Anopheles gambiae s.s. is a highly anthropophilic mosquito and is the major vector for human malaria in sub-Sahara Africa, while Anopheles quadriannulatus, largely due to its zoophilic behaviour, is considered a non-vector species in the same region. Careful studies of olfaction in these sibling species may lead to insights about the mechanisms that drive host preference behaviour. In the present study, the external anatomy of the antenna, the principle olfactory organ in the female mosquito of both species, was examined as an initial step toward more detailed comparisons.Volatile odors are important sensory inputs that shape the behaviour of insects, including agricultural pests and disease vectors. An. gambiae s.s. and An. quadriannulatus. Sensory structures, called sensilla, were categorized and counted; their distributions are reported here as well as densities calculated for each species.Scanning electron and light microscopy were used to examine the antennae ultrastructures of adult female An. gambiae s.s. and An. quadriannulatus bear five classes of sensilla on their antennae: chaetica (bristles), trichodea (hairs), basiconica (pegs), coeloconica (pitted pegs), and ampullacea (pegs in tubes). Female An. quadriannulatus antennae have approximately one-third more sensilla, and a proportionally larger surface area, than female An. gambiae s.s. antennae.Both An. quadriannulatus has greater numbers of each sensilla type, sensilla densities are very similar for each species, suggesting that other factors may be more important to such olfactory-driven behaviours as host preference.The same types of sensilla are found on the antennae of both species. While  An. gambiae s.s. and An. quadriannulatus, display very different patterns of blood meal host preference. An. gambiae s.s. exhibits a high degree of anthropophily, while An. quadriannulatus exhibits strong zoophily [An. gambiae s.s. females is a major contributing factor to human malaria transmission, a disease that afflicts more than 200 million people and causes as many as 3 million deaths annually [An. quadriannulatus, because of its preference for cattle, is not considered a malaria vector [Odors are the principle sensory signals that direct female mosquitoes to their preferred blood meal hosts ,2. Antena vector .An. gambiae s.s. Accordingly, comparisons of the peripheral and central olfactory events between behaviourally divergent sibling species, such as An. gambiae s.s. and An. quadriannulatus, should provide valuable insights into the mechanisms that determine their respective host preferences. Understanding the basic molecular events that underlie blood feeding may ultimately lead to the design of new ways to interfere with the human/mosquito interaction and thereby reduce the associated disease burden.In order to understand the specifics of the attractiveness of humans to the mosquito, a major focus of this laboratory is the study of components in the olfactory signal transduction and coding process of An. gambiae s.s. and An. quadriannulatus, which may suggest areas of future investigation. As such, a comparative examination of the olfactory apparatus of An. gambiae s.s. and An. quadriannulatus as a prelude to future comparative molecular studies has been undertaken.Antennal sensilla have previously been described for several mosquito species , inclAn. gambiae s.s. (G3) and An. quadriannulatus (SANGQUA) were reared as described previously [An. gambiae s.s. was received from Dr. Mark Benedict at the Centers for Disease Control and Prevention and is described in more detail at: . An. quadriannulatus (SANGQUA) was the kind gift of Dr. Willem Takken .eviously . The G3 An. gambiae s.s. or An. quadriannulatus were fixed with 4% paraformaldehyde, 0.1% Triton X-100 in phosphate buffered saline (PBS). Heads were then dehydrated; first in an ethanol series from 50% to 100% in 10% increments, followed by ethanol:hexamethyldisilazane (HMDS) at 75:25, 50:50, 25:75 and 0:100. HMDS was decanted and heads were dried in a fume hood. Heads were then glued onto aluminum pin mounts with colloidal silver paint and sputter coated for 30 seconds with gold-palladium. Samples were viewed using a Hitachi S-4200 scanning electron microscope and digital micrographs of each flagellomere were collected using Quartz PCI version 6.0 image acquisition software .Heads from 4- to 6-day-old adult An. gambiae s.s. (G3 strain) or An. quadriannulatus were hand dissected from cold-anesthetized animals and placed in 25% sucrose, 0.1% Triton X-100 in water. These antennae were mounted on glass slides with a cover slip and sealed with clear enamel nail polish. Slides were stored at 4°C and observed under an Olympus BX-60  microscope at 400× magnification. Photomicrographs were captured using an Olympus DP70 digital camera.Antennae from 4- to 6-day-old adult n [t-tests (two-tailed) were performed as described [Antennae were initially observed by SEM on both the dorsal and ventral aspects to look for any bias in sensilla arrangements. Thereafter, heads were mounted such that the antennae were most often observed from the lateral aspect, with some variation between individuals. Sensilla on each micrograph were classified by type and counted. Mean values for each sensillum type were calculated for 10 individuals per species and then multiplied by a factor of 2, assuming that only half the sensilla could be seen in each micrograph. For brightfield counting, all sensilla of a given type could be observed on each flagellomere by continuous focus adjustment through the specimen. Mean values for each type were calculated for 20 individuals. Standard deviations were calculated for each data set as described previously [t-tests (two-tailed) were performed to determine whether the mean surface areas differed significantly between species. Sensilla densities were calculated by dividing the mean number of sensilla by the total mean surface area where the particular type of sensilla was found.Flagellomeres 1–13 were viewed using brightfield optics, as described above, at 400× magnification. The length of each flagellomere was measured with an Olympus DP70 digital scale bar, and the width (diameter) was measured at the midpoint of the length Figure . The meaAn. gambiae s.s. and An. quadriannulatus adults are equipped with three types of appendages – the antennae, maxillary palps, and proboscis (labellum) – each with associated chemosensory functions . Externally, male and female proboscises appear identical. However, males often lack or have highly modified forms of stylets enclosed within the proboscis [The proboscis, or modified labium in mosquitoes, is also longer than the antennae and is the most ventral of the head appendages Figure . At the roboscis .An. gambiae s.s. and An. quadriannulatus, this study focuses on a structural comparison of their antennae. The following are detailed descriptions of the types, numbers, distributions, and densities of female antennal sensilla.Because of the major importance of the antennae on host seeking and other behaviours in female An. gambiae s.s. and An. quadriannulatus. The large sensilla chaetica are arranged in a whorl on the basal end of each flagellomere 2–13 and distributed evenly around the circumference . Similarly, An. quadriannulatus had 6–8 on the first seven flagellomeres, 4–6 on the next five flagellomeres, and 1 or 2 on the 13th flagellomere. The number of small sensilla chaetica averaged 50.5 in An. gambiae s.s. and 57.5 in An. quadriannulatus.Small sensilla chaetica were generally found on the dorsal surface and nearer the distal edge of flagellomeres 2–13 . The sharp sensilla trichodea seemed to be randomly distributed around the circumference of each flagellomere 2–13. An. gambiae s.s. females had, on average, about 615 trichoid sensilla per antenna, and An. quadriannulatus females had about 770 per antenna in this study.The most numerous sensilla found along the flagellum of ed Table . Two dised Table , 4A, C. tephensi . While gtephensi . The numAn. gambiae s.s. and An. quadriannulatus, blunt sensilla trichodea were the least numerous class of sensilla quantified in this study  small sensilla coeloconica , or environmental factors are more likely to contribute to the host preference divergence of An. gambiae s.s. and An. quadriannulatus. A recent comparison of candidate olfactory receptor genes in An. gambiae s.s. and An. quadriannulatus showed an extremely high level of conservation between their amino acid sequences . This study provides an essential foundation for these and other future comparative analyses that will focus on the molecular genetics and physiology of olfaction in these two species.There are at least six species in the recently . Furtherentified . While tl organs . ClearlyAn. gambiae s.s. and An. quadriannulatus mosquitoes carry the same morphological types of sensilla and the densities of each type are effectively equal between the two species. Therefore, the lack of specialization at the gross morphological level of the antennae implies that other factors are more likely to account for the olfactory-driven host preference difference between An. gambiae s.s. and An. quadriannulatus. This study establishes a foundation for future neurological, physiological, and molecular comparative studies aimed at elucidating potential differences in olfaction between these sibling species.The antennae of adult female RJP and LJZ conceived and designed the study. RJP carried out all microscopy and analysis (with some assistance from undergraduate researchers as listed in the acknowledgements) and drafted the manuscript. Both authors read and approved the final manuscript.


