Evolution of HTLV-1 proviral load in patients from Salvador, Brazil

INTRODUCTION: Variations in human T cell lymphotropic virus type 1 (HTLV-1) proviral load (PVL) in infected individuals over time are not well understood. Objective: To evaluate the evolution of proviral load in asymptomatic individuals and HAM/TSP patients in order to help determine periodicity for measuring proviral load. METHODS: A group of 104 HTLV-1 infected patients, followed at the HTLV reference center in Salvador, Brazil, were included in the study (70 asymptomatic and 34 HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) patients). HTLV-1 PVL was measured using real-time polymerase chain reaction (PCR) at baseline and again at another point, either < 12 months, between 12-24 months, or > 24 months. RESULTS: HAM/TSP patients had higher PVL (ranging from 11,041 to 317,009 copies/10(6) PBMC) when compared to asymptomatic individuals (ranging from 0 to 68,228 copies/10(6) PBMC). No statistically significant differences were observed in the medians of PVL in HAM/TSP patients or asymptomatic individuals over time. However, in asymptomatic individuals with a PVL below 50,000 copies/10(6) PBMC, a statistically significant two-fold increase was observed over time. CONCLUSION: HTLV-1-PVL remained stable in both asymptomatic individuals and HAM/TSP patients over time. Frequent monitoring of asymptomatic individuals with low PVLs is recommended and further studies should be conducted to assess the course of PVL in these patients over extended periods of time.

Epidemiología genómica y paraparesia espástica tropical asociada a la infección por el virus linfotrópico humano de células T tipo 1 / Genome epidemiology and tropical spastic paraparesis associated with human T-cell lymphotropic virus type 1

OBJETIVO: Caracterizar el ambiente genómico de las secuencias adyacentes al virus linfotrópico humano de células T tipo 1 (HTLV-1) en pacientes con paraparesia espßstica tropical y mielopatía asociada a la infección con HTLV-1 (PET/MAH) de diferentes regiones de Colombia y del Japón. MÉTODOS: Se enfrentaron 71 clones recombinantes con secuencias del genoma humano adyacentes al 5'-LTR de pacientes con PET/MAH, a las bases de datos del Genome Browser y del Gen-Bank. Se identificaron y analizaron estadísticamente 16 variables genómicas estructurales y composicionales mediante el programa informßtico R, versión 2.8.1, en una ventana de 0,5 Mb. RESULTADOS: El 43,0 por ciento de los provirus se localizaron en los cromosomas del grupo C; 74 por ciento de las secuencias se ubicaron en regiones teloméricas y subteloméricas (P < 0,05). Un anßlisis de conglomerados permitió establecer las relaciones jerßrquicas entre las características genómicas incluidas en el estudio; el anßlisis de componentes principales identificó las componentes que definieron los ambientes genómicos preferidos para la integración proviral en casos de PET/MAH. CONCLUSIONES: El HTLV-1 se integró con mayor frecuencia en regiones de la cromatina ricas en islas de citocina fosfato guanina (CpG), de alta densidad de genes y de repeticiones tipo LINE (elemento disperso largo [long interspersed element]) y transposones de ADN que, en conjunto, conformarían los ambientes genómicos blanco de integración. Este nuevo escenario promoverß cambios sustanciales en el campo de la salud pública y en el manejo epidemiológico de las enfermedades infecciosas, y permitirß desarrollar potentes herramientas para incrementar la eficiencia de la vigilancia epidemiológica.

OBJECTIVE: Characterize the genomic environment of the sequences adjacent to human T-cell lymphotropic virus type 1 (HTLV-1) in patients with HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) in different regions of Colombia and Japan. METHODS: A total of 71 recombinant clones with human genome sequences adjacent to 5' LTR in patients with HAM/TSP were compared to the Genome Browser and GenBank databases. Sixteen structural and compositional genome variables were identified, and statistical analysis was conducted in the R computer program, version 2.8.1, in a 0.5 Mb window. RESULTS: A total of 43.0 percent of the proviruses were located in the group C chromosomes; 74 percent of the sequences were located in the telomeric and subtelomeric regions (P < 0.05). A cluster analysis was used to establish the hierarchical relations between the genome characteristics included in the study. The analysis of principal components identified the components that defined the preferred genome environments for proviral integration in cases of HAM/TSP. CONCLUSIONS: HTLV-1 was integrated more often in chromatin regions rich in CpG islands with a high density of genes and LINE type repetitions, and DNA transposons which, overall, would form the genomic environments targeted for integration. This new scenario will promote substantial changes in the field of public health and in epidemiological management of infectious diseases. It will also foster the development of powerful tools for increasing the efficiency of epidemiological surveillance.

