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Sample images from the dataset analysed in the report : "Single-cell heterogeneity and cell-cycle-related viral gene bursts in the human leukaemia virus HTLV-1" Complete dataset of raw images can be obtained from Charles Bangham on request. c.bangham@imperial.ac.uk Filename Format: Date - Coverslip Number - Clone_Replicate - Different-Probes-Used - Image Number .tif Channel 1 (Blue) - DAPI / nuclei Channel 2 (Green) - gag probe / autofluorescence Channel 3 (Yellow) - hbz probe / autofluorescence Channel 4 (Red) - tax probe

Additional file 1: Table S1. Toxicity.

Adult T‐cell leukemia/lymphoma (ATL), a human T‐lymphotropic virus type 1 (HTLV‐1)‐associated disease, has a highly variable clinical course and four subtypes with therapeutic and prognostic implications. However, there are overlapping features between ATL subtypes and between ATL and nonmalignant (non‐ATL) HTLV‐1 infection complicating diagnosis and prognostication. To further refine the diagnosis and prognosis of ATL, we characterized the immunophenotype of HTLV‐1‐infected cells in ATL and non‐ATL. A retrospective study of peripheral blood samples from 10 HTLV‐1‐uninfected subjects (UI), 54 HTLV‐1‐infected patients with non‐ATL, and 22 with ATL was performed using flow cytometry. All patients with ATL had CD4+ CCR4+ CD26− immunophenotype and the frequency of CD4+ CCR4+ CD26− T cells correlated highly significantly with the proviral load in non‐ATL suggesting CD4+ CCR4+ CD26− as a marker of HTLV‐1‐infected cells. Further immunophenotyping of CD4+ CCR4+ CD26− cells revealed that 95% patients with ATL had a CD7− (≤30% CD7+ cells), whereas 95% HTLV+ non‐ATL had CD7+ (>30% CD7+ cells) immunophenotype. All patients with aggressive ATL had a CCR7+ (≥30%), whereas 92% with indolent ATL and 100% non‐ATL had a CCR7− (

Human T-cell lymphotropic virus type 1 (HTLV-1) and hepatitis B virus coinfection is frequent in certain Indigenous Australian populations, but its outcome remains unknown. We report a higher degree of HTLV-1 clonal expansion than in those with HTLV-1 alone., The prevalence of human T-cell lymphotropic virus type 1 (HTLV-1) and hepatitis B virus (HBV) coinfection is high in certain Indigenous Australian populations, but its impact on HTLV-1 has not been described. We compared 2 groups of Indigenous adults infected with HTLV-1, either alone or coinfected with HBV. The 2 groups had a similar HTLV-1 proviral load, but there was a significant increase in clonal expansion of HTLV-1–infected lymphocytes in coinfected asymptomatic individuals. The degree of clonal expansion was correlated with the titer of HBV surface antigen. We conclude that HTLV-1/HBV coinfection may predispose to HTLV-1–associated malignant disease.

Summary The human retrovirus HTLV-1 causes a hematological malignancy or neuroinflammatory disease in ∼10% of infected individuals. HTLV-1 primarily infects CD4+ T lymphocytes and persists as a provirus integrated in their genome. HTLV-1 appears transcriptionally latent in freshly isolated cells; however, the chronically active anti-HTLV-1 cytotoxic T cell response observed in infected individuals indicates frequent proviral expression in vivo. The kinetics and regulation of HTLV-1 proviral expression in vivo are poorly understood. By using hypoxia, small-molecule hypoxia mimics, and inhibitors of specific metabolic pathways, we show that physiologically relevant levels of hypoxia, as routinely encountered by circulating T cells in the lymphoid organs and bone marrow, significantly enhance HTLV-1 reactivation from latency. Furthermore, culturing naturally infected CD4+ T cells in glucose-free medium or chemical inhibition of glycolysis or the mitochondrial electron transport chain strongly suppresses HTLV-1 plus-strand transcription. We conclude that glucose metabolism and oxygen tension regulate HTLV-1 proviral latency and reactivation in vivo., Graphical Abstract, Highlights • Physiological (1%) hypoxia enhances HTLV-1 plus-strand transcription • HTLV-1 transcription is hypoxia regulated but HIF independent • Inhibition of glycolysis or the mitochondrial ETC suppresses HTLV-1 transcription • Extracellular glucose concentration regulates HTLV-1 reactivation from latency, The human leukemia virus HTLV-1 remains dormant most of the time in the infected person, but is intermittently reactivated by unknown mechanisms. Kulkarni et al. show that fluctuations in glucose metabolism and oxygen availability are two major factors that govern the reactivation of HTLV-1 from dormancy.

