Provirus Mutations of Human T-Lymphotropic Virus 1 and 2 (HTLV-1 and HTLV-2) in HIV-1-Coinfected Individuals

Provirus mutations of human T-lymphotropic virus 1 (HTLV-1), mostly the lack of the 5= long terminal repeat (LTR) genomic region, have been described and associated with severe adult T cell leukemia/lymphoma (ATLL), non-sense point mutations with low proviral load, and Western blotting indeterminate results. Until now, no information concerning provirus mutations of HTLV-2 and its consequences, as well as those of HTLV-1/2 in HIV-coinfected individuals, had been described. Therefore, we searched for these mutations in provirus samples of 44 HIV/HTLV-1- and 25 HIV/HTLV-2-coinfected individuals. Using protocols well established for amplification and sequencing of segments of the LTR, env, and tax regions, we searched for defective type 1 particles that retain LTRs and lack internal sequences and type 2 particles that lack the 5=LTR region. In addition, using as references the prototypes ATK (HTLV-1) and Mo (HTLV-2), we searched for point mutations in the LTR and synonyms and nonsynonymous mutations and non-sense mutations in env and tax regions. Defective HTLV-1 and HTLV-2 provirus type 1 or 2 was detected in 31.8% of HIV/HTLV-1- and 32.0% of HIV/HTLV-2-coinfected individuals. Synonymous and nonsynonymous mutations were identified mostly in HTLV-2 and associated with lower levels of specific antibodies. No non-sense mutations that resulted in premature termination of Env and Tax proteins were detected. On the contrary, mutation in the stop codon of Tax2a produced a long protein characteristic of the HTLV-2c subtype. The clinical significance of these mutations in coinfected individuals remains to be defined, but they confirmed the lower sensitivity of serological and molecular diagnostic tests in HIV/HTLV-1/2 coinfections. IMPORTANCE HTLV-1 and HTLV-2 are endemic to Brazil, and they have different effects in HIV/AIDS disease progression. HIV/HTLV-1 has been described as accelerating the progression to AIDS and death, while HIV/HTLV-2 slows the progression to AIDS. Provirus mutations of HTLV-1 were implicated in severe leukemia development and in problems in the diagnosis of HTLV-1; in contrast, provirus mutations of HTLV-2 had not been confirmed and associated with problems in HTLV-2 diagnosis or disease outcome. Nevertheless, data obtained here allowed us to recognize and understand the false-negative results in serologic and molecular tests applied for HTLV-1 and HTLV-2 diagnosis. Defective proviruses, as well as synonymous and nonsynonymous mutations, were associated with the diagnosis deficiencies. Additionally, since HIV-1 and HTLV-1 infect the same cells (CD4 positive), the production of HIV-1 pseudotypes with HTLV-1 envelope glycoprotein during HIV/HTLV-1 coinfection cannot be excluded. Defective provirus of HTLV-2 and Tax2c is speculated to influence progression to AIDS. (AU)

Regulation of porcine endogenous retrovirus by dual LTR1+2 (Long Terminal Region) miRNA in primary porcine kidney cells

Porcine endogenous retroviruses (PERVs) integrate into germline DNA as proviral genome that enables vertical transmission from parents to their offspring. The provirus usually survives as part of the host genome rather than as an infectious agent, but may become pathogenic if it crosses species barriers. Therefore, replication-competent PERV should be controlled through selective breeding or knockout technologies. Two microRNAs (miRNAs), dual LTR1 and LTR2, were selected to inhibit the expression of PERV in primary porcine kidney cells. The inhibition efficiency of the miRNAs was compared based on their inhibition of different PERV regions, specifically long terminal repeats (LTRs), gag, pol, and env. Gene expression was quantified using real-time polymerase chain reaction and the C-type reverse transcriptase (RT) activity was determined. The messenger RNA (mRNA) expression of the PERV LTR and env regions was determined in HeLa cells co-cultured with primary porcine kidney cells. The mRNA expression of the LTR, gag, pol, and env regions of PERV was dramatically inhibited by dual miRNA from 24 to 144 h after transfection, with the highest inhibition observed for the LTR and pol regions at 120 h. Additionally, the RT activity of PERV in the co-culture experiment of porcine and human cells was reduced by 84.4% at the sixth passage. The dual LTR 1+2 miRNA efficiently silences PERV in primary porcine kidney cells.