High HTLV-1 proviral load, a marker for HTLV-1 associated myelopathy/tropical spastic paraparesis, is also detected in patients with infective dermatitis associated with HTLV-1

Salvador (BA, Brazil) is an endemic area for human T-cell lymphotrophic virus type 1 (HTLV-1). The overall prevalence of HTLV-1 infection in the general population has been estimated to be 1.76%. HTLV-1 carriers may develop a variety of diseases such as adult T-cell leukemia/lymphoma, HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) and infective dermatitis associated with HTLV-1 (IDH). IDH is a chronic and severe form of childhood exudative and infective dermatitis involving mainly the scalp, neck and ears. It has recently been observed that 30% of patients with IDH develop juvenile HAM/TSP. The replication of HTLV-1 has been reported to be greater in adult HAM/TSP patients than in asymptomatic HTLV-1 carriers. In the current study, the proviral load of 28 children and adolescents with IDH not associated with HAM/TSP was determined and the results were compared to those obtained in 28 HTLV-1 adult carriers and 28 adult patients with HAM/TSP. The proviral load in IDH patients was similar to that of patients with HAM/TSP and much higher than that found in HTLV-1 carriers. The high levels of proviral load in IDH patients were not associated with age, duration of illness, duration of breast-feeding, or activity status of the skin disease. Since proviral load is associated with neurological disability, these data support the view that IDH patients are at high risk of developing HAM/TSP.

Factors Associated with HIV-1 Proviral DNA Loads in Patients with Undetectable Plasma RNA Load

To evaluate factors associated with human immunodeficiency virus type 1 (HIV-1) proviral DNA load, we conducted a cross-sectional study of 36 chronically HIV-1- infected individuals with undetectable plasma viral RNA. We used real-time polymerase chain reaction to determine the number of HIV-1 proviral DNA copies per 10(6) peripheral blood mononuclear cells. The mean level of plasma viral RNA when the CD4+ T cell count was above 500 cells/microliter without highly active antiretroviral therapy (HAART) was significantly associated with proviral DNA load at the time of undetectable plasma HIV RNA with HAART. Strategies to reduce the level of plasma viral RNA when patients' CD4+ T cell counts are above 500 cells/microliter without HAART could help reduce HIV-1 proviral DNA load.

Existence of proviral porcine endogenous retrovirus in fresh and decellularised porcine tissues.

PURPOSE: Swine are expected to be utilized as xenograft donors for both whole organ and cellular transplantation. A major concern in using porcine organs for transplantation is the potential of transmission of porcine endogenous retrovirus (PERV). Tissue-engineered or decellularised heart valves have already been implanted in humans and have been marketed by certain companies after Food and Drug Administration (FDA) approval. The aim of this study was to examine the existence of porcine endogenous retrovirus (PERV) in fresh and decellularised porcine tissues. METHODS: Porcine tissues (both fresh and decellularised) were analysed using validated assays specific for PERV: polymerase chain reaction (PCR), reverse transcriptase polymerase chain reaction (RT-PCR). RESULTS: PERV specific GAG sequences were found in the porcine heart tissue samples using PCR for DNA and RT- PCR for RNA. All tissue samples (both fresh and treated tissues) like aortic valve, pulmonary valve and heart muscle showed the presence of PERV DNA. RT PCR for PERV was positive in all fresh tissues and was found to be negative in decellularised treated tissues. CONCLUSIONS: PCR is a rapid, specific test for the detection of PERV virus in xenografts. These findings have demonstrated that the presence of proviral DNA form of PERV in porcine tissues needs to be carefully considered when the infectious disease potential of xenotransplantation is being assessed.