International audience; HTLV-1 causes Adult T cell Leukemia/Lymphoma (ATLL) in humans. We describe an ATL-like disease in a 9 year-old female baboon naturally infected with STLV-1 (the simian counterpart of HTLV-1), with a lymphocyte count over 10(10)/L, lymphocytes with abnormal nuclear morphology, and pulmonary and skin lesions. The animal was treated with a combination of AZT and alpha interferon. Proviral load (PVL) was measured every week. Because the disease continued to progress, the animal was euthanized. Abnormal infiltrates of CD3(+)CD25(+) lymphocytes and Tax-positive cells were found by histological analyses in both lymphoid and non-lymphoid organs. PVL was measured and clonal diversity was assessed by LM-PCR (Ligation-Mediated Polymerase Chain Reaction) and high throughput sequencing, in blood during treatment and in 14 different organs. The highest PVL was found in lymph nodes, spleen and lungs. One major clone and a number of intermediate abundance clones were present in blood throughout the course of treatment, and in organs. These results represent the first multi-organ clonality study in ATLL. We demonstrate a previously undescribed clonal complexity in ATLL. Our data reinforce the usefulness of natural STLV-1 infection as a model of ATLL.

Even though an estimated 10–20 million people worldwide are infected with the oncogenic retrovirus, human T-lymphotropic virus type 1 (HTLV-1), its epidemiology is poorly understood, and little effort has been made to reduce its prevalence. In response to this situation, the Global Virus Network launched a taskforce in 2014 to develop new methods of prevention and treatment of HTLV-1 infection and promote basic research. HTLV-1 is the etiological agent of two life-threatening diseases, adult T-cell leukemia and HTLV-associated myelopathy/tropical spastic paraparesis, for which no effective therapy is currently available. Although the modes of transmission of HTLV-1 resemble those of the more familiar HIV-1, routine diagnostic methods are generally unavailable to support the prevention of new infections. In the present article, the Taskforce proposes a series of actions to expand epidemiological studies; increase research on mechanisms of HTLV-1 persistence, replication and pathogenesis; discover effective treatments; and develop prophylactic and therapeutic vaccines.

Erythrocyte count, total blood haemoglobin and haematocrit levels at the first and fifth clinical visit.

Protein peaks associated with brain atrophy: identification by liquid chromatography-electrospray ionization tandem mass spectrometry Relative molecular mass, protein score and identity of the genes with sequence matches to peptide fragments from the protein peaks that were significantly correlated with the rate of brain atrophy. The common contaminant keratin and partial matches to contaminating bacterial sequences were excluded.

Raw normalised data from SELDI-TOF at low range following Expression Difference Mapping (ProteinChip Data Manager. Bio-Rad). Columns contain sample name, sample group (corresponds to longitudinal measurements for each patients at 1,3,4 and 5), peak number (all peaks detected at low range at each time point), peak intensity and mass/charge ratio.

Human T cell lymphotropic virus-1 (HTLV-1) primarily infects CD4+ T cells, causing inflammatory disorders or a T cell malignancy in 5% to 10% of carriers. The cytotoxic T lymphocyte (CTL) response is a key factor that controls the viral load and thus the risk of disease. The ability to detect the viral protein Tax in primary cells has made it possible to estimate the rate at which Tax-expressing infected cells are eliminated by CTLs in persistently infected people. However, most HTLV-1-infected cells are Tax– at a given time, and their immunophenotype is poorly defined. Here, we aimed to identify a cell-surface molecule expressed by both Tax+ and Tax– HTLV-1-infected cells and use it to analyse the CTL response in fresh peripheral blood mononuclear cells. Cell adhesion molecule 1 (CADM1/ TSLC1) was the best single marker of HTLV-1 infection, identifying HTLV-1-infected cells with greater sensitivity and specificity than CD25, CCR4 or ICAM-1. CADM1+ CD4+ T cells carried a median of 65% of proviral copies in peripheral blood. In a cohort of 23 individuals, we quantified the rate of CTL-mediated killing of Tax+ and Tax− CADM1+ cells. We show that CADM1 expression is associated with enhanced susceptibility of infected cells to CTL lysis: despite the immunodominance of Tax in the CTL response, Tax+ CADM1– cells were inefficiently lysed by CTLs. Upregulation of the CADM1 ligand CRTAM on CD8+ T cells correlated with efficient lysis of infected cells. Tax– CADM1+ cells were lysed at a very low rate by autologous CTLs, however, were efficiently killed when loaded with exogenous peptide antigen. High expression of CADM1 on most HTLV-1-infected cells in the face of enhanced CTL counterselection implies that CADM1 confers a strong benefit on the virus., 論文

