUMO
We measured plasma human herpesvirus 8 (HHV8) DNA load in consecutive patients presenting with HIV-associated multicentric Castleman disease (MCD) and in contemporaneous patients who had Kaposi sarcoma (KS), lymphoma or other diagnoses. All 11 patients with MCD had detectable plasma HHV8 DNA compared with 18 (72%) of 25 patients with KS, none with lymphoma and one of 38 patients with other diagnoses. Detectable plasma HHV8 DNA levels were higher among MCD patients, median (interquartile range [IQR]) = 43,500 (5200-150,000) copies/mL, when compared with those with KS, median (IQR) = 320 (167-822) copies/mL and those with lymphoma and other diagnoses (one-way analysis of variance; P = 0.0303). Using receiver operating characteristic analysis, a cut-off of >1000 copies HHV8 DNA/mL of plasma helped to discriminate between MCD and other diagnoses, with a specificity of 94.7% and a negative predictive value of 97.3%. The level of HHV8 viraemia, while not diagnostic, may aid discrimination between patients with MCD and those with KS and other systemic illnesses.
Assuntos
Hiperplasia do Linfonodo Gigante/diagnóstico , DNA Viral/sangue , Diagnóstico Diferencial , Herpesvirus Humano 8/fisiologia , Sarcoma de Kaposi/diagnóstico , Infecções Oportunistas Relacionadas com a AIDS/diagnóstico , Infecções Oportunistas Relacionadas com a AIDS/virologia , Hiperplasia do Linfonodo Gigante/virologia , Feminino , HIV , Infecções por HIV/complicações , Infecções por HIV/virologia , Herpesvirus Humano 8/genética , Humanos , Linfoma/diagnóstico , Linfoma/virologia , Masculino , Valor Preditivo dos Testes , Sarcoma de Kaposi/virologia , Sensibilidade e Especificidade , Carga ViralRESUMO
OBJECTIVES: To determine whether the recently identified multiple sclerosis-associated retrovirus, MSRV, is detectable in the serum and synovial fluid of patients with rheumatoid arthritis (RA). METHODS: A reverse transcription-polymerase chain reaction (RT-PCR) assay was used to seek evidence of particle-associated MSRV/HERV-W RNA in the plasma and synovial fluid of patients with RA and controls. Stringent precautions were taken to avoid detection of contaminating human genomic DNA and cellular RNA sequences. RESULTS: Thirty-seven plasma samples were tested (20 from RA patients and 17 from controls) but none had detectable MSRV/HERV-W RNA. Synovial fluid samples were available from nine patients with RA and 10 controls. Particle-associated MSRV/HERV-W RNA was reproducibly detected in two of nine synovial fluid samples from RA patients and in one control sample. The identity of RT-PCR products was confirmed by sequencing. CONCLUSION: MSRV/HERV-W RNA sequences are detectable in the synovial fluid of a small proportion of RA patients, but this phenomenon may not be specific to RA.
Assuntos
Artrite Reumatoide/virologia , Esclerose Múltipla/virologia , RNA/análise , Retroviridae/genética , Líquido Sinovial/química , HumanosRESUMO
The recognition that both human and murine retroviruses can cause motor neurone disease-like syndromes has raised the possibility that a retrovirus may be involved in the aetiology of motor neurone disease. This possibility was explored by looking for evidence of reverse transcriptase in the serum of motor neurone disease patients. Sera from 56 patients with motor neurone disease and 58 controls were tested by the product-enhanced reverse transcriptase assay, a technique that is approximately a million fold more sensitive than conventional reverse transcriptase assays and capable of detecting very low numbers of retroviral particles. Cell-free reverse transcriptase activity was detected in the serum of 33 of the 56 motor neurone disease patients (59%) but in only 3 of the controls (P < 0.00001). The reverse transcriptase activity was detectable in the presence of a large excess of an effective inhibitor of human cellular DNA polymerases and was therefore tentatively considered to be compatible with a retroviral origin. The reverse transcriptase activity, however, was not found to be due to the presence of known human exogenous retroviruses including HIV-1, HIV-2, HTLV-I, HTLV-II, HRV-5 or human foamy virus, as assessed by PCR-based assays. Further investigations will be required to determine the source of the reverse transcriptase activity observed in these motor neurone disease patient sera.