HTLV-I causes the disabling inflammatory disease HAM/TSP: there is no vaccine, no satisfactory treatment and no means of assessing the risk of disease or prognosis in infected people. Like many immunopathological diseases with a viral etiology the outcome of infection is thought to depend on the virus-host immunology interaction. However the dynamic virus-host interaction is complex and current models of HAM/TSP pathogenesis are conflicting. The CD8+ cell response is thought to be a determinant of both HTLV-I proviral load and disease status but its effects can obscure other factors.We show here that in the absence of CD8+ cells, CD4+ lymphocytes from HAM/TSP patients expressed HTLV-I protein significantly more readily than lymphocytes from asymptomatic carriers of similar proviral load (P = 0.017). A high rate of viral protein expression was significantly associated with a large increase in the prevalence of HAM/TSP . Additionally, a high rate of Tax expression and a low CD8+ cell efficiency were independently significantly associated with a high proviral load .per se.These results disentangle the complex relationship between immune surveillance, proviral load, inflammatory disease and viral protein expression and indicate that increased protein expression may play an important role in HAM/TSP pathogenesis. This has important implications for therapy since it suggests that interventions should aim to reduce Tax expression rather than proviral load  Human T-Lymphotropic Virus Type I (HTLV-I) is a persistent retrovirus. The majority of infected individuals remain lifelong, asymptomatic carriers of the virus (ACs). However, 2–3% of infected individuals develop an aggressive malignancy named Adult T cell Leukemia. A further 2–3% develop inflammatory disease of one or more organs. The best characterised inflammatory disease is HTLV-I-associated myelopathy/ tropical spastic paraparesis (HAM/TSP), a chronic inflammatory condition of the central nervous system. It is not understood why most HTLV-I infected individuals remain asymptomatic yet some develop inflammatory disease.The two factors most often associated with HAM/TSP are high proviral load  and highex vivo  = -A(CTL lysic rate) + b(if rate of Tax expression = high).Ln [Using this model the fraction of the observed variation in proviral load that could be explained by the variation in CTL lysis rate and rate of Tax expression was calculated. The significance of predictors quoted is the significance of that variable given the other variable in the regression equation.To estimate the probability of an infected (provirus-positive) cell expressing Tax in 18 h we expressed the fraction of CD4+ cells that were Tax+ after 18 h culture as the fraction of infected cells that were Tax+ after 18 h using the formulaIn this calculation we made the simplifying assumption that all proviral load was carried in CD4+ cells.A theoretical model Fig.  predicteAC: asymptomatic carrier, CTL: cytotoxic T lymphocyte, HAM/TSP: HTLV-I associated myelopathy/ tropical spastic paraparesis, HTLV-I: Human T Lymphotropic Virus-I.The author(s) declare that they have no competing interests.BA conceived of and designed the study, performed the analysis and wrote the manuscript. AJM performed the Tax staining. AH & ARM contributed to the data interpretation. YT provided reagents. GPT recruited and monitored the subjects. CRMB helped design the study and draft the manuscript and contributed to data interpretation.in vivo.Description of the general model to describe the relationship between Tax+ and Tax- infected cells Click here for fileFigure illustrating the classification of the subject group into individuals whose provirus-positive cells had a high or low rate of Tax expression.Click here for file


Levels of proviral load in HTLV-1 infected patients correlate with clinical outcome and are reasonably prognostic. Adaptation of proviral load measurement techniques is examined here for use in an experimental rabbit model of HTLV-1 infection. Initial efforts sought to correlate proviral load with route and dose of inoculation and with clinical outcome in this model. These methods contribute to our continuing goal of using the model to test treatments that alleviate virus infection.A real-time PCR assay was used to measure proviral load in blood and tissue samples from a series of rabbits infected using HTLV-1 inocula prepared as either cell-free virus particles, infected cells or blood, or by naked DNA injection. Proviral loads from asymptomatically infected rabbits showed levels corresponding to those reported for human patients with clinically silent HTLV-1 infections. Proviral load was comparably increased in 50% of experimentally infected rabbits that developed either spontaneous benign or malignant tumors while infected. Similarly elevated provirus was found in organs of rabbits with experimentally induced acute leukemia/lymphoma-like disease. Levels of provirus in organs taken at necropsy varied widely suggesting that reservoirs of infections exist in non-lymphoid organs not traditionally thought to be targets for HTLV-1.Proviral load measurement is a valuable enhancement to the rabbit model for HTLV-1 infection providing a metric to monitor clinical status of the infected animals as well as a means for the testing of treatment to combat infection. In some cases proviral load in blood did not reflect organ proviral levels, revealing a limitation of this method for monitoring health status of HTLV-1 infected individuals. HTLV-I was the first human retrovirus discovered and was isolated from cell lines derived from patients with cutaneous T cell lymphoma or adult T cell leukemia (ATL) ,2. LaterPrevious studies of infected human subjects suggest that high proviral load is associated with increased tendency to develop HTLV-I-associated HAM/TSP, while ATL is associated with extremely high levels of provirus -8. High Rabbit experimental infection has proven to be an excellent model of human HTLV-I infection -31. ReseIn this paper we report adaptation of techniques  to measugag, pol, env and rex genes were analyzed. For each isolate 4,486 bases were compared and no differences from the original K30 clone were detected for the period of observation. These data gave confidence that proviral load studies may be conducted with little concern for effects of mutations on primer recognition of the provirus.An HTLV-I producing cell line BH24 was derived from rabbit BH24 inoculated with HTLV-I molecular clone K30p naked DNA. HTLV-I env protein gp46 was detected on the surface of BH24 cells and HTLV-I virions isolated from BH24 cell line have normal size and density Figure . Cell frenv gene was selected as an amplification target. To determine the sensitivity for this assay, scalar dilutions of K30p clone DNA ranging from 1 to one billion (109) copies were analyzed. The results indicate that a positive signal was consistently detected at HTLV-I DNA concentrations above 1 copy per ng of DNA. Based on these results the limitation of this assay was considered to be 1 copy of HTLV-1 proviral DNA per ng of genomic DNA.The proviral load was determined using a real time PCR-based QPCR assay, in which HTLV-I Fifty-seven rabbits infected by different routes and using different sources of HTLV-I were monitored for proviral load over a period of 75 weeks at two to four weekly intervals Figure . The higAverage proviral load measured for HTLV-I infected asymptomatic rabbits was compared with reported data for human samples Table . When alIn the course of collecting proviral load data one rabbit (TO9) showed an unexpected increase from about 3 copies/ng in a sample taken at 4 months to more than 10 copies/ng at 8 months post infection with cell-free virus. Examination of the rabbit revealed an enlarged kidney, which upon necropsy, was a nephroblastoma. Tissues selected from rabbit TO9 and tested for provirus revealed elevated levels in the thymus, spleen and the tumor dissected from the kidney Cell-free viruses were prepared from the culture supernatant of HTLV-I producing cell lines. Cells and debris were removed from supernatants by centrifugation at 800 g for 10 min, and then passed through a 0.22 mm filter . The filtrates were concentrated to15 to 20 fold through a centrifugal filter device with 100 NMWL membrane . The virus stock preparation was stored at -80°C until use. The virus quantitation was measured by a real-time QRT-PCR assay. Thawed virus preparations lost binding activity within several hours unless kept at 4°C . HTLV-I The PBMC genomic DNAs were isolated from EDTA-treated blood samples using Wizard Genomic DNA Purification Kit . The organ DNAs were prepared using DNeasy Tissue Kit .env region were used to amplify a 185 bp fragment. The sequences of HTLV-I env primers are: 5'-ATC CAC TTG GCA CGT CCT ATA-3'  and 5'-GCA GGA TGA GGG AGT TAT GTC-3' (nt 6054–6074). The dual-labeled fluorescent probe was FAM -5'-CTT TAC CCA TCG TTA GCG CTT CCA GCC CCC-3'-BHQ1 (nt 5954–5983). Rabbit beta-globin DNA quantitation was performed in parallel on all samples in order to determine the amount of cellular DNA present and was used as an endogenous reference to normalize variations due to differences in the PBMC count or DNA extraction. A 187 bp fragment of the rabbit beta-globin gene was amplified by forward primer 5'-GGT ATC CTT TTT ACA GCA CAA C-3'  and reverse primer 5'-CAG GTC CCC AAA GGA CTC G-3' (nt 531–549) in a real-time PCR assay. The fluorogenic probe used to detect rabbit beta-globin gene was 5'Quasar 670 - CCT GGG CTG TTT TCA TTT TCT CAG G - BHO-2, 3' (nt 471–495). Both the primers and probes were synthesized by a commercial company Two sequence-specific primers that detect HTLV-I env and rabbit beta-globin gene fragments were amplified separately with an Mx3000P Real-Time PCR System  in 50 μl reaction mixture consisting of 10 μl of DNA sample, 25 μl of Brilliant QPCR Master Mix  , 10 pmol of each primer, and 5 pmol of TaqMan probe. Thermal cycling conditions were as follows: 95°C for 10 min, and 45 cycles of 95°C for 30 s, 55°C for 1 min, and 72°C for 30 s. Each sample was analyzed in duplicate, and HTLV-I proviral load was calculated at the copy number of each per ng of genomic DNA.HTLV-I ATLL, adult T-cell leukaemia/lymphomaHAM/TSP, HTLV-I -associated myelopathy/ tropical spastic paraparesisPBMC, peripheral blood mononuclear cellsQPCR, quantitative polymerase chain reactionQRT-PCR, quantitative reverse transcription-polymerase chain reactionThe author(s) declare that they have no competing interests.


