RESUMO
During Epstein-Barr virus (EBV) latency, the EBV genome is largely silenced by methylation. This silencing is overturned during the switch to the lytic cycle. A key event is the production of the viral protein Zta which binds to three Zta-response elements (ZRE) from the Rta promoter (Rp), two of which (ZRE2 and ZRE3) include three CpG motifs methylated in the latent genome. The bisulphite pyrosequencing reaction was used to quantify the methylation of ZRE2, ZRE3a, and ZRE3b in EBV-positive cell lines and in ex vivo samples of EBV-related diseases, in order to assess whether the level of methylation in these ZREs could provide additional information to viral DNA load and serology in the characterization of EBV-associated diseases. In PBMC from two patients with infectious mononucleosis, over time Rp became increasingly methylated whereas EBV load decreased. In tonsil from patients with chronic tonsillitis, the methylation was less than in EBV-associated tumors, regardless of the viral load. This was even more striking when only the ZRE3a and ZRE3b were considered since some samples presented unbalanced profiles on ZRE2. EBV reactivation in cell culture showed that the reduction in the overall level of methylation was closely related to the production of unmethylated virions. Thus, an assessment of the level of methylation may help to better characterize EBV replication in PBMC and in biopsies with high EBV load, during infectious mononucleosis and EBV-associated cancers. J. Med. Virol. 88:1814-1820, 2016. © 2016 Wiley Periodicals, Inc.
Assuntos
Metilação de DNA , Infecções por Vírus Epstein-Barr/virologia , Proteínas Imediatamente Precoces/química , Proteínas Imediatamente Precoces/genética , Regiões Promotoras Genéticas , Transativadores/química , Transativadores/genética , Carga Viral , Linhagem Celular , DNA Viral/genética , Infecções por Vírus Epstein-Barr/sangue , Infecções por Vírus Epstein-Barr/imunologia , Regulação Viral da Expressão Gênica , Genoma Viral , Herpesvirus Humano 4/genética , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Proteínas Imediatamente Precoces/metabolismo , Mononucleose Infecciosa/virologia , Leucócitos Mononucleares/virologia , Tonsila Palatina/virologia , Saliva/virologia , Transativadores/metabolismo , Latência Viral/genéticaRESUMO
BACKGROUND: Hepatitis C virus (HCV) is underdiagnosed and therefore increasing the opportunities for HCV testing without venipuncture may be useful. OBJECTIVES: We evaluated the analytical performance of a modified, commercially available, combined HCV antigen-antibody assay (cEIA) (Monolisa(®) HCV-Ag-Ab-ULTRA) and a commercially available point-of-care (POC) device (OraQuick(®) HCV) on fingerstick blood (FSB) and oral mucosal transudate (OMT). STUDY DESIGN: FSB, OMT and serum samples were collected from 113 cases of HCV-antibody-positive patients and 88 HCV-antibody-negative controls. The HCV-antibody-positive group included 63 patients with quantifiable HCV-RNA (56%) and 17 HIV/HCV co-infected patients (15%). FSB and OMT specimens were collected as dried blood spots (DBSs) or with the OraSure collection system, before testing with cEIA. RESULTS: With FSB specimens, the cEIA and the POC device exhibited 100% specificity and 98.2% and 97.4% sensitivity, respectively. The specificity of the cEIA in FSB sharply decreased if stored 3days at room temperature. With OMT specimens, the cEIA sensitivity (71.7%) and specificity (94.3%) were significantly lower than the performance of OraQuick(®) HCV (sensitivity, 94.6%; specificity, 100%). The optical densities obtained with the cEIA in FSB and OMT were lower in HIV/HCV co-infected patients compared with HCV monoinfected patients. CONCLUSION: The cEIA using FSB specimens collected on DBSs preserved in appropriate storage conditions was a reliable alternative, equivalent to the POC assay, for HCV testing without venipuncture. The cEIA was not adapted for HCV testing on OMT.
Assuntos
Antígenos Virais/sangue , Técnicas de Laboratório Clínico/métodos , Anticorpos Anti-Hepatite C/sangue , Hepatite C/diagnóstico , Flebotomia , Sistemas Automatizados de Assistência Junto ao Leito , Adulto , Idoso , Sangue/imunologia , Sangue/virologia , Feminino , Humanos , Imunoensaio/métodos , Masculino , Pessoa de Meia-Idade , Saliva/imunologia , Saliva/virologia , Sensibilidade e Especificidade , Adulto JovemRESUMO
To monitor multiple Epstein-Barr virus (EBV) infections during the early and convalescent stages of infectious mononucleosis (IM), a cloning and sequencing study of the LMP1 gene was conducted in saliva and peripheral blood mononuclear cells (PBMCs) from 23 patients with IM at day 0 (D0) and day 180 (D180) after the onset of the disease. Multiple EBV strains were detected in 9 (39%) of the patients during follow-up, with 7 of 9 cases detected as early as D0. Six of the nine patients harbored the same dominant strain in saliva and PBMCs during follow-up, with a trend toward a restriction of the number of EBV strains in saliva but not in PBMCs at D180. Furthermore, transmission of a minor strain was observed between partners in a heterosexual couple. There was no correlation between multiple infections and EBV DNA load in either compartment.
Assuntos
Herpesvirus Humano 4/genética , Herpesvirus Humano 4/isolamento & purificação , Mononucleose Infecciosa/virologia , Leucócitos Mononucleares/virologia , Saliva/virologia , Clonagem Molecular , Feminino , Genótipo , Herpesvirus Humano 4/classificação , Humanos , Mononucleose Infecciosa/transmissão , Masculino , Análise de Sequência de DNA , Carga Viral , Proteínas da Matriz Viral/genéticaRESUMO
Epstein-Barr virus (EBV) DNA loads in peripheral blood mononuclear cells (PBMCs), plasma, and saliva, as well as infectivity of the virus in saliva, were evaluated in 20 patients for 6 months after the onset of infectious mononucleosis (IM). All patients displayed sustained high EBV DNA loads in the saliva, associated with a persistent infectivity of saliva at day 180. EBV DNA load in PBMCs decreased significantly from day 0 to day 180 (in spite of a viral rebound between day 30 and day 90 in 90% of the patients), and EBV DNA rapidly disappeared from plasma. These data show that patients with IM remain highly infectious during convalescence.