Assuntos
Doença dos Neurônios Motores/sangue , DNA Polimerase Dirigida por RNA/sangue , Adulto , Idoso , Esclerose Lateral Amiotrófica/sangue , Esclerose Lateral Amiotrófica/virologia , Linhagem Celular/virologia , DNA Viral/análise , DNA Polimerase Dirigida por DNA/genética , DNA Polimerase Dirigida por DNA/metabolismo , Reações Falso-Positivas , Feminino , Humanos , Lentivirus/isolamento & purificação , Masculino , Pessoa de Meia-Idade , Doença dos Neurônios Motores/virologia , Reação em Cadeia da Polimerase , Distribuição Aleatória , Retroviridae/isolamento & purificação , Spumavirus/isolamento & purificaçãoRESUMO
The partial molecular characterization of multiple sclerosis (MS)-associated retrovirus (MSRV), a novel retrovirus previously called LM7, is reported. MSRV has been isolated repeatedly from leptomeningeal, choroid plexus and from Epstein-Barr virus-immortalized B cells of MS patients. A strategy based on reverse transcriptase PCR with RNA-purified extracellular virions yielded an initial pol fragment from which other regions of the retroviral genome were subsequently obtained by sequence extension. MSRV-specific PCR primers amplified a pol region from RNA present at the peak of reverse transcriptase activity, coinciding with extracellular viral particles in sucrose density gradients. The same sequence was detected in noncellular RNA from MS patient plasma and in cerebrospinal fluid from untreated MS patients. MSRV is related to, but distinct from, the endogenous retroviral sequence ERV9. Whether MSRV represents an exogenous retrovirus with closely related endogenous elements or a replication-competent, virion-producing, endogenous provirus is as yet unknown. Further molecular epidemiological studies are required to determine precisely the apparent association of virions containing MSRV RNA with MS.
Assuntos
Esclerose Múltipla/virologia , RNA Viral/genética , Retroviridae/genética , Retroviridae/isolamento & purificação , Sequência de Aminoácidos , Sequência de Bases , Humanos , Dados de Sequência Molecular , Filogenia , Análise de SequênciaRESUMO
INTRODUCTION: Although recent claims implicating HTLV-1 in multiple sclerosis (MS) have been refuted, several reports suggest that another, hitherto uncharacterised, retrovirus may be involved. We have developed and applied a novel PCR-based strategy to explore this possibility. METHODS: Degenerate oligonucleotides were used in a semi-nested format to amplify, from reverse-transcribed RNA, a region of the pol gene which is well conserved amongst all known retroviruses. RESULTS: The 'pan-retrovirus' detection system was shown to be capable of detecting diverse retroviruses including human lentivirus, human oncovirus, simian D-type virus and murine oncovirus. The 'pan-retrovirus' technique identified a novel retroviral sequence, designated MSRV-cpol, in the serum of an MS patient and also in purified virions from MS patient-derived tissue cultures. Sequence comparisons suggest that in the pol gene MSRV is related (approximately 75% homology) to the endogenous retroviral element ERV9. CONCLUSION: These findings lend further support to the concept of retroviral involvement in MS.
Assuntos
Esclerose Múltipla/virologia , Reação em Cadeia da Polimerase/métodos , Infecções por Retroviridae/diagnóstico , Retroviridae/genética , Sequência de Aminoácidos/genética , Animais , Sequência de Bases/genética , Produtos do Gene pol/genética , Produtos do Gene pol/isolamento & purificação , Genes pol/genética , Humanos , Dados de Sequência Molecular , Retroviridae/isolamento & purificação , Infecções por Retroviridae/virologia , Homologia de SequênciaRESUMO
Hepatitis C virus (HCV) has as yet no practical culture system so any antigen or antibody studies must be carried out using recombinant antigens. In this study, HCV core sequence was amplified by PCR, inserted into pRSET, and expressed in E. coli. The resultant core protein was purified using nickel affinity chromatography which bound the six histidine tag attached to the N-terminus of the protein. After elution in imidazole buffer, the core protein was used to immunise Balb/c mice and monoclonal antibodies against HCV core were raised. Six monoclonals were examined in a variety of assays. All of them recognised the p27 kDa protein which they were raised against and 2D2 and 3D7 recognised the core component of an HCV Recombinant Immunoblot Assay (RIBA). None of the antibodies recognised the linear peptides in an Innolia HCV assay. 2D2 showed cytoplasmic granular staining in 1-5% of cells in frozen section of HCV infected livers. Cross-competition assays between themselves and human anti-HCV core positive sera divided the antibodies into two main groups (I and II), with a sub-division of group I into a and b. Group I antibodies were unable to be inhibited by human anti-HCV sera whereas group II antibodies were inhibited by these sera (up to 62%). Epitopes recognised by all the monoclonals were probably conformational with the group I epitope being located within the first 105 amino acids of the core sequence and the group II epitope between amino acids 105 and 160.