Using new and highly cross-reactive env primer pairs, we observed the presence of Env quasispecies in HTLV-1 infected individuals and STLV-1 naturally infected macaques, irrespective of the clinical status. These intrahost variants helped us to define highly conserved residues and motifs in the RBD. The new highly sensitive env PCR described here appears suitable for the screening of all known variants of the different PTLV types and should, therefore, be useful for the analysis of seroindeterminate samples.Four primate (PTLV), human (HTLV) and simian (STLV) T-cell leukemia virus types, have been characterized thus far, with evidence of a simian zoonotic origin for HTLV-1, HTLV-2 and HTLV-3 in Africa. The PTLV envelope glycoprotein surface component (SUgp46) comprises a receptor-binding domain (RBD) that alternates hypervariable and highly conserved sequences. To further delineate highly conserved motifs in PTLV RBDs, we investigated the intrahost variability of HTLV-1 and STLV-1 by generating and sequencing libraries of DNA fragments amplified within the RBD of the SUgp46  Retroviridae family and are globally referred to as primate T-cell lymphotropic viruses (PTLV). Thus far, four distinct groups of PTLV have been discovered: PTLV-1, -2 and -3 include both human  and simian  viruses while the fourth type (HTLV-4) has only been described in humans [Human T-cell lymphotropic viruses (HTLV) and their simian T-cell lymphotropic virus (STLV) counterparts belong to the ype HTLV- has onlyn humans ,6. In adn humans ,8. Transn humans  and by sn humans ,11.The close relationship between HTLV and STLV suggests a simian origin for HTLV. The HTLV-I strains can be classified into six different subtypes according to their geographic origin . Moreovein vivo mainly via clonal expansion of infected cells [tax or env [env in samples obtained from 2 asymptomatic patients [Unlike other retroviruses, which in general show a high rate of nucleotide substitutions, PTLV exhibit a remarkable genetic stability . This ised cells -17. Desped cells -20, a fex or env ,21-23. Opatients . Thus, tThe extracellular surface component (SU) of retroviral envelopes is involved in cellular tropism, target cell infection, and induction of host viral immunity. For any retrovirus, the SU exhibits the highest level of protein variability . The proenv gene.In order to delineate conserved motifs that are likely to be involved in binding or post-binding events, we have investigated the intrahost variability of HTLV-1 and STLV-1 RBD by sequencing intrahost libraries of DNA fragments amplified from the RBD-encoding part of the tax amplicons. In parallel, we amplified the equivalent regions in samples obtained from 2 STLV-1 naturally-infected Celebes macaques (Macaca tonkeana) from Indonesia [DNA directly isolated from blood samples obtained from three unrelated infected individuals, one asymptomatic seropositive donor, one ATL patient, and one TSP/HAM patient, were used to derive a fragment library of SU RBD and ndonesia .env-like sequences. We delineated a 195 nt sequence surrounding the env gene codon corresponding to Tyr114 in the HTLV-1 SU RBD, previously shown to be a critical determinant for HTLV Env receptor binding [env PCR was highly sensitive when tested on several blood samples obtained from HTLV infected individuals as well as from STLV infected monkeys. As a highly conserved control sequence we also amplified and sequenced intrahost fragments corresponding to a 219 bp fragment of the HTLV-1 tax gene. For our study, we used degenerate PCR primers in order to match all different sequences present in the template. One μg of DNA from fresh PBMCs for each sample was amplified by nested PCR using the primers and cycling conditions as follows(using standard abbreviations for degenerate positions) : 83VS, 5' TAYBTATTYCCNCATTGG 3'; and 240VAS, 5' RTANAGNACRTGCCA 3', located in the Y/LFPHW motif and WHVLY motif, respectively (positions 5452 to 5926 in the ATK-1 reference strain [® Taq DNA Polymerase (Invitrogen) including a hot start (94°C for 2 min), with the following cycle conditions: 26 cycles of 94°C for 30 s, 50°C decreasing by 0.5°C per cycle, and 72°C for 45 s; this was followed by 12 cycles of 94°C for 30 s, 48°C for 30 s, and 72°C for 45 s, with a final elongation at 72°C for 5 min before cooling to 4°C. Then, 1/15th of the first PCR volume was used as the source of templates in a semi-nested amplification performed with the same cycling conditionsexcept for the annealing step (50°C decreasing 0.5°C per cycle for the first 10 cycles followed by 30 cycles at 50°C). The tax-rex fragments were amplified using previously described generic primers and PCR conditions [tax and env regions, respectively. Nucleotide and amino acid sequences (corresponding to the PCR fragment without primers) were aligned with ClustalW(1.7) [env sequences as described by Nei and Gojobori [Using a multiple envelope alignment of all available PTLV strain types, we designed degenerate PCR primers spanning the RBD in order to allow the detection of all PTLV  binding . This PTe strain ) for thenditions . PCR ampalW(1.7) , and anaGojobori .tax primer pairs, previously shown to match sequences from a variety of divergent HTLV and STLV strains, to amplify our samples. Sequencing and subsequent analyses of the corresponding 183 bp fragments of 50 independent PTLV-1 tax clones derived independently from one healthy carrier, one ATL patient and 2 macaques (Mac1 and Mac2), resulted in only a single nucleotide change in 2 clones. One change was observed in a clone derived from Mac1, while the second change was observed in a clone derived from the ATL patient sample. Each change translated into a different amino acid substitution when compared to the consensus sequence. Thus, in our experimental conditions, the frequency of bases changes that could be attributed to the Taq polymerase remained under 0.4%.We used highly cross-reactive tax sequencing, a significant degree of variability was seen with env. Indeed, there was an important sequence heterogeneity within each isolate, indicative of a quasispecies nature of HTLV-1/STLV-1 infections, as revealed by intrahost variations in the Env RBD  and the rate of synonymous changes per synonymous site (ds) were calculated for the RBD sequences of each patient and macaque. A nonsynonymous substitution rate higher than the synonymous substitution rate indicates positive selection for advantageous mutations, whereas a nonsynonymous rate lower than the synonymous rate indicates « purifying selection » that prevents the spread of detrimental mutations [dn/ds ratio we calculated were relatively low (< 0.5) for all blood samples, irrespective of the host species and the clinical status. Therefore, despite the significant level of intrahost variations observed in the RBD sequences, strong constraints against sequence variation prevailed in the RBD region of the PTLV-1 env genes.The rate of nonsynonymous changes per nonsynonymous site , suggestive of a robust selectivity conferred env sequence variation found between isolates does not reflect the significant degree of env sequence variation found within individuals. However, the same dominant HTLV-1 sequence was found independently in the three unrelated infected patients and the two STLV-1 macaques, in agreement with a strong positive pressure on this highly conserved consensus. Altogether, our results point to the selective transmission of an optimally adapted form, rather than to an absence of replication or to a stricter polymerase fidelity of PTLV-1.Interestingly, the low degree of env PCR protocol, based on degenerate primers matching conserved motifs in the RBD, allows the detection of all known PTLV types (data not shown). This property will help elucidate further the detection of undescribed PTLV divergent variants as well as that of potentially undescribed PTLV types which would be masked under conventional PTLV PCR screening.Importantly, our new highly sensitive Env: envelope glycoproteinHTLV : Human T-cell lymphotropic virusSTLV : Simian T-cell lymphotropic virusPTLV:Primate T-cell lymphotropic virusMLV: Murine leukemia virusnt: nucleotideLTR: Long Terminal RepeatPCR: Polymerase Chain ReactionRBD: receptor-binding domainSU: Env extracellular surface componentenv accession number for the sequences determined in this study are: Genbank DQ530557 to DQ530596.The The author(s) declare that they have no competing interests.FJK set up the initial design and experiments and participated to the writing of the manuscript. ML performed some of the PCR, cloning and sequencing experiments. AG and SG provided some of the DNA samples and corrected the final draft of the manuscript. JLB participated to the design of the study, helped with the interpretation of the data and corrected the manuscript. MS initiated the project, co-participated in the design of the study, co-coordinated its realization and co-wrote the manuscript. VC was the principal designer and experimentator of this study, coordinated its realization, wrote the first draft of the manuscript and co-wrote the following versions. All authors read and approved the final manuscript.


The Deltaretrovirus genus comprises viruses that infect humans (HTLV), various simian species (STLV) and cattle (BLV). HTLV-I is the main causative agent in adult T-cell leukemia in endemic areas and some of the simian T-cell lymphotropic viruses have been implicated in the induction of malignant lymphomas in their hosts. BLV causes enzootic bovine leukosis in infected cattle or sheep. During the past few years several new Deltaretrovirus isolates have been described in various primate species. Two new HTLV-like viruses in humans have recently been identified and provisionally termed HTLV-III and HTLV-IV. In order to identify a broad spectrum of Deltaretroviruses by a single PCR approach we have established a novel consensus PCR based on nucleotide sequence data obtained from 42 complete virus isolates . The primer sequences were based on highly interspecies-conserved virus genome regions. We used this PCR to detect Deltaretroviruses in samples from adult patients with a variety of rare T-cell neoplasms in Germany.-2 and 10-3 with 100% specificity as demonstrated by serial dilutions of cell lines infected with either HTLV-I, HTLV-II or BLV. Fifty acute T-cell lymphoblastic leukemia (T-ALL) samples and 33 samples from patients with various rare mature T-cell neoplasms  were subsequently investigated. There were no cases with HTLV-I, HTLV-II or any other Deltaretroviruses.The sensitivity of the consensus PCR was at least between 10The results rule out a significant involvement of HTLV-I or HTLV-II in these disease entities and show that other related Deltaretroviruses are not likely to be involved. The newly established Deltaretrovirus PCR may be a useful tool for identifying new Deltaretroviruses. Papio Hamadryas) [Retroviruses are the main etiologic agents in a variety of malignant diseases in animals . Bovine madryas) -10. Untimadryas) ,12. Moretax and rex genes that act as transcription factors, thereby promoting cell growth and malignant transformation However, the etiology of many human malignant T-cell and T-/NK-cell disorders is still not well understood. On the other hand a great deal of knowledge has been gained in the last years on the molecular biology of Deltaretroviruses, since a large number of new isolates have been described. To investigate the possible involvement of Deltaretroviruses in various human T-cell neoplasms, we have constructed a novel Deltaretrovirus consensus PCR based on nucleotide sequence alignments of all 42 complete Deltaretrovirus isolates published to date. Highly conserved virus genome regions were identified that allowed the construction of a generic PCR, capable of detecting all known Deltaretroviruses.HTLV-I and STLV-I are etiologically linked to the induction of certain T-cell lymphomas/leukemias in their hosts ,14. The pol ORF of HTLV-I/-II/BLV is expressed by using two ribosomal -1 frame shifts, and the second frame shift with the transcription start site of the pol ORF lies within this region [-2 – 10-3 for our PCR. This sensitivity appeared highly sufficient for our purpose. The PCR produced a faint 657 bp sideband when testing human DNA or cell line DNA diluted in human DNA  and a successive BLAST search revealed that it originated from amplification of a sequence on chromosome 3  and 11  by primer delta-F.A total of 42 complete Deltaretrovirus isolates could be retrieved from the EMBL/Genbank/DDBJ nucleotide sequence database Table . These is region -18. We t-3 – 10-4 (while retaining specificity) by lowering the annealing temperature to 60°C and increasing the number of