Background: Human T-lymphotropic virus type 1 (HTLV-1) infects an estimated 10 million persons globally with transmission resulting in lifelong infection. Disease, linked to high proviral load, occurs in a minority. In established infection HTLV-1 replicates through infectious spread and clonal expansion of infected lymphocytes. Little is known about acute HTLV-1 infection. The kinetics of early HTLV-1 infection, following transplantation-acquired infection in three recipients from one HTLV-1 infected donor, is reported. The recipients were treated with two HTLV-1 enzyme inhibitors 3 weeks post exposure following the detection of HTLV-1 provirus at low level in each recipient. HTLV-1 infection was serially monitored by serology, quantification of proviral load and HTLV-1 2LTR DNA circles and by HTLV-1 unique integration site analysis. Results: HTLV-1 antibodies were first detected 16–39 days post-transplantation. HTLV-1 provirus was detected by PCR on day 16–23 and increased by 2–3 log by day 38–45 with a peak proviral doubling time of 1.4 days, after which steady state was reached. The rapid proviral load expansion was associated with high frequency of HTLV-1 2LTR DNA circles. The number of HTLV-1 unique integration sites was high compared with established HTLV-1 infection. Clonal expansion of infected cells was detected as early as day 37 with high initial oligoclonality index, consistent with early mitotic proliferation. Conclusions: In recipients infected through organ transplantation HTLV-1 disseminated rapidly despite early antiHTLV-1 treatment. Proviral load set point was reached within 6 weeks. Seroconversion was not delayed. Unique integration site analysis and HTLV-1 2LTR DNA circles indicated early clonal expansion and high rate of infectious spread.

Background Human T-lymphotropic virus type 1 (HTLV-1) infects an estimated 10 million persons globally with transmission resulting in lifelong infection. Disease, linked to high proviral load, occurs in a minority. In established infection HTLV-1 replicates through infectious spread and clonal expansion of infected lymphocytes. Little is known about acute HTLV-1 infection. The kinetics of early HTLV-1 infection, following transplantation-acquired infection in three recipients from one HTLV-1 infected donor, is reported. The recipients were treated with two HTLV-1 enzyme inhibitors 3 weeks post exposure following the detection of HTLV-1 provirus at low level in each recipient. HTLV-1 infection was serially monitored by serology, quantification of proviral load and HTLV-1 2LTR DNA circles and by HTLV-1 unique integration site analysis. Results HTLV-1 antibodies were first detected 16–39 days post-transplantation. HTLV-1 provirus was detected by PCR on day 16–23 and increased by 2–3 log by day 38–45 with a peak proviral doubling time of 1.4 days, after which steady state was reached. The rapid proviral load expansion was associated with high frequency of HTLV-1 2LTR DNA circles. The number of HTLV-1 unique integration sites was high compared with established HTLV-1 infection. Clonal expansion of infected cells was detected as early as day 37 with high initial oligoclonality index, consistent with early mitotic proliferation. Conclusions In recipients infected through organ transplantation HTLV-1 disseminated rapidly despite early anti-HTLV-1 treatment. Proviral load set point was reached within 6 weeks. Seroconversion was not delayed. Unique integration site analysis and HTLV-1 2LTR DNA circles indicated early clonal expansion and high rate of infectious spread.