Assuntos
Anticorpos Monoclonais/imunologia , Hepacivirus/imunologia , Anticorpos Anti-Hepatite C/imunologia , Antígenos da Hepatite C/imunologia , Hepatite C/imunologia , Proteínas do Core Viral/imunologia , Animais , Hepatite C/sangue , Antígenos da Hepatite C/química , Antígenos da Hepatite C/genética , Antígenos da Hepatite C/isolamento & purificação , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Coelhos , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/isolamento & purificação , Proteínas do Core Viral/química , Proteínas do Core Viral/genética , Proteínas do Core Viral/isolamento & purificaçãoRESUMO
OBJECTIVES: To determine the prevalence of antibodies to the human T cell leukaemia/lymphoma viruses (HTLV-I and HTLV-II) in blood donors in north London in order to assess the economic impact and the logistic effects that routine screening would have on the blood supply. DESIGN: All donations collected by the north London blood transfusion centre between January 1991 and June 1991 were screened for antibodies to HTLV-I and HTLV-II by modified, improved Fujirebio gel particle agglutination test. Positive samples were titrated and retested as necessary. SUBJECTS: 96,720 unpaid volunteers, who gave 105,730 consecutive donations of blood and plasma. SETTING: North London blood transfusion centre. MAIN OUTCOME MEASURE: Observed numbers of donors confirmed to be seropositive for HTLV by reference laboratories. RESULTS: Of 2622 (2.5%) initially reactive samples, 414 (0.4% of all samples) gave a titre of > or = 1 in 16 on the modified agglutination test. Thirty five of the 414 serum samples yielded positive results on one of two enzyme linked immunosorbent assays (ELISA (Cambridge Biotech and Abbot)), and none of these results were confirmed by either reference laboratory. Five samples yielded positive results on both ELISAs and all five of these were confirmed to contain antibodies to HTLV. One of the five contained antibodies to HTLV-II and the others antibodies to HTLV-I. Four seropositive donors were white women whose only risk factor for infection was sexual contact. The fifth (positive for antibodies to HTLV-II) was an Anglo-Caribbean man who admitted to previous misuse of intravenous drugs. CONCLUSION: The prevalence of antibodies to HTLV in blood donors in north London was one in 19,344 (0.005%). Up to 100 donors a year might be identified in the United Kingdom as being infected with HTLV, although prevalence in different regions may vary considerably.
Assuntos
Doadores de Sangue , Anticorpos Anti-HTLV-I/análise , Infecções por HTLV-I/epidemiologia , Anticorpos Anti-HTLV-II/análise , Infecções por HTLV-II/epidemiologia , Adulto , Western Blotting , Ensaio de Imunoadsorção Enzimática , Estudos de Viabilidade , Feminino , Infecções por HTLV-I/imunologia , Infecções por HTLV-I/prevenção & controle , Infecções por HTLV-II/imunologia , Infecções por HTLV-II/prevenção & controle , Humanos , Londres/epidemiologia , Masculino , Programas de Rastreamento , Pessoa de Meia-Idade , PrevalênciaRESUMO
A 'nested' polymerase chain reaction (PCR) assay is described which is capable of detecting single copies of human T-cell lymphotropic virus (HTLV) in genomic DNA extracted from peripheral blood mononuclear cells (PBMCs). A single set of 'nested' oligonucleotide primers, based on the highly conserved tax/rex region of the viral genome, was able to detect both HTLV-I and HTLV-II proviral sequences in clinical samples of diverse geographical origins, from the United States, Great Britain, Japan, the Caribbean, Italy, Greece, Iraq and West Africa. Rapid discrimination between HTLV-I and HTLV-II infections was achieved by restriction enzyme analysis of unpurified second-round PCR products, even in those cases in which serological assays had failed to provide a definitive result. Over a 2-year period, a total of 53 HTLV infections (37 HTLV-I and 16 HTLV-II) were identified by this technique and complete concordance with serological typing, available in 41 cases, was observed.
Assuntos
Infecções por HTLV-I/diagnóstico , Infecções por HTLV-II/diagnóstico , Reação em Cadeia da Polimerase , Sequência de Bases , Linhagem Celular , DNA Viral/análise , Desoxirribonucleases de Sítio Específico do Tipo II , Diagnóstico Diferencial , Infecções por HTLV-I/microbiologia , Infecções por HTLV-II/microbiologia , Vírus Linfotrópico T Tipo 1 Humano/genética , Vírus Linfotrópico T Tipo 1 Humano/isolamento & purificação , Vírus Linfotrópico T Tipo 2 Humano/genética , Vírus Linfotrópico T Tipo 2 Humano/isolamento & purificação , Humanos , Leucemia-Linfoma de Células T do Adulto/diagnóstico , Leucemia-Linfoma de Células T do Adulto/microbiologia , Leucócitos Mononucleares/microbiologia , Dados de Sequência Molecular , Paraparesia Espástica Tropical/diagnóstico , Paraparesia Espástica Tropical/microbiologia , Provírus/genética , Provírus/isolamento & purificação , Sensibilidade e Especificidade , SorotipagemRESUMO
Sera from 30 patients with community-acquired, biopsy-proven chronic non-a,non-B hepatitis (NANBH) were tested for antibodies to the C100 protein of hepatitis C virus (HCV). The 20 patients who showed reactivity in this assay were followed prospectively for 6 months, during which time seven were treated with recombinant alpha-interferon. HCV RNA was detected by "nested" polymerase chain reaction (PCR) in 19 of the 20 anti-C100-positive sera taken at the onset of the study and also in five of the ten anti-C100-negative sera. Pretreatment viraemia levels ranged from 2 x 10(3) to 2 x 10(8) HCV genomes/ml. After 6 months of interferon, elevated serum alanine aminotransferase (ALT) levels had fallen to normal in four of the seven treated patients. In each case the response to interferon was accompanied by either a disappearance of or a decline (1 log to 8 log reduction) in viraemia. HCV genome titres in the three nonresponders and in the 13 untreated anti-C100-positive patients did not change significantly over this 6 month period. These findings confirm the aetiological role of HCV in community-acquired NANBH and suggest that quantitative PCR will become a valuable technique for monitoring the antiviral effect of interferon and other experimental treatments.