PCR cycles but at the expense of a stronger 657 bp sideband.It should be noted that the PCR sensitivity could be further increased to 10A total of 83 samples were obtained from patients with various rare mature T-cell (N = 31) and precursor T-cell neoplasms (N = 50) and from 2 patients with NK-cell disorders. All samples had been thoroughly characterized immunologically and genetically and contained a high percentage (>= 50%) of malignant cells , MJ [G1ymphoma, ) and Mo eukemia, ). All cePureGene kit  and dissolved in Tris/EDTA buffer at a concentration of 60 ng/μl.DNA was isolated from sample material or cell lines using the Serial dilutions of cell line DNA in DNA from buffy coats of blood donors were prepared as recently described .All samples were obtained for diagnostic purposes, and we retrospectively investigated archived material. The patients had given their consent for scientific investigations. The T-precursor samples were obtained within the German Multicenter Study Group for Adult Acute Lymphoblastic Leukemia (GMALL). Our study complied with the Helsinki Declaration.delta-F 5'-CARKTGGGGGCTCGTCCGGGAT-3' and delta-R 5'-GGCCTGGAGGCGYTCHRGTTTAA-3', buffer conditions and polymerase mix as recommended by the supplier. The primers were optimized derivatives of those previously published [The HotStarTaq kit  was used with 200 ng sample DNA, 400 nM of each PCR primer ublished  and HPLCAll available complete nucleotide sequences from Deltaretrovirus isolates were collected from the EMBL/Genbank/DDBJ database Table , convertdnacomp and drawgram was used with the default parameters to construct a phylogenetic tree from the aligned sequences.The PHYLIP program package , versionImmunophenotyping by FACS analysis was done essentially by standard methods described elsewhere .HTLV human T-cell lymphotropic virusSTLV simian T-cell lymphotropic virusBLV bovine leukemia virusPTLV primate T-cell lymphotropic virusPCR polymerase chain reactionPLL prolymphocytic leukemiaNHL Non Hodgkin lymphomaALL acute lymphoblastic leukemiaThe author(s) declare that they have no competing interests.TB designed and performed the laboratory work, particularly the PCR, did the alignments and phylogenetic analysis and wrote the paper. SS and ET performed immunophenotyping of samples. MH characterized specific samples by analysis of T-cell receptor clonality. DH is chairman of the German Multicenter Study Group for Adult Acute Lymphoblastic Leukemia (GMALL) which provided the T-ALL samples. All authors have read and approved the manuscript.delta-F and delta-R).contains an alignment of the 42 retrovirus sequences from Table Click here for file


The scientific conference, sponsored by the International Retrovirology Association, is held biennially at rotating international venues around the world. The meeting brings together basic scientists, epidemiologists and clinical researchers to discuss findings to prevent HTLV infection or develop new therapies against HTLV-mediated diseases. The Association fosters the education and training of young scientists to bring new approaches to the complex problems of HTLV research, such as translational research to bring findings from the laboratory into clinical trials that benefit HTLV-infected patients. The breadth and quality of research presentations and workshops at the 12th International Conference indicate that these goals are being accomplished. As HTLV research enters its third decade a new generation of scientists face many challenges. However, HTLV scientists and clinicians displayed exciting new approaches and discoveries during plenary talks and poster sessions. The conference encouraged research in HTLV infections and disease, fostered collaborations, and stimulated new partnerships between clinicians and scientists to encourage clinical trials and novel therapeutic interventions.The 12 The Association was established in May 1994 to promote informal discussions among established human T-lymphotropic viruses (HTLV) scientists to enhance the efforts of scientists and clinicians to form interdisciplinary groups to study HTLV and its related diseases in a cooperative and creative manner. The Association sponsors its biennial general scientific conferences at rotating international venues, generally in areas with endemic HTLV infection. This unique meeting brings together basic scientists, epidemiologists and clinical researchers in a free form exchange of data to discuss approaches to prevent HTLV infection or develop new therapies against HTLV-mediated diseases. The Association has focused its international meeting on HTLV and other related human and nonhuman primate retroviruses, to promote excellent science and to facilitate the communication of scientific results. Equally important, the Association fosters the education and training of young scientists to bring new approaches to the complex problems of HTLV research, such as translational research to bring findings from the laboratory into clinical trials that benefit HTLV-infected patients. Judging from the breadth and quality of research presentations and workshops at the 12th International Conference, these goals are being accomplished.The International Retrovirology Association in conjunction with the National Institutes of Health and the University of the West Indies, Jamaica welcomed over 300 scientists from diverse disciplines to the 12th anniversary of the discovery of the first identified human retrovirus, HTLV-1. Scientists in this field of research have many new discoveries to energize their work, many of which were on display during the meeting as plenary talks or during poster sessions. The conference encouraged research in HTLV infections and disease and fostered collaborations between research groups. Importantly, the conference through its many workshops provided a setting to stimulate new partnerships between clinicians and scientists to encourage the development of clinical trials and novel interventions.HTLV research enters its third decade with the challenge to bring a new generation of scientists to the challenges facing the field . Many ofth International Conference on Human Retrovirology, but is by no measure a comprehensive overview of all of the exciting findings from the conference and we apologize in advance to those investigators we have not mentioned in an effort to provide a concise summary of the meeting.Human T-lymphotropic virus type 1 (HTLV-1) and the closely related HTLV-2 were the first human retroviruses discovered . HTLV-1 Reports of well characterized cohorts by Dr. Edward Murphy of the University of San Francisco (United States) began the meeting with a clear challenge to carefully examine the relationship between viral loads and disease outcomes. Data from these groups suggest that a "set point" of viral load is determined in each individual studied based on their ability to form an effective early immune response to the infection. Risk factors for the transmission of HTLV-1 from infected mothers to their children based on HLA haplotype was presented by Dr. Robert Biggar and colleagues , who illustrated how immune recognition may determine rates of viral transmission.A highlight of this session included Dr. Masao Matsuoka's and colleagues'  recent findings related to the functional significance of an anti-sense message that codes for the protein HBZ and the role of methylation in the control of HTLV-1 gene expression. His group provided data to indicate that proviral DNA in cell lines derived from ATLL patients was highly methylated correlating with low or absent HTLV-1 gene expression. Unlike the 5' LTR of proviral DNA and the coding region and the 3' LTR was not heavily methylated in these same cells. The expression of HBZ mRNA, but not those encoding other viral genes such as tax, was detected in all the fresh ATL samples, strongly suggesting that HBZ has a significant role in leukemic cell survival in vivo. These findings were further supported subsequently in the meeting by Dr. Eric Wattel  who also found correlation between proviral loads in patients with HBZ mRNA expression. In addition, it appears that HBZ expression had a growth stimulating influence on ATL cells. Transfection of HBZ expressing plasmids in an IL-2 dependent human T-cell line augmented the cell line growth and inhibition of HBZ expression by siRNA interference reduced cell growth of ATL-derived cell lines. Finally, transgenic mice containing HBZ gene under the control of a CD4 promoter exhibited an increased number of CD4+ spleen-derived T-cells. Dr. Matsuoka proposed that HBZ and Tax act synergistically to transform CD4+ T-cells eventually leading to the development of ATLL. Since HBZ, but not Tax is expressed in ATL cells in vivo, this unique anti-sense encoded nonstructural protein may represent a promising therapeutic target in ATLL patients. Later in the meeting, the HBZ encoding region was further characterized and mapped to reveal new start sites for the anti-sense transcript by Dr. M. Cavanagh and colleagues . Collectively, these presentations provide new insights into the complex interaction between HTLV-1 and virus replication and lymphocyte survival.An exciting summary of recently published findings about novel nucleotide sequences of human retroviruses, HTLV-3 and HTLV-4 were summarized by Dr. William Switzer of the United States Centers for Disease Control and Prevention. These researchers used polymerase chain detection to discover evidence of two related HTLVs (named HTLV-3 and HTLV-4). The first group directed by Switzer investigated 930 central Africans with contact with nonhuman primate blood through hunting and butchering. Serological tests and partial DNA sequencing of blood samples showed that two humans were infected with novel viruses, designated HTLV-3 and HTLV-4. Phylogenetic analysis showed that HTLV-3 is originated from a known monkey virus STLV-3 through cross-species transmission, while a corresponding monkey virus to HTLV-4 has not been reported yet and genetically equidistant from all known HTLVs/STLVs. The second group directed by Dr. Antoine Gessain  tested serum specimens or DNA from 240 subjects from Cameroon to provide suggestive evidence that an HTLV-3 related to STLV-3 was present in individuals exposed to nonhuman primate blood or tissues. Importantly, these findings suggest that cross species transmission between nonhuman primates and humans is ongoing where people are exposed to primate blood and tissues ,8. A chaThe study of HTLV-1 Tax initiated the concept that complex retroviruses regulate their expression by producing transactivating proteins. The mechanisms used by HTLV-1 Tax to alter cell cycle regulation or cell division, as in past meetings, continued to dominate the basic biology sessions of the 2005 meeting. Dr. Jennifer Nyborg  provided an overview of how the well-studied transactivating protein of HTLV-1, Tax, promotes viral transcription from the chromosomally-integrated HTLV-1 promoter. By using chromatin immunoprecipitation analysis, Dr. Nyborg's group provided data that the promoter activation by Tax1 correlated with the apparent loss of nucleosomes from the promoter and coding region. These results suggest that the Tax functions by causing nucleosome removal from the HTLV-1 promoter. Later in the meeting, by using in vitro transcription using "chromatinized" templates, Dr. Fatah Kashanchi  presented complementary data indicating that Tax converts the randomly-assembled nucleosomes into periodic assembled ones, and this periodic assembly of the nucleosomes correlated with the activation of HTLV-1 promoter by Tax. His group has previously demonstrated that Tax recruits SWI/SNF complex containing BRG1 on the HTLV-1 promoter, and the recruitment is essential for the transcriptional activation of HTLV-1 promoter . Thus, SDr. Kuan-Teh Jeang  presented data that continued this theme of the meeting. His new findings suggest that Tax interacts with the Ran-GTP network to cause abnormal amplification of cellular centrosomes, which may be an initial event of cancer caused by the virus. Dr. Patrick Green  furthered this subject area by providing new information about the role of the anti-sense encoded protein, PDZ-binding motif of Tax in induction of micronuclei, a hallmark of genomic instability. Dr. Chou-Zen Giam  reported that "unscheduled" activation by the Cdc20-associated anaphase promoting complex by HTLV-1 Tax induces mitotic dysfunction and inactivation of critical regulators of mitosis. This presentations overall solidified the concept that the acquisition of genetic and epigenetic changes in Tax-expressing T cells favors the selection of cells with mutated proviral DNA in a manner to favor eventual outgrowth of the transformed