Techniques used to identify the Brazilian variant of HIV-1 subtype B

The purpose of the present study was to compare the sensitivity and specificity of V3 enzyme immunoassay (solid phase EIA and EIA inhibition) and restriction fragment length polymorphism (RFLP) with the DNA sequencing "gold standard" to identify the Brazilian HIV-1 variants of subtype B and B"-GWGR. Peripheral blood mononuclear cells were collected from 61 HIV-1-infected individuals attending a clinic in São Paulo. Proviral DNA was amplified and sequentially cleaved with the Fok I restriction enzyme. Plasma samples were submitted to a V3-loop biotinylated synthetic peptide EIA. Direct partial DNA sequencing of the env gene was performed on all samples. Based on EIA results, the sensitivity for detecting B-GPGR was 70 percent, compared to 64 percent for the Brazilian variant B"-GWGR while, the specificity of B-GPGR detection was 85 percent, compared to 88 percent for GWGR. The assessment of RFLP revealed 68 percent sensitivity and 94 percent specificity for the B-GPGR strain compared to 84 and 90 percent for the B"-GWGR variant. Moreover, direct DNA sequencing was able to detect different base sequences corresponding to amino acid sequences at the tip of the V3 loop in 22 patients. These results show a similar performance of V3 serology and RLFP in identifying the Brazilian variant GWGR. However, V3 peptide serology may give indeterminate results. Therefore, we suggest that V3 serology be used instead of DNA sequencing where resources are limited. Samples giving indeterminate results by V3 peptide serology should be analyzed by direct DNA sequencing to distinguish between B-GPGR and the Brazilian variant B"-GWGR.

Carga proviral do HTLV-1 e HTLV-2: um método simples através da PCR quantitativa em tempo real / HTLV-1 and HTLV-2 proviral load: a simple method using quantitative real-time PCR

Os vírus linfotrópicos de células T humanas, quando integrados ao genoma da célula hospedeira, provírus, têm como marcador de replicação seu DNA proviral. A carga proviral parece ser um importante fator no desenvolvimento de patologias associadas a estes retrovírus. Neste estudo foi desenvolvida uma metodologia para quantificação absoluta da carga proviral dos HTLV-1 e HTLV-2 através da PCR em tempo real. Cinqüenta e três amostras de doadores de sangue com teste de ELISA reagente foram submetidas à metodologia, que utilizou o sistema TaqMan® para três seqüências alvo: HTLV-1, HTLV-2 e albumina. A quantificação proviral absoluta foi determinada através da proporção relativa entre o genoma do HTLV e o genoma da célula hospedeira, levando em consideração o número de leucócitos. O método apresentado é sensível (215 cópias/mL), prático e simples para quantificação proviral, além de eficiente e adequado para confirmação e discriminação da infecção pelos tipos virais.

When the human T cell lymphotropic virus (HTLV) is integrated with the host cell genome (provirus), its proviral DNA is a replication marker. Proviral load appears to be an important factor in the development of diseases related to these retroviruses. In this study, a methodology for absolute quantification of the HTLV-1 and HTLV-2 proviral load using real-time PCR was developed. Fifty-three blood donor samples with positive ELISA test result were subjected to this methodology, which utilized the TaqMan® system for three target sequences: HTLV-1, HTLV-2 and albumin. The absolute proviral load was quantified using the relative ratio between the HTLV genome and the host cell genome, taking into consideration the white blood cell count. The method presented is sensitive (215 copies/ml), practical and simple for proviral quantification, and is efficient and appropriate for confirming and discriminating infections according to viral type.

Human T-cell lymphotropic virus type I (HTLV-I) proviral DNA viral load among asymptomatic patients and patients with HTLV-I-associated myelopathy/tropical spastic paraparesis

To evaluate the human T-cell lymphotropic virus type I (HTLV-I) proviral DNA load among asymptomatic HTLV-I-infected carriers and patients with HTLV-I-associated myelopathy/tropical spastic paraparesis (HAM/TSP), real time PCR using TaqMan probes for the pol gene was performed in two million peripheral blood mononuclear cells (PBMC). The albumin gene was the internal genomic control and MT2 cells were used as positive control. The results are reported as copies/10,000 PBMC, and the detection limit was 10 copies. A total of 89 subjects (44 HAM/TSP and 45 healthy HTLV-I-infected carriers) followed up at the Institute of Infectious Diseases "Emilio Ribas" and in the Neurology Division of Hospital of Clínicas were studied. The asymptomatic HTLV-I-infected carriers had a median number of 271 copies (ranging from 5 to 4756 copies), whereas the HAM/TSP cases presented a median of 679 copies (5-5360 copies) in 10,000 PBMC. Thus, HAM/TSP patients presented a significantly higher HTLV-I proviral DNA load than healthy HTLV-I carriers (P = 0.005, one-way Mann-Whitney test). As observed in other persistent infections, proviral DNA load quantification may be an important tool for monotoring HTLV-I-infected subjects. However, long-term follow-up is necessary to validate this assay in the clinical setting.