Distinctive Drug-resistant Mutation Profiles and Interpretations of HIV-1 Proviral DNA Revealed by Deep Sequencing in Reverse Transcriptase / 生物医学与环境科学（英文）

<p><b>OBJECTIVE</b>To investigate distinctive features in drug-resistant mutations (DRMs) and interpretations for reverse transcriptase inhibitors (RTIs) between proviral DNA and paired viral RNA in HIV-1-infected patients.</p><p><b>METHODS</b>Forty-three HIV-1-infected individuals receiving first-line antiretroviral therapy were recruited to participate in a multicenter AIDS Cohort Study in Anhui and Henan Provinces in China in 2004. Drug resistance genotyping was performed by bulk sequencing and deep sequencing on the plasma and whole blood of 77 samples, respectively. Drug-resistance interpretation was compared between viral RNA and paired proviral DNA.</p><p><b>RESULTS</b>Compared with bulk sequencing, deep sequencing could detect more DRMs and samples with DRMs in both viral RNA and proviral DNA. The mutations M184I and M230I were more prevalent in proviral DNA than in viral RNA (Fisher's exact test, P<0.05). Considering 'majority resistant variants', 15 samples (19.48%) showed differences in drug resistance interpretation between viral RNA and proviral DNA, and 5 of these samples with different DRMs between proviral DNA and paired viral RNA showed a higher level of drug resistance to the first-line drugs. Considering 'minority resistant variants', 22 samples (28.57%) were associated with a higher level of drug resistance to the tested RTIs for proviral DNA when compared with paired viral RNA.</p><p><b>CONCLUSION</b>Compared with viral RNA, the distinctive information of DRMs and drug resistance interpretations for proviral DNA could be obtained by deep sequencing, which could provide more detailed and precise information for drug resistance monitoring and the rational design of optimal antiretroviral therapy regimens.</p>

Serological and virological evaluation of human T-lymphotropic virus type 1 infection in family groups from Tumaco, Colombia / Evaluación serológica y virológica de la infección por el virus linfotrópico de células T humanas de tipo 1 en grupos familiares de Tumaco, Colombia

Introduction: To date there has been no statistical evaluation of the profiles of immunoglobulin classes and viral replication as variables in the study of HTLV-1 infection and circulation among families in virus-endemic areas of Colombia. Objective: To evaluate the correlation of several immunological and molecular characteristics with the transmission and circulation of HTLV-1 among families in the town of Tumaco. Materials and methods: Plasma levels of HTLV-1 specific immunoglobulin classes IgG, IgM and IgA1, as well as IgG and sIgA in oral fluids, were calculated for 32 members of 10 family groups from Tumaco in which the mother and at least one child were infected with the virus. Levels of the different immunoglobulin classes were correlated with viral RNA circulating in plasma or oral fluids and the proviral burden as detected by RT-PCR. Results: Significant differences were determined between mothers and carrier children for immunoglobulin levels (p=0.037) and proviral burden (p=0.002). The overall estimate of IgG in plasma and sIgA in oral fluids could be correlated with the circulation of free viral RNA in both fluids and high proviral burden, and associated with HAM/TSP mothers. The detection of anti- tax IgG in plasma revealed differences between HAM/TSP mothers and their offspring. Conclusion: The study of immunological and molecular variables permitted the analysis of HTLV-1 circulation among families of Tumaco. The strong correlation between levels of IgM specific for the virus and viral RNA circulating in fluids indirectly confirmed the transmission of HTLV-1 among families.