cell in a way that avoids immune system recognition. Collectively, the precise role of Tax in cellular transformation was a major theme of the basic science sections of the meeting.vif-like gene in its genome, but data presented from Dr. Derse suggested that the HTLV-1 structural gene, gag contained sequences that act to inhibit A3G incorporation into HTLV-1 virions. Thus, HTLV-1 may overcome A3G anti-retroviral activity through gag-mediated exclusion of A3G. Consistently with these results are findings that indicate that there are rare dG-dA hypermutations among HTLV-1 isolates.Dr. Warner Greene  provided a stimulating plenary talk about the role of APOBEC 3G (A3G), a cellular deoxycytidine deaminase with broad anti-retroviral activity. Dr. Greene summarized what is currently understood about A3G, which is inactive as a high-molecular-weight ribonucleoprotein complex in activated CD4 T-cells, but is active in low-molecular-weight complexes in resting CD4 T-cells. Resting CD4+ T-cells are typically resistance for HIV-1 infection, but the resistance was greatly relieved by A3G-specific siRNAs that block A3G function. Surprisingly, sequence analysis showed rare dG-dA hypermutations, a signature of A3G-mediated inhibition, in infected CD4 T-cells. Thus, A3G may have another mechanism to inhibit HIV infection. Dr. Greene suggested that deaminase activity may have a broader role and perhaps may influence HTLV-1's ability to infected CD4+ T-cells, a preferred natural target of HTLV-1 infection in vivo. In this regard, Dr. David Derse and colleagues  at the meeting reported that exogenous overexpression of APOBEC 3B (A3G) also inhibited HTLV-1 infectivity. A3G was incorporated into HTLV-1 virions, but the amount was much less than that of HIV-1 stains that lack the ability to inactive the cellular co-factor (HIV Vif defective). Interestingly, HTLV-1 does not appear to have The meeting brought together a variety of clinicians seeking better treatments against ATLL and HAM/TSP. Dr. Thomas Waldmann , a pioneer in the field of therapeutic approaches against ATLL, provided a summary of the role of IL-15 in T-cell signaling and his groups recent findings involving Hu-Mik-Beta-1 (HM-beta) to block β-chain signaling following IL-15 receptor engagement. This approach may provide exciting new avenues to inhibit lymphocyte-mediated disorders such as HAM/TSP. Clinical trials using the potent immunosuppresive agent Campath-1H (anti-CD52) was presented by Dr. J.C. Morris . HTLV-1 Leukemic patients appeared to respond well to Campath-1H, despite its adverse side effects. A key factor in non-responsive ATL lymphoma patients appears to be the difficulties with drug penetration of solid tissues.HTLV-1 Tax has been shown to interact directly with different members of the NF-κB family . InterfeNovel treatments continue to be a focus of researchers in the HTLV and related retrovirus field. Dr. Luc Willems and colleagues  using their established bovine leukemia virus infection of sheep model, revealed new approaches to treatment of leukemia using the drug, valproate. This treatment was shown to be effective in decreasing lymphocyte numbers and tumor regression in this model system.A particularly interactive portion of the meeting were the educational and technical workshops held offsite at the Barnett Estates, a serene setting that promoted scientific exchange. Morning workshops discussed the epidemiology, animal models, immunology, and basic virology of HTLV and related viruses. These group sessions were spirited and were lead by scientist who provided an excellent overview of their fields before leading the discussion of selected topics of interest to the group. These sessions allowed a more informal exchange of ideas and unpublished data, a goal of any scientific meeting. New virus strains including HTLV-3 and HTLV-4 characterized by molecular signatures were the subject of many conversations. Current strengths of weakness or gaps in the literature dominated many sessions as participants provided their interpretation of recent published or new data at the early plenary sessions. These workshops were extended in the afternoon to include important and clinically focused sessions based on HTLV disease associations. Thus, workshops devoted to adult T-cell leukemia/lymphoma, infectious dermatitis, and HTLV-1 neurologic disease allowed scientist to debate current approaches and how clinical trials could be evaluated and improved. Overall, these workshops provided a dynamic interaction among scientists and clinicians, which were apparent when patient case studies were included to focus the discussions.Dr. Masahiro Fujii and colleagues  reported data using co-transfection assays of Tax expression plasmids with luciferase reporters to test the functional differences between HTLV-1 and HTLV-2 Tax . This grDr Claudine Pique and colleagues  presented new findings that neuropilin 1 (NP-1), a receptor for Sematophorin 3a and vascular endothelial growth factor (VEGF), acts as a co-factor for HTLV-1 entry. NP-1 directly interacted with HTLV-1 envelope protein. There appears to be two separable domains in the HTLV-1 envelope that are required for the virus entry. While one domain is a binding surface for GLUT-1, a recently identified HTLV-1 receptor, the other was that for NP-1. NP-1, GLUT-1 and the envelope proteins appear to form a ternary complex on the cell surface. These results argued that HTLV-1 has two cellular receptors for the viral entry into host cells similar to HIV-1.foxp3 expression, which would be predicted to suppress the function of these important regulatory T-cells, perhaps contributing to lymphocyte-driven diseases. Dr. Charles Bangham  and his research group continue to lead the field of cellular immunity against HTLV-1 infection. His presentation provided novel insights into the efficiency of cytotoxic T-cell killing as a key determinant of patient viral load outcome and data describing unique lymphocyte labeling techniques -glucose) to monitor the kinetics of lymphocyte proliferation and death in HTLV-1-infected subjects.Dr. Steven Jacobson  presented new information about the intriguing findings related to regulatory T-cells  in the immunopathogenesis of HTLV-1-associated neurologic disease. Interestingly, his research group found that Tax specifically inhibits I is recruited to the immunological synapse and inhibits signaling from the T-cell receptor. Dr. Vincenzo Ciminale  provided an update of his research of a novel mitochondrial localizing protein p13II in modifying lymphocyte survival and apoptotic cell death. Dr. Michael Lairmore and his colleagues  provided new information on the role of p30II in modifying G2 exit of the cell cycle furthering implicating this protein in lymphocyte survival.The role of nonstructural proteins including proteins included in pX ORFs 1 and 2 were featured by a number of investigators. Dr. Franchini's research group  presented work indicating that p12th International Conference on Human Retrovirology illustrated the dedication of the many scientists, clinicians, and patient advocates to address these challenges with a new determination and energy, as they all reluctantly left the gracious atmosphere of Jamaica to travel back to their homes, laboratories and offices throughout the world.The meeting concluded with poignant remarks by one of the pioneering epidemiologist in the field of HTLV research, Dr. Nancy Mueller . She provided appropriate context to past studies of cohorts of HTLV-1 infected subjects, disease associations, and the useful applications of carefully controlled population-based studies. As the meeting came to a close, it was clear that HTLV and related retrovirus researchers faces many challenges, which confound and frustrate scientists in the HTLV field. However, the 12HTLV, human T-lymphotropic virusesHTLV-1, human T-lymphotropic virus type 1HTLV-2, human T-lymphotropic virus type 2HAM/TSP, HTLV-1-associated myelopathy/tropical spastic paraparesisATLL, adult T-cell lymphoma/leukemiaThe authors have no competing financial or other interests involved in the data, methods, or writing of this manuscript.Each author (MF and ML) have each met the definition of author as outlined by the Retrovirology journal. Each has made substantive intellectual contributions to the commentary. Each author has given final approval of the version to be published. Each author has participated sufficiently in the work to take public responsibility for appropriate portions of the content.


We previously identified the glucose transporter Glut-1, a member of the multimembrane-spanning facilitative nutrient transporter family, as a receptor for both HTLV-1 and HTLV-2. However, a recent report concluded that Glut-1 cannot serve as a receptor for HTLV-1 on CD4 T cells: This was based mainly on their inability to detect Glut-1 on this lymphocyte subset using the commercial antibody mAb1418. It was therefore of significant interest to thoroughly assess Glut-1 expression on CD4 and CD8 T cells, and its association with HTLV-1 and -2 envelope binding.RBD or H2RBD). Using antibodies raised against the carboxy-terminal peptide of Glut-1, we found that Glut-1 expression was significantly increased in both CD4 and CD8 cells following TCR stimulation. Corresponding increases in the binding of H1RBD as well as H2RBD, not detected on quiescent T cells, were observed following TCR engagement. Furthermore, increased Glut-1 expression was accompanied by a massive augmentation in glucose uptake in TCR-stimulated CD4 and CD8 lymphocytes. Finally, we determined that the apparent contradictory results obtained by Takenouchi et al were due to their monitoring of Glut-1 with a mAb that does not bind cells expressing endogenous Glut-1, including human erythrocytes that harbor 300,000 copies per cell.As previously reported, ectopic expression of Glut-1 but not Glut-3 resulted in significantly augmented binding of tagged proteins harboring the receptor binding domains of either HTLV-1 or HTLV-2 envelope glycoproteins (H1Transfection of Glut-1 directly correlates with the capacities of HTLV-1 and HTLV-2 envelope-derived ligands to bind cells. Moreover, Glut-1 is induced by TCR engagement, resulting in massive increases in glucose uptake and binding of HTLV-1 and -2 envelopes to both CD4 and CD8 T lymphocytes. Therefore, Glut-1 is a primary binding receptor for HTLV-1 and HTLV-2 envelopes on activated CD4 as well as CD8 lymphocytes. We identified the ubiquitous glucose transporter Glut-1 as a receptor for deltaretrovirus HTLV-1 and HTLV-2 envelopes (Env), mediating viral binding and entry . The eluin vivo, leading these authors to question the role of Glut-1 as primary binding receptor for HTLV glucose, was at the limits of detection in both quiescent CD4 and CD8 T cells and increased by >20-fold in both subsets following stimulation  molecule, which is not a Glut-1 ligand, did not alter H2RBD-EGFP binding . This molecule inhibits Glut-1 function by directly binding to its sugar export site ,35. In ting Fig. . Moreoveing Fig.  and 4C. Induction of surface Glut-1, both upon exogenous transfection and stimulation of the endogenous protein, directly correlates with the capacities of HTLV-1 and HTLV-2 envelope-derived ligands to bind cells. We have previously reported a physical interaction between HTLV-1 and HTLV-2 RBDs and Glut-1 and mapped the primary binding site to ECL6 -12.RBD in conjunction with Glut-1 ECL1 and 5 are likely to depend on other co-factors. In this regard, it is interesting to note that neuropilin-1 and HSC70 have been shown to interact with the SU region located immediately downstream of HRBD glucose  to an unlabelled deoxy-glucose concentration of 0.1 mM and incubating cells for 45 min at 37°C. Alternatively, glucose uptake was performed in a 50 μl volume in the presence of 5 μM 2-deoxy-D [1-3H]glucose (2 μCi) for 10 min at RT. Cells were then washed in cold serum/glucose-free RPMI, and solubilized in 0.1% SDS. Radioactivity was measured by liquid scintillation and statistical analyses were performed using Student's t test.5 × 10RBD-EGFP is commercially available without any private interest to the authors of the manuscript.The H2SK and LS participated in the design of the study, performed significant numbers of the experiments and helped with the draft of the manuscript; LM, CM, AMH, and MC participated in the design and validation of antibodies and receptor binding domain constructs as well as binding studies; NM helped to conceive the studies, participated in their design and performed the initial comparisons of RBDs; NT is responsible for the overall study and drafted the manuscript together with MS and JLB. All authors read and approved the final manuscript.