Investigation of the Bovine Leukemia Virus Proviral DNA in Human Leukemias and Lung cancers in Korea

The bovine leukemia virus (BLV) is the causative agent of enzootic bovine leucosis. This study investigated the presence of the BLV in leukemia (179 acute lymphoblastic leukemia, 292 acute myeloid leukemia and 46 chronic myelogenous leukemia cases) and 162 lung cancer patients (139 adenocarcinoma, 23 squamous cell carcinoma) to determine if the BLV is a causative organism of leukemia and lung cancer in Koreans. A BLV infection was confirmed in human cells by PCR using a BLV-8 primer combination. All 517 cases of human leukemia and 162 lung cancer were negative for a PCR of the BLV proviral DNA. In conclusion, although meat has been imported from BLV endemic areas, the BLV infection does not appear to be the cause of human leukemia or lung cancer in Koreans. These results can be used as a control for further studies on the BLV in Koreans.

A novel molecular method for HIV-1 proviral DNA detection: non-radioactively--reversed probe hybridization and nested PCR.

A novel molecular method for HIV-1 proviral DNA detection comprising two main techniques: nested PCR, amplifying a target sequence of the ENV-gene of HIV-1, and nonradioactively-reversed probe hybridization for the detection of the amplified target sequence. The dual amplification of inserted HIV-1 proviral DNA in each DNA sample to be tested was performed by nested PCR in two steps: firstly with two outer primers covering the target sequence of the ENV-gene of HIV-1; secondly with two 5'-biotinylated primers specific to the target sequence. The biotinylated PCR product could be visualized as a single band of 141bps in length on agarose gel stained with ethidium bromide. For the confirmation of the primary result, a method of reversed probe hybridization, using a nylon membrane immobilized with the oligonucleotide probe specific to the target sequence, was established. The oligonucleotide probe was given a homopolymer tail with terminal deoxyribonucleotidyl-transferase; the tail was spotted onto a nylon membrane and bound covalently by UV irradiation. Owing to its length, the tail bound to the nylon, leaving the oligonucleotide probe free to hybridize. Hybridization of the amplified target sequence to the immobilized probe was accomplished by a simple colorimetric reaction involving the enzymatic oxidation of a colorless chromogen that yielded a purple color wherever hybridization occurred.

Biología molecular del virus de la inmunodeficiencia humana y los recientes progresos en el tratamiento del SIDA / Molecular biology of HIV and recent progress in the treatment of AIDS

El virus de inmunodeficiencia humana (VIH), causante del síndrome de inmunodeficiencia adquirida (SIDA), ha constituido desde su emergencia hace 2 décadas, un enorme desafío a la investigación biomédica. Utilizando una amplia gama de recursos para interferir y evadir la respuesta inmune normal, infecta las células CD4+, ingresa a ellas a través de sus receptores de superficie, y expresa una alta frecuencia de mutación lo que le permite cambiar repetidamente sus determinantes antígenos. Los VIH tipo 1 y 2 corresponden a lentivirus, los cuales, junto a los oncornavirus y espumavirus integran la familia de retrovirus RNA humanos. En este artíulo se revisan los conocimientos actuales de la biología molecular del virus, se comentan modernas técnicas de diagnóstico como la reacción de transcriptasa reversa y polimerasa en cadena, usadas actualmente para medir la replicación del virus en la sangre del huésped infectado, y se discuten las actuales líneas de tratamiento exploradas, las que básicamente se dirigen a blancos moleculares susceptibles de intervención farmacológica que no afecten la funcionalidad celular, como son la interacción virus-receptor celular, , transcripción reversa del RNA viral, integración del provirus, procesamiento proteolítico del precursor gag-pol, y la regulación transcripcional virus específica por los productos tat y rev. Finalmente se comentan aspectos relacionados a la investigación en el campo de la inmunización VIH, desafío pendiente para la primera década de este nuevo siglo

Síndrome de inmunodeficiencia adquirida (SIDA): definición; historia; epidemiología; etiología; dimensiones sociales; prevención / Acquired immunodeficiency syndrome (AIDS): definition; history; epidemiology; ethiology; social dimension; prevention

Este trabajo de revisión, que fue expuesto durante el Simposio SIDA`90, incluye los conceptos fundamentales sobre la historia de la epidemia del síndrome de inmunodefiencia adquirida (SIDA), los últimos datos epidemiológicos, tanto a nivel mundial como de la Argentina, los grupos poblacionales de riesgo y las formas de transmisión. Se describe también al agente etiológico del síndrome, el virus de la inmunodeficiencia humana (HIV), su estructura, su arquitectura genética y su forma de replicación. Por último, se analizan los alcances sociales de la enfermedad, los principales métodos de prevención y las medidas sanitarias, legales y educativas necesarias, que permitan una eficaz atención del paciente y eficientes acciones preventivas