Introducción. Todavía no hay una evaluación estadística de los perfiles de las clases de inmuno- globulina s y la replicación viral, como variables para estudiar la infección y la circulació n del HTLV-1 en familias de zonas endémicas en Colombia. Objetivo. Evaluar la correlación de varias características inmunológicas y moleculares, con la transmisión y circulación del virus en familias del municipio de Tumaco. Materiales y métodos. Se calcularon los niveles de IgG, IgM e IgA1 en plasma, e IgG y IgA secretoria en fluido oral, de 32 miembros de 10 grupos familiares de Tumaco, en los que la madre y, al menos, un hijo estaban infectados con el virus. La concentración de las diferentes clases de inmunoglobulinas se pudo correlacionar con la circulación de ARN viral libre en plasma y fluido oral, y la carga proviral, según su detección mediante reacción en cadena de la polimerasa de transcripción inversa. Resultados. Se encontraron diferencias significativas en los niveles de inmunoglobulinas (p=0,037) y en la carga proviral (p=0,002) entre madres e hijos portadores. La estimación total de IgG en plasma e IgA secretoria en fluido oral, se pudo correlacionar con la circulación de ARN viral libre en ambos fluidos y una alta carga proviral, y se asoció con las madres paraparesia espástica tropical o mielopatía asociada con el HTLV-1. La detección en plasma de IgG anti-Tax reveló diferencias entre ellas y sus hijos. Conclusión. El estudio de las variables inmunológicas y moleculares permitió analizar la circulación del HTLV-1 en familias de Tumaco. La fuerte asociación entre los niveles de IgM específica para el virus y el ARN viral circulante en los fluidos y la carga proviral, confirmó indirectamente la transmisión intrafamiliar del virus.

Cloning and sequencing analyses of the complete genome of the provirus of the Inner-Mongolia pandemic strain of the Jaagsiekte sheep retrovirus / 病毒学报

To investigate the kinship between the Inner Mongolia pandemic strain and representative strains of the Jaagsiekte sheep retrovirus (JSRV), total DNA from the lung tissue of a JSRV-infected sheep in Inner Mongolia was used to clone fragments of gag, pro and pol genes. The recombinant plasmid pMD-JSRV (including complete genomic sequence of the JSRV strain isolated from Inner Mongolia) was constructed by linking all the cloned fragments with long terminal repeat (LTR) and env gene fragments (cloned previous and reserved by our research team). Sequence analyses revealed that the genome was 7690 bp in length and contained several typical molecular markers for exogenous form of JSRV. These included the Sca I restriction site in the gag gene, two predicted "CCHC" motifs of zinc finger in the encoded nucleocapsid protein and the predicted "YXXM" motif in the TM region of Env. Homology analyses showed that the virus strain belonged to the JSRV type II. pMD-JSRV and AF105220 strains shared a nucleotide identification of 95%. The full length genomic clone of JSRV could provide a molecular basis for an infectious JSRV molecular clone as well as an experimental platform to study the detection and pathogenesis of JSRV.

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.

Simultaneous detection and subtyping of porcine endogenous retroviruses proviral DNA using the dual priming oligonucleotide system

The purpose of this study was to develop a multiplex PCR that can detect porcine endogenous retrovirus (PERV) proviral genes (pol, envA, envB, envC) and porcine mitochondrial DNA, using a dual priming oligonucleotide (DPO) system. The primer specifically detected the PERV proviral genes pol, envA, envB, envC, and porcine mitochondrial DNA only in samples of pig origin. The sensitivity of the primer was demonstrated by simultaneous amplification of all 5 target genes in as little as 10 pg of pig DNA containing PERV proviral genes and mitochondrial DNA. The multiplex PCR, when applied to field samples, simultaneously and successfully amplified PERV proviral genes from liver, blood and hair root samples. Thus, the multiplex PCR developed in the current study using DPO-based primers is a rapid, sensitive and specific assay for the detection and subtyping of PERV proviral genes.

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.

Construction and characterization of a new simian/human immunodeficiency viruses clone carrying an env gene derived from a CRF07_BC strain / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>The CRF07_BC recombinant strain has been one of the most predominantly circulated HIV-1 strains in China, it is therefore necessary and urgent to develop a relevant animal model to evaluate candidate vaccines targeting HIV-1 CRF07_BC. A highly replication-competent simian/human immunodeficiency viruses (SHIV) construct containing the Chinese CRF07_BC HIV-1 env gene with the ability to infect Chinese rhesus monkeys would serve as an important tool in the development of HIV vaccines. The aim of this study was to examine whether SHIV XJDC6431 with the env fragment from a Chinese HIV-1 isolate virus could infect the human and monkey peripheral blood mononuclear cell (PBMC), establish infection in Chinese rhesus macaque.</p><p><b>METHODS</b>A SHIV strain was constructed by replacing the rev/env genes of SHIV KB9 with the corresponding fragment derived from the HIV-1 CRF07_BC strain. The infectious activity of the SHIV clones was determined in vitro in PBMCs from both non-human primate animals and humans. Finally, one Chinese rhesus macaques (Macaca mulatta) was infected with one SHIV via intravenous infusion.</p><p><b>RESULTS</b>One SHIV clone designated as SHIV XJDC6431, was generated that could infect macaque and human PBMC. The virus produced from this clone also efficiently infected the CCR5-expressing GHOST cell lines, indicating that it uses CCR5 as its coreceptor. Finally, the virus was intravenously inoculated into one Chinese rhesus macaque. Eventually, the animal became infected as shown by the occurrence of viremia within 3 of infection. The viral load reached 105 copies of viral RNA per ml of plasma during the acute phase of infection and lasted for 10 weeks post infection.</p><p><b>CONCLUSIONS</b>We conclude that SHIV XJDC6431 is an R5-tropic chimeric virus, which can establish infection not only in vitro but also in vivo in the Chinese rhesus macaque. Although the animal inoculated with SHIV XJDC6431 became infected without developing a pathologic phenotype, the virus efficiently replicated with a persistent level of viral load in the plasma. This suggested that the SHIV could be used as a tool to test candidate AIDS vaccines targeting the Chinese HIV-1 CRF_07BC recombinant strain.</p>