Type A prostaglandins  were found to block the proliferation of HTLV-I infected cord blood lymphocytes (CBL) in vitro, thus preventing the clonal immortalisation that is considered as a predisposing condition to HTLV-I positive leukaemia. PGA1 and di-M-PGA2 did not affect the long-term survival of normal non-infected CBL, whereas they suppressed the proliferation of an established cord-blood derived HTLV-I positive cell line, MT-2. As shown by the number of HTLV-I infected p19+ cells, the block of the selection of immortalised, infected clones by PGAs did not appear to be due to an inhibition of early stages of HTLV-I infection. The possibility that the effect of PGAs could be mediated by an action on the immune response was also examined. PGAs regulated the cell-mediated cytotoxic function of CBL to a different extent when normal non-infected or HTLV-I exposed CBL were compared. In fact, PGAs down-regulated the natural killing and macrophage/lymphocyte cytotoxic response of normal CBL, whereas they did not modify the already depressed immune response of CBL challenged with HTLV-I. These results suggest that the protective effect of PGAs against HTLV-I infection in vitro is mostly related to the direct suppression of the clonal expansion of virus-infected cells, rather than to the anti-viral activity or modulation of the cell-mediated immunity.


Accurate, rapid, and economic on-line analysis of ethanol is very desirable. However, available biosensors achieve saturation at very low ethanol concentrations and thus demand the time and labour consuming procedure of sample dilution.Hansenula polymorpha (Pichia angusta) mutant strains resistant to allyl alcohol in methanol medium were selected. Such strains possessed decreased affinity of alcohol oxidase (AOX) towards methanol: the KM values for AOX of wild type and mutant strains CA2 and CA4 are shown to be 0.62, 2.48 and 1.10 mM, respectively, whereas Vmax values are increased or remain unaffected. The mutant AOX alleles from H. polymorpha mutants CA2 and CA4 were isolated and sequenced. Several point mutations in the AOX gene, mostly different between the two mutant alleles, have been identified. Mutant AOX forms were isolated and purified, and some of their biochemical properties were studied. An amperometric biosensor based on the mutated form of AOX from the strain CA2 was constructed and revealed an extended linear response to the target analytes, ethanol and formaldehyde, as compared to the sensor based on the native AOX.The described selection methodology opens up the possibility of isolating modified forms of AOX with further decreased affinity toward substrates without reduction of the maximal velocity of reaction. It can help in creation of improved ethanol biosensors with a prolonged linear response towards ethanol in real samples of wines, beers or fermentation liquids. The detection and quantification of alcohols with high selectivity and accuracy is required in many different areas. Accurate and rapid measurement of ethanol is important in clinical and forensic practice to analyse human body fluids, e.g. blood, serum, saliva, urine, breath and sweat, among others. The food, beverage , and pulp industries also require simple, correct, fast, and economic analytical methods to control fermentation processes and the quality of products.2 oxidoreductase, EC 1.1.3.13) has been extensively used for the determination of lower primary alcohols and formaldehyde [e.g. ethanol in real samples of wines, beers or fermentation cultures, is complicated and economically detrimental due to the necessity of diluting the samples. This is especially important in automatic electrochemical devises such as biosensors since the additional step of sample dilution causes significantly increasing costs of analysis. In order to overcome this inconvenience, obtaining modified forms of AOX with decreased affinity towards substrates and a wide range of linear response is highly desirable.Enzymatic and biosensor approaches are among the most convenient analytical methods for this purpose. Yeast alcohol oxidase  which stained in the presence of 15 mM ethanol, but not 5 mM or 10 mM, were selected.For isolation of the mutant forms of AOX (mAOX) with decreased affinity to substrates, a positive selection procedure was developed. Allyl alcohol was used as the selective agent in the medium with methanol as sole carbon and energy source. The rationale for the developed selection scheme was as follows. Allyl alcohol is oxidized by AOX with the formation of highly toxic acrolein. In the medium with the mixture of methanol (0.5%) and allyl alcohol , mutants defective in AOX could not arise. Mutants producing lower amounts of acrolein and enough formaldehyde for growth could be selected. One of the reasons for such an event would be the decrease of affinity of AOX toward substrates while retaining high enough reaction velocity. This selection scheme would produce few or no mutants with decreased rate of AOX reaction as they would not grow efficiently due to the negligible amount of formaldehyde produced. Altogether, 125 mutants of M values towards methanol for the parental strain and the CA2 and CA4 mutants were calculated as 0.62, 2.48, and 1.10 mM, respectively , E > K (131), P > L (148), K > R (150), N > D (306), F > S (532), and W > R (567). The mutant protein in strain CA4 has a lower substitution number which includes: I > V (232), P > S (245), M > V (246), and L > P (602). The level of the substrate affinity reduction correlates with the average quantity of amino acid substitutions.-1·mg-1, respectively. The average yield of chromatographically pure AOX samples was close to 20% of the initial amount (Table The AOXs were purified. Samples of CA2 and CA4 mAOXs as well as native AOX (nAOX) were obtained with quantities close to 100 mg with specific activities of 16, 23 and 19 μmol·minHRP/Os-AP59//AOX(CA2)/CP59, was constructed according to [An amperometric biosensor based on mAOX (from the CA2 mutant) and peroxidase (HRP), architected as rding to . CharactIn addition to alcohols (e.g. ethanol or methanol), formaldehyde is also a substrate for AOX . The calM value towards methanol in solution: the mutant enzyme possesses 4-fold increased KM value and, thus, decreased substrate affinity  was characterised by decreased affinity towards analyzed substrates without altered operational stability of the sensor.max. Furthermore, the operational stability of the mAOX-immobilised electrode was not affected and remained similar to an electrode based on the natural enzyme. The described methodology opens up the possibility for construction of biosensors appropriate for precise, rapid, and cheap analysis of target analytes, e.g. ethanol in real samples of wines, beers or fermentation cultures. For further improvement of mAOX as a biosensor element, various strategies could be used: 1) direct design of mutant forms of AOX based on results obtained from site-specific mutagenesis; 2) evolutionary engineering of mutant forms of AOX through continuous cultivation of the corresponding strains in medium with increased concentrations of allyl alcohol; 3) determination of crystal structures of an AOX of H. polymorpha, elucidation of the structural bases involved in the enzyme-substrate interaction and subsequent direct modification of enzyme characteristics.In this study, we describe a novel selection procedure for isolation of the mutant forms of AOX, and their use as a suitable bioelement for biosensor technologies. The created biosensor based on mAOX (from the strain CA2) was characterised by a decreased affinity towards analyzed substrates and slightly increased VEscherichia coli strain DH5α , supE44, relA1, deoR, Δ(lacZYA-argF)U169) was used in experiments which required a bacterial host. DH5α was grown at 37°C in rich (LB) medium as described in Sambrook et al. [E. coli cells were maintained on a medium containing 100 mg·L-1 of ampicillin.k et al. . TransfoH. polymorpha DL-1-356 (leu2) [-1 if required. For solid media, agar was added to 1.5% (w/v). Yeast cells were grown in Erlenmeyer flasks with continuous shaking (200 rpm) at 37°C.6 (leu2)  derived 6 (leu2)  supplemeH. polymorpha DL-1-356 were cultivated in liquid medium with glucose until midexponential growth phase, washed with distilled water, diluted to 106 cell·mL-1 and irradiated with a dose of UV light that allows 10% cell survival. Then, cells were diluted and plated on the solid Burkholder medium containing methanol (0.5%) and allyl alcohol (0.3 mM). Allyl alcohol was applied as a selective agent; it is oxidized by AOX to acrolein. This compound is toxic for yeast and results in the death of cells with a high AOX activity. On the other hand, AOX activity is essential for growth in medium with methanol as a sole carbon source. Thus, the medium with a mixture of methanol and allyl alcohol will provide a growth of the cells with decreased AOX activity and/or decreased AOX affinity to alcohols. Mutant colonies grown after 21–25 days of incubation were replica-plated into the same medium for confirmatory selection. Mutants with decreased enzyme affinity towards ethanol were screened by a plate colony assay, on the basis of visualization of AOX activity by the rate of hydrogen peroxide formation in reaction with ethanol as monitored by the peroxidative oxidation of o-dianisidine in the presence of peroxidase resulted purple colour of colonies [-1), o-dianisidine (0.5 mg·mL-1), peroxidase Sigma RZ 1.1 (0.13 mg·mL-1), and different (0–15 mM) concentrations of ethanol. The overlying assay mixture was allowed to set, and the plates were incubated at 37°C for up to 1 h. Colonies that stained purple only in the presence of 15 mM ethanol, but not 5 mM or 10 mM, were selected.For mutant isolation, cells of the strain colonies . For thiH. polymorpha strains CA2 and CA4 in the presence of different methanol concentrations (0–100 mM) in the peroxidase reaction with ABTS . KM was calculated from the initial reaction rates, depending on substrate concentration, via the equation:AOX affinity towards methanol was evaluated via a kinetic study of enzyme activity in crude cell-free extracts of parental  and mutamax and KM – kinetic constants. Constants were calculated with a double-reciprocal method of Lineweaver-Burk [where V – reaction rate, S – substrate concentration, Vver-Burk  using ExAAG CTT ATG GCC ATT CCT GAC GAA TTC-3'); LV2 (5'-CCC AAG CTT TTA GAA TCT GGC AAG TCC G-3') and DNA was extracted from the H. polymorpha DL-1-356, and mutant strains CA2 and CA4. The primers were designed based on the AOX sequence of the H. polymorpha CBS 4732 [HindIII and ligated. The resulting plasmids were sequenced. Sequencing of these DNA fragments was performed on both strands. Alignments of putative amino acid sequences were carried out by the programs MultAlin [H. polymorpha AOX wild type (DL-1-356), mutant alleles CA2 and CA4 were deposited in GenBank and were assigned the accession nos AM690088, AM690089 and AM690090, respectively.The 1995 bp coding regions for the AOX was amplified by PCR using the following primers LV1 . Plasmid DNA isolations from E. coli were carried out by using NucleoSpin® Plasmid .Preparations of total DNA from yeast were carried out by using the DNeasyHighly purified AOX was isolated from a cell-free extract of the isolated mutants, as described elsewhere ,18, by mAmperometric biosensors based on mAOX (CA2) were evaluated using constant-potential amperometry in a three-electrode configuration with a Ag/AgCl/KCl (3 M) reference electrode and a Pt-wire counter electrode. The applied working potential was -50 mV vs Ag/AgCl. Amperometric measurements were carried out using a bipotentiostat  connected to a personal computer via a RS232 port for data acquisition. Between experiments, the enzyme electrodes were stored in 20 mM phosphate buffer, pH 7.2, at 4°C.HRP/Os-Ap59//AOX(CA2)/CP9 and included two layers: the inner, with horse radish peroxidase (HRP) electrochemically precipitated in the presence of carboxylate-containing polymer modified with an osmium-pyridyl complex (Os-Ap59), and the outer, with mAOX (CA2) immobilized via cathodic precipitation in the presence of an amino-containing polymer (CP9). The Os-containing polymer served as a redox-mediator in the electrochemical reaction. Platinized graphite rods  were used as working electrodes which were sealed in a glass tube using epoxy thus forming disk electrodes.Bioselective layer of sensor was constructed according to . The strThe determination of operational stability and inactivation kinetics of the constructed biosensors was performed by using the analyzing system "Olga"  .KVD carried out the molecular genetic studies, participated in the sequence alignment and drafted the manuscript. OVS constructed and characterised the amperometric biosensors. OBR performed mutant isolation and evaluation of kinetic parameters of the AOX mutants. GZG purified enzymes. OVS, OBR and MVG co-drafted the manuscript. VAS, WS and MVG supervised some of the work and participated in the design of the experiments. MVG, WS and AAS provided guidance and suggestions for experimental design, analyzed data, and edited the manuscript. VAS and AAS conceived the presented study. AAS is the author of the method for selection of mutants with affected affinity of alcohol oxidase toward substrates. All authors read and approved the final version of the manuscript.


