A simple and rapid approach for human herpesvirus type 8 subtype characterization using single base extension.

Sequence analysis of the ORFK1 of human herpesvirus type 8 (HHV-8) allows the identification of six major subtypes (A-F), which are related to human migrations and the clinical progression of Kaposi's sarcoma. Sequencing and subsequent phylogenetic analysis of ORFK1 is considered to be the most reliable method for HHV-8 genotyping. However, it exhibits challenges and limitations. Herein, we designed and validated a single base extension (SBE) protocol for characterization of HHV-8 ORFK1 subtypes. A nested polymerase chain reaction (PCR) protocol was carried out to amplify a small 294-bp PCR product encompassing four single nucleotide polymorphisms at positions 360, 406, 465 and 527 of the HHV-8 genome. Finally, a multiplex SBE technique was developed and validated in 20 samples previously genotyped by phylogenetic analysis. The patterns obtained in this reaction could successfully discriminate between ORFK1 subtypes. The typing results obtained completely matched with those of the 'gold standard' method in all analysed samples. This method can reliably identify HHV-8 subtypes A, B and C, which are the most prevalent ones worldwide, and the remaining subtypes (D, E and F). SBE can be useful as an efficient, rapid and low-cost screening method for viral genotyping in a single tube, particularly samples with low-quality DNA, and with easy data interpretation.

Genetic diversity and phylogeographic analysis of human herpesvirus type 8 (HHV-8) in two distant regions of Argentina: Association with the genetic ancestry of the population.

BACKGROUND: The genetic diversity of persistent infectious agents, such as HHV-8, correlates closely with the migration of modern humans out of East Africa which makes them useful to trace human migrations. However, there is scarce data about the evolutionary history of HHV-8 particularly in multiethnic Latin American populations. OBJECTIVES: The aims of this study were to characterize the genetic diversity and the phylogeography of HHV-8 in two distant geographic regions of Argentina, and to establish potential associations with pathogenic conditions and the genetic ancestry of the population. STUDY DESIGN: A total of 101 HIV-1 infected subjects, 93 Kaposi's Sarcoma (KS) patients and 411 blood donors were recruited in the metropolitan (MET) and north-western regions of Argentina (NWA). HHV-8 DNA was detected by ORF-26 PCR in whole blood, saliva and FFPE tissues. Then, ORF-26 and ORF-K1 were analyzed for subtype assignment. Mitochondrial DNA and Y chromosome haplogroups, as well as autosomal ancestry markers were evaluated in samples in which subtypes could be assigned. Phylogeographic analysis was performed in the ORF-K1 sequences from this study combined with 388 GenBank sequences. RESULTS: HHV-8 was detected in 50.7%, 59.2% and 8% of samples from HIV-1 infected subjects, KS patients and blood donors, respectively. ORF-K1 phylogenetic analyses showed that subtypes A (A1-A5), B1, C (C1-C3) and F were present in 46.9%, 6.25%, 43.75% and 3.1% of cases, respectively. Analyses of ORF-26 fragment revealed that 81.95% of strains were subtypes A/C followed by J, B2, R, and K. The prevalence of subtype J was more commonly observed among KS patients when compared to the other groups. Among KS patients, subtype A/C was more commonly detected in MET whereas subtype J was the most frequent in NWA. Subtypes A/C was significantly associated with Native American maternal haplogroups (p = 0.004), whereas subtype J was related to non-Native American haplogroups (p < 0.0001). Sub-Saharan Africa, Europe and Latin America were the most probable locations from where HHV-8 was introduced to Argentina. CONCLUSIONS: These results give evidence of the geographic circulation of HHV-8 in Argentina, suggest the association of ORF-26 subtype J with KS development and provide new insights about its relationship with ancient and modern human migrations and identify the possible origins of this virus in Argentina.

Identificação do gene EBER1 e EBNA1 do vírus de Epstein Barr (EBV) em tecidos de pacientes com doença de Hodgkin na região Norte do Brasil / Identification of gene EBER1 and EBNA 1 of Epstein Barr virus (EBV) in tissues of patients with Hodgkin’s disease in Northern Brazil

O vírus de Epstein Barr (EBV) é o agente causador da mononucle¬ose infecciosa e está associado a várias desordens proliferativas malignas tais como: linfoma de Burkitt, linfoma de Hodgkin e lin¬fomas não Hodgkin. Objetivo: detectar o genoma do EBV mediante a identificação dos genes EBER1 e EBNA1 em casos de doença de Hodgkin. Métodos: um total de 65 casos de linfomas diagnosti¬cados no Hospital Ophir Loyola no período de 1996 e 2005 foram analisados no Instituto Evandro Chagas, Ananindeua, Brasil. Todos os espécimes parafinizados foram analisados por hibridização in situ (gene EBER1) e PCR em tempo real (EBNA1). Resultados: do total, 64,6% (42/65) dos pacientes eram do sexo masculino e 35,4% (23/65) do sexo feminino. O EBV foi identificado por HIS nas células Reed Sternberg e variantes em 76,9% (50/65) dos casos com idade média de 28,3 anos (variação 2-84 anos). Os subtipos histológicos de casos EBV-positivos foram os seguintes: esclerose nodular em 50% (25/50), celularidade mista em 28% (14/50), depleção linfocitária em 14% (7/50) e predominância linfocitária em 8% (4/50). O DNA do EBV foi detectado em 53% (26/49) dos casos de doença de Hodgkin com um coeficiente de regressão para a curva padrão de 0,99. Conclusão: este estudo foi a primeira descrição do vírus de Epstein Barr em casos de linfoma de Hodgkin na Amazônia Brasileira, reforçando a hipótese de que o EBV seja um co-fator no processo de transformação neoplásica em conjunto com a predisposição genética e imunidade do paciente

Introduction: EBV is the causative agent of infectious mononucleosis and is associated with several malignant proliferative disorders such as Burkitt’s lymphoma, Hodgkin’s lymphoma, some B and T cell non-Hodgkin’s lymphomas. Objective: The main objective of the study was to determine the prevalence of EBER 1 gene and EBNA1 gene in cases of Hodgkin’s disease. Material and Methods: A total of 65 cases of lymphomas diagnosed between 1996 and 2005 were obtained from “Instituto Ofir Loyola” and analyzed at the “Instituto Evandro Chagas” Ananindeua, Brazil. The EBV antigens using EBER 1 probe in situ hybridization (HIS) and real time quantitative PCR. Results: From the total obtained, 64.6% (42/65) were male and 35.4% (23/65) female. EBV was identified in the Reed- Sternberg cells and variants in 76.9% (50/65) of Hodgkin’s disease cases, the median age were 28.3 years (range 2-84). The histologic subtypes of EBV-positive cases were as follows: nodular sclerosis in 50% (25/50), mixed cellularity in 28% (14/50), lymphocyte depletion in 14% (7/50) and lymphocyte predominance in 8% (4/50). We detected EBV DNA in 53% (26/49) with a coefficient of regression for the standard curve of a minimum of 0.99. Conclusion: These results were the first demonstration of the role of Epstein Barr virus in cases of Hodgkin diseases in northern Brazil and are consistent with the hypothesis that the presence of EBV during neoplasic transformation could be an additional cofactor acting together with both genetic predisposition and immunity of the patient