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.

Sequence analysis for the complete provial genome of endogenous avian leukosis virus strain SD0501 / 病毒学报

The genomic DNA extracted from chicken embryo fibroblasts (CEF) of SPF chickens from three chicken farms was used as template to amplify the ALV proviral DNA by PCR with four pairs of primers, high positive detection rates of gag - gene (29/46), pol - gene (27/46), env - gene (24/46) and LTR fragment (31/46) were achieved. Eight continuous and overlapping fragments were amplified from one DNA sample with 8 pairs of primers according to published sequences, then cloned into the TA vector and se quenced. The complete sequence of the whole genome of ALV strain SD0501 was established and analyzed with DNAstar software. Comparisons of SD0501 sequence with that of other representative endogenous avian virus strains demonstrated that the genomes of ALV were relatively conservative, the nucleotide identity of all the strains was over 99.1%, and env - gene was over 98.5%. However, a low identity was demonstrated among the representative strains of different subgroups, especially, the env - gene showed obvious difference, the corresponding identity was as low as 56.3% - 91.5%.

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.

A Case of Adult T-cell Leukemia/Lymphoma / 대한피부과학회지

Adult T-cell leukemia/lymphoma (ATLL) is a human malignancy associated with human T-cell leukemia virus type I (HTLV-I), which frequently involves the skin. ATLL can be diagnosed based on clinicopathological findings and the presence of anti-HTLV-I serum antibodies and monoclonal integrated HTLV-I provirus in the DNA of tumor cells. It is characterized by leukemia, lymphadenopathy, hypercalcemia, and lytic bone lesions. We report a case of ATLL in a 59-year-old man who developed multiple, scattered papules on the face and trunk. He had a 3-month history of melena. The physical examination showed multiple cervical and axillary lymph node enlargements. On laboratory investigation, the white blood cell count was 113,900/mm(3) with 70% atypical lymphocytes. Histopathological and immunohistochemical analyses of a skin and stomach biopsy confirmed the diagnosis of T-cell lymphoma. Final diagnosis of ATLL was made based on HTLV-I positivity. The patient underwent multiple cycles of combination chemotherapy and combination therapy of zidovudine and interferon-alpha which produced some improvement, but he died of pulmonary complications 3 months after the initial diagnosis.

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.

Expression and Packaging of a Human Endogenous Retrovirus-K Genomic DNA Clone

Human contains large number of human endogenous retroviruses (HERVs) in its genome. One of the HERV families, HERV-K, entered human genome most recently and includes many members with full-length intact proviruses. Normally, these proviruses do not express but infrequently they seem to express in cancers or autoimmune disease patients. To investigate expression mechanisms of these endogenous retroviruses, a DNA copy of HERV-K was cloned and its expression was studied. The transfection of the full-length clone into human cell lines did not produce any detectable viral capsid protein, Gag, and the transcription from its own promoter in LTR was extremely poor. The transcription was less than 10 percent compare to the exogenous retrovirus. However, when the Gag coding region was cloned under CMV promoter, Gag could be expressed efficiently and secreted as particles, probably virus like particles. The efficient expression also required a nuclear export signal. The expressed Gag could also package its own genomic RNA. These results indicate that the LTR of HERV-K is normally not active but its genes have a potential to express and possibly produce infectious particles.

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.