The clinical entity of adult T-cell leukemia (ATL) was established around 1977, and human T-cell leukemia virus type 1 (HTLV-I) was subsequently identified in 1980. In the 25 years since the discovery of HTLV-I, HTLV-I infection and its associated diseases have been extensively studied, and many of their aspects have been clarified. However, the detailed mechanism of leukemogenesis remains unsolved yet, and the prognosis of ATL patients still poor because of its resistance to chemotherapy and immunodeficiency. In this review, I highlight the recent progress and remaining enigmas in HTLV-I infection and its associated diseases, especially ATL. In 1977, Takatsuki et al. reported adult T-cell leukemia (ATL) as a distinct clinical entity . This diCPZ) [HTLV-I is a member of the Deltaretroviruses, which include HTLV-II, bovine leukemia virus and simian T-cell leukemia virus (STLV). The latter two viruses also cause lymphoid malignancies in the host, similar to the case with HTLV-I. HTLV and STLV are thought to originate from common ancestors, and share molecular, virological and epidemiological features. Therefore, they have been designated primate T-cell leukemia viruses (PTLVs). Phylogenetical analyses have revealed that HTLV-Ic first diverged from simian leukemia virus around 50,000 ± 10,000 years ago, while the spread of PTLV-I in Africa is estimated to have occurred at least 27,300 ± 8,200 years ago. Subsequently, HTLV-Ia, which is the most common subtype in Japan, diverged from the African strain 12,300 ± 4,900 years ago [There are approximately 10–20 million HTLV-I carriers in the world . In partgag, pol and env genes. A characteristic of HTLV-I is the presence of the pX region, which exists between env and the 3'-LTR. This region encodes several accessory genes, which include the tax, rex, p12, p21, p30, p13 and HBZ genes. Among these, the tax gene plays central roles in viral gene transcription, viral replication and the proliferation of HTLV-I-infected cells. Tax enhances viral gene transcription from the 5'-LTR via interaction with cyclic AMP responsive element binding protein (CREB). Tax also interacts with cellular factors and activates transcriptional pathways, such as NF-κB, AP-1 and SRF [bcl-xL and survivin [in vitro and in vivo [in vitro [The HTLV-I provirus has a similar structure to other retroviruses: a long terminal repeat (LTR) at both ends and internal sequences such as the  and SRF ,17-20. Fsurvivin . The act in vivo ,25. On tin vitro , suggestTax also interferes with the functions of p53, p16 and MAD1 -30. Thesin vivo. Moreover, excess expression of Tax protein is considered to be harmful to infected cells. Therefore, HTLV-I has redundant mechanisms to suppress Tax expression. Rex binds to Rex-responsive element (RxRE) in the U3 and R regions of the 3'-LTR, and enhances the transport of the unspliced gag/pol and the singly spliced env transcripts. By this mechanism, double-spliced tax/rex mRNA decreases, resulting in suppressed expression of Tax [tax/rex transcripts, and retains them in the nucleus [HBZ gene is encoded by the complementary strand of HTLV-I, and contains a leucine zipper domain. HBZ directly interacts with c-Jun or JunB [Although Tax promotes the proliferation of infected cells, it is also the major target of cytotoxic T-lymphocytes (CTLs) n of Tax . On the  nucleus . The HBZ or JunB , or enha or JunB , resultiTransforming growth factor-β (TGF-β) is an inhibitory cytokine that plays important roles in development, the immune system and oncogenesis. Since TGF-β generally suppresses the growth of tumor cells, most tumor cells acquire escape mechanisms that inhibit TGF-β signaling, including mutations in its receptor and in the Smad molecules that transduce the signal from the receptor. Tax has also been reported to inhibit TGF-β signaling by binding to Smad2, 3 and 4 or CBP/p300 ,39. InhiATL cells have been reported to show remarkable chromosomal abnormalities , which sin vivo. Although it was first discovered in a patient with variant hairy cell leukemia, HTLV-II is less likely to have oncogenic properties since there is no obvious association between HTLV-II infections and cancers. Regardless of the homology of their tax sequences, the oncogenic potential of Tax1 (HTLV-I Tax) is more prominent than that of Tax2 (HTLV-II Tax). The most striking difference is that Tax2 lacks the binding motif at C-terminal end to PDZ domain proteins, while Tax 1 retains it [In contrast to HTLV-I, HTLV-II promotes the proliferation of CD8-positive T-lymphocytes tains it . When thtains it .To understand the pleiotropic actions of Tax protein more clearly, transcriptome analyses are essential. The transcriptional changes induced by Tax expression have been studied using DNA microarrays, which revealed that Tax upregulated the expression of the mixed-lineage kinase MLK3. MLK3 is involved in NF-κB activation by Tax as well as NIK and MEKK1 . In addiin vivo by enhanced proliferation and suppressed apoptosis.HTLV-I can infect various types of cells, such as T-lymphocytes, B-lymphocytes, monocytes and fibroblasts . GlucoseIn vitro analyses of HTLV-I-infected cells revealed that HTLV-I-infected cells form "virological synapses" with uninfected cells. Contact between an infected cell and a target cell induces the accumulation of the viral proteins Gag and Env, viral RNA and microtubules, and the viral complex subsequently transfers into the target cell [in vivo.In HTLV-I-infected individuals, no virions are detected in the serum. In addition, the infectivity of free virions is very poor compared with that of infected cells. These findings suggest that HTLV-I is spread by cell-to-cell transmission, rather than by free virions. get cell . HTLV-I HTLV-I is mainly transmitted via three routes: 1) mother-to-infant transmission (mainly through breast feeding) ; 2) sexuThe provirus load varies more than 1000-fold among asymptomatic carriers . Since mtax, facilitates transmission. Therefore, HTLV-I has strategies that increase the number of HTLV-I-infected cells via the action of accessory gene products, thereby increasing the chance of transmission.Why does HTLV-I increase the number of infected cells by the pleiotropic actions of Tax? The provirus load in peripheral blood mononuclear cells (PBMCs) is well correlated with that in breast milk, and a higher provirus load in breast milk increases the risk of vertical transmission of HTLV-I ,56. Simiin vivo. Analyses using inverse PCR, which can identify the integration sites of the HTLV-I provirus, revealed that the proliferation of infected cells is oligoclonal, and that infected cells persistently survive in vivo [After HTLV-I infection, viral proteins such as Tax protein promote the proliferation of infected cells and also inhibit apoptosis by their pleiotropic actions. Since the HTLV-I provirus is randomly integrated into the host genome, the identification of integration sites enables to identify each infected clone, and to trace the kinetics of infected cells  in vivo -59. Impo in vivo . Thus, t in vivo  or HTLV- in vivo . Moreovetax gene  [tax gene transcript. Interestingly, ATL cells with genetic changes of the tax gene expressed its transcripts, suggesting that ATL cells do not silence the transcription when the tax gene is abortive [As mentioned above, Tax expression confers advantages and disadvantages on HTLV-I-infected cells. Although the proliferation of infected cells is promoted by Tax expression, CTLs attack the Tax-expressing cells since Tax is their major target . In HTLVertions) ,65; 2) Dertions) ; and 3) ertions) . Among fLymphoid malignancy with a T-cell origin is rare compared with B-cell malignancy. ATL shares hematological, pathological and immunological features with cutaneous T-cell lymphoma . The frequency of CTCL in Japan is estimated to be one/million/year. On the other hand, the frequency of ATL among carriers is estimated to be 1000/million/year. From these data, HTLV-I infection is estimated to increase the risk of T-cell malignancy by up to 1000-fold in carriers.in vivo is thought to increase the incidence of cancer in T-cells. What is the mechanism for this oncogenesis? DNA methylation is known to be associated with aging. Some genes are hypermethylated in older people, indicating that DNA hypermethylation is a physiological phenomenon in some genes. Under normal circumstances, T-lymphocytes survive for several years, and long-lived T-lymphocytes with disordered methylation should be replaced. However, HTLV-I-infected T-cells are considered to survive and accumulate abnormal methylation. The process of oncogenesis is similar to that of evolution [in vivo, and epigenetic and genetic changes of the genome play critical roles in this selection. Accumulating alterations of the host genome transform the HTLV-I-infected cells into ATL cells, and also enable ATL cells to proliferate in the absence of Tax expression  and 30% (3/10) [p16 gene is an inhibitor of cyclin-dependent kinase 4/6, and blocks the cell cycle. Genetic changes in this gene (deletion in most cases) have been described in many types of cancer cells. Deletion of the p16 gene has also been reported in ATL cells [p16 gene is also implicated in the suppression of p16 [p27KIP1, RB1/p105 and RB2/p130 genes have been reported in ATL, although they are relatively rare: 2/42 (4.8%) for the p27KIP1 gene; 2/40 (5%) for the RB1/p105 gene; and 1/41 (2.4%) for the RB2/p130 gene) [As described, some ATL cells can proliferate without functional Tax protein, suggesting that somatic (genetic and epigenetic) alterations cause transcriptional or functional changes to the host genes. The % (3/10) -71. The TL cells . Moreoven of p16 . In addi30 gene) . The facFas gene in ATL cells, which confer resistance to the Fas-mediated signal, have been reported [EGR3 gene as a hypermethylated gene compared to PBMCs from carriers [FasL gene [EGR3 gene transcription is silenced in ATL cells could account for the loss of FasL expression, and the escape of ATL cells from AICD. Thus, alterations of the Fas (genetic) and EGR3 (epigenetic) genes are examples of ATL cell evolution in vivo.Fas antigen was the first identified death receptor. It transduces the death signal by binding of its ligand, Fas ligand (FasL). ATL cells highly express Fas antigen on their cell surface , and arereported ,77. Normreported . Althougcarriers . EGR3 isasL gene . The finMEL1S gene [p16 [EGR3 and KLF4 genes as well as many others [Disordered DNA methylation has been identified in the genome of ATL cells compared with that of PBMCs from carriers: hypomethylation is associated with aberrant expression of the L1S gene , while hene [p16 , EGR3 any others . It is rtumor suppressor in lung cancer 1 (TSLC1), caveolin 1 and prostaglandin D2 synthase genes were increased more than 30-fold in fresh ATL cells compared with normal CD4+ and CD4+, CD45RO+ T-cells [Tgat gene [Tgat gene is observed in aggressive forms of ATL, and in vitro experiments showed that its expression is associated with an invasive phenotype.Transcriptome analyses using DNA microarrays have revealed transcriptional changes that are specific to ATL cells. Among 192 up-regulated genes, the expressions of the  T-cells . TSLC1 igat gene . Ectopicin vivo [The host immune system, especially the cellular response, against HTLV-I exerts critical control over virus replication and the proliferation of infected cells . CTLs agin vivo . HTLV-I-in vivo . Hence, in vivo . Thus, CTNF-α, SDF-1, HLA-B54, HLA-DRB-10101 and IL-15) are also associated with the provirus load, although their associations are not as significant compared with HLA-A02, and Cw08 [TNF-α gene was reported to show an association [As mentioned above, the provirus load is considered to be controlled by host factors. Considering that the cellular immune responses are critically implicated in the control of HTLV-I infection, human leukocyte antigen (HLA) should be a candidate for such a host genetic factor. From analyses of HAM/TSP patients and asymptomatic carriers, HLA-A02, and Cw08 are independently associated with a lower provirus load and a lower risk of HAM/TSP. In addition, polymorphisms of other genes  [Although the mechanism of immunodeficiency remains unknown, some previous reports have provided important clues. One mechanism for immunodeficiency is that HTLV-I infects CD8-positive T-lymphocytes, which may impair their functions . Indeed,17; 59%) . Such ATATL cells are derived from activated helper T-lymphocytes, which play central roles in the immune system by elaborating cytokines and expressing immunoregulatory molecules. ATL cells are known to retain such features, and this cytokine production or surface molecule expression may modify the pathogenesis.ATL is well known to infiltrate various organs and tissues, such as the skin, lungs, liver, gastrointestinal tract, central nervous system and bone . This inHypercalcemia is frequently complicated in patients with acute ATL  . In hypebcl-xL and survivin. A proteasome inhibitor, bortezomib, is currently used for the treatment of multiple myeloma. One of its mechanisms is suppression of the NF-κB pathway by inhibiting the proteasomal degradation of IκB protein. Several groups have shown that bortezomib is effective against ATL cells both in vitro and in vivo [Regardless of intensive chemotherapies, the prognosis of ATL patients has not so improved. The median survival time of acute or lymphoma-type ATL was reported to be 13 months with the most intensive chemotherapy . Such a  in vivo -119. Sin in vivo .During chemotherapy for ATL, chemotherapeutic agents worsen the immunodeficient state of ATL patients. In this regard, antibody therapy against ATL cells has advantages due to its decreased adverse effects. A humanized monoclonal antibody to CD25 has been clinically administered to patients with ATL ,122. In tax gene status is correlated with the effect of allogeneic stem cell transplantation, and whether the effectiveness of the anti-HTLV-I immune response is against leukemic cells or non-leukemic HTLV-I-infected cells. Nevertheless, these data suggest that potentiation of the immune response against viral proteins such as Tax may be an attractive way to treat ATL patients [Advances in the treatment of ATL were brought about by allogeneic bone marrow or stem cell transplantation ,126. Abspatients . Such stin vivo, and the maximum production of HIV-1 virions in the body can reach 1010 per day. Since reverse transcriptase is an error-prone enzyme due to its lack of proof-reading activity, it produces about one mistake per replication, resulting in tremendous errors in the proviral sequence during replication. Although most of these variations ruin the virus replication due to nonsense mutations or impairment of viral gene functions, some become capable of replicating under different circumstances such as the presence of anti-HIV drugs and activation of the host immune system. This can account for why HIV-1 acquires resistance against anti-HIV drugs, and escape from CTLs. On the other hand, HTLV-I increases its copy number in two ways, namely replication of HTLV-I itself and the proliferation of HTLV-I-infected cells in vivo. Although immune responses  against viral proteins suggest the presence of active viral replication in vivo, most of increased HTLV-I provirus load (the number of infected cells) is considered to be due to proliferation of infected cells since CTLs efficiently eliminate virus-expressing cells. Therefore, there is much less variation in the HTLV-I provirus sequence compared with HIV-1 [As described in the first section, HTLV-I has resided in humans for a long time. On the other hand, HIV-1 has only been recently transmitted to humans, probably from chimpanzees. Due to the comparatively small genomic differences between humans and chimpanzees, this virus can quickly adapt to human cells. These two human retroviruses are opposite in many aspects. HIV-1 vigorously replicates th HIV-1 . However


Retroviruses called human T-lymphotropic viruses (HTLVs) are found in two types—HTLV-1 and HTLV-2—in people all over the world. Genetic evidence suggests that they crossed into humans from simian T-lymphotropic viruses (STLVs) and that each type, plus various subtypes, have crossed independently. Now, two more types of HTLV have been found in humans living in central Africa.At least 22 million humans are infected with HTLV-1 or HTLV-2, and the viruses are endemic in several areas. About 2–5% of those infected with HTLV-1 develop adult T cell leukemia. HTLV-1 also causes a neurologic disease called tropical spastic paraparesis/HTLV-1 associated myelopathy. HTLV-2 is less pathogenic but is thought to cause similar neurologic illnesses and increase susceptibility to opportunistic infection.Proceedings of the National Academy of Sciences.William Switzer, a researcher at the Centers for Disease Control and Prevention, and his colleagues sequenced HTLV strains from a high-risk population: people in Cameroon who reported contact with nonhuman primate tissues through hunting and butchering or keeping primate pets. The study uncovered many previously unknown subtypes of HTLV-1, most with known correlates in nonhuman primates. The team also found that two people carried previously unknown HTLV types. One, HTLV-3, is similar to the nonhuman primate virus STLV-3. The other, HTLV-4, is genetically different from any known virus in humans or other primates. The findings appear in the 31 May 2005 issue of the Because HTLV-4 is so divergent from other HTLVs, this virus may have evolved in humans over quite some time, Switzer says. It’s possible, though, that primates are infected with an equally divergent simian version that just hasn’t been found yet. “We’re screening primates in that same area to see if we can answer that question,” Switzer says.Retrovirology.A group led by Antoine Gessain, head of the Epidemiology and Physiopathology of Oncogenic Viruses Unit at the Pasteur Institute, also recently found a subtype of HTLV-3 in a human, but it’s somewhat different from the subtype Switzer and his colleagues found, which suggests “another example of multiple independent, cross-species transmission events,” Switzer says. The HTLV-3 strain Gessain found is extremely similar to a strain reported in the red-capped mangabey, which suggests that it crossed to humans very recently, Switzer says. Gessain’s findings were published 9 May 2005 in The current dogma surrounding retroviruses is that cross-species transmission is rare, but finding so many near-identical strains between humans and nonhuman primates suggests this is not a rare event. Benign retroviruses probably cross from nonhuman primates to humans frequently, but we don’t notice them because we don’t get sick, says Bernard Poiesz, a professor of medicine at SUNY Upstate Medical University. “But every once in a while,” he says, “one of them will jump and we may not handle it so well.”