Human T-lymphotropic virus type 1 (HTLV-1) and type 2 (HTLV-2) both cause lifelong persistent infections, but differ in their clinical outcomes. HTLV-1 infection causes a chronic or acute T-lymphocytic malignancy in up to 5% of infected individuals whereas HTLV-2 has not been unequivocally linked to a T-cell malignancy. Virus-driven clonal proliferation of infected cells both in vitro and in vivo has been demonstrated in HTLV-1 infection. However, T-cell clonality in HTLV-2 infection has not been rigorously characterized. In this study we used a high-throughput approach in conjunction with flow cytometric sorting to identify and quantify HTLV-2-infected T-cell clones in 28 individuals with natural infection. We show that while genome-wide integration site preferences in vivo were similar to those found in HTLV-1 infection, expansion of HTLV-2-infected clones did not demonstrate the same significant association with the genomic environment of the integrated provirus. The proviral load in HTLV-2 is almost confined to CD8+ T-cells and is composed of a small number of often highly expanded clones. The HTLV-2 load correlated significantly with the degree of dispersion of the clone frequency distribution, which was highly stable over ∼8 years. These results suggest that there are significant differences in the selection forces that control the clonal expansion of virus-infected cells in HTLV-1 and HTLV-2 infection. In addition, our data demonstrate that strong virus-driven proliferation per se does not predispose to malignant transformation in oncoretroviral infections., Author Summary The two human retroviruses HTLV-1 and HTLV-2 are similar in their structure, replication cycle and the manner through which they spread between and within individuals. They differ in their preferred host T-cell type and in their possible clinical outcomes. HTLV-2 has not been linked with a specific disease, whereas HTLV-1 infection can cause leukemia and profound neuropathology. It is well established that HTLV-1-infected cells undergo clonal expansion in infected individuals, but little is known about clonality in HTLV-2 infection. In this work, we demonstrate that the extent of HTLV-2-infected cell expansion significantly exceeds that of HTLV-1-infected cells in healthy carriers, approximating instead to that observed in patients with HTLV-1-associated leukemia. Furthermore, we show that HTLV-2 characteristically resides in a small number of expanded clones that persist over time, and that the degree of oligoclonality significantly correlates with viral burden in HTLV-2-infected individuals. These results highlight the distinction between in vivo clonal proliferation and malignant transformation, and suggest that the infected cell type may be a more important determinant of clinical outcome in retroviral infections.

p. 1-11 Submitted by Edileide Reis (leyde-landy@hotmail.com) on 2014-12-03T11:52:55Z No. of bitstreams: 1 Nicolas A. Gillet.pdf: 1059336 bytes, checksum: 232799195c15409c16eb6fad2b1d9470 (MD5) Approved for entry into archive by Delba Rosa (delba@ufba.br) on 2015-06-11T19:05:26Z (GMT) No. of bitstreams: 1 Nicolas A. Gillet.pdf: 1059336 bytes, checksum: 232799195c15409c16eb6fad2b1d9470 (MD5) Made available in DSpace on 2015-06-11T19:05:26Z (GMT). No. of bitstreams: 1 Nicolas A. Gillet.pdf: 1059336 bytes, checksum: 232799195c15409c16eb6fad2b1d9470 (MD5) Previous issue date: 2013 Human T-lymphotropic Virus-1 (HTLV-1) is a retrovirus that persists lifelong by driving clonal proliferation of infected T-cells. HTLV-1 causes a neuroinflammatory disease and adult T-cell leukemia/lymphoma. Strongyloidiasis, a gastrointestinal infection by the helminth Strongyloides stercoralis, and Infective Dermatitis associated with HTLV-1 (IDH), appear to be risk factors for the development of HTLV-1 related diseases. We used high-throughput sequencing to map and quantify the insertion sites of the provirus in order to monitor the clonality of the HTLV-1-infected T-cell population (i.e. the number of distinct clones and abundance of each clone). A newly developed biodiversity estimator called “DivE” was used to estimate the total number of clones in the blood. We found that the major determinant of proviral load in all subjects without leukemia/lymphoma was the total number of HTLV-1-infected clones. Nevertheless, the significantly higher proviral load in patients with strongyloidiasis or IDH was due to an increase in the mean clone abundance, not to an increase in the number of infected clones. These patients appear to be less capable of restricting clone abundance than those with HTLV-1 alone. In patients co-infected with Strongyloides there was an increased degree of oligoclonal expansion and a higher rate of turnover (i.e. appearance and disappearance) of HTLV-1-infected clones. In Strongyloides co-infected patients and those with IDH, proliferation of the most abundant HTLV-1+ T-cell clones is independent of the genomic environment of the provirus, in sharp contrast to patients with HTLV-1 infection alone. This implies that new selection forces are driving oligoclonal proliferation in Strongyloides co-infection and IDH. We conclude that strongyloidiasis and IDH increase the risk of development of HTLV-1-associated diseases by increasing the rate of infection of new clones and the abundance of existing HTLV-1+ clones.

Human T-cell leukemia virus type-1 (HTLV-1) is a replication-competent human retrovirus associated with two distinct types of disease only in a minority of infected individuals: the malignancy known as adult T-cell leukemia (ATL) and a chronic inflammatory central nervous system disease HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). HAM/TSP is a chronic progressive myelopathy characterized by spastic paraparesis, sphincter dysfunction, and mild sensory disturbance in the lower extremities. Although the factors that cause these different manifestations of HTLV-1 infection are not fully understood, accumulating evidence from host population genetics, viral genetics, DNA expression microarrays, and assays of lymphocyte function suggests that complex virus-host interactions and the host immune response play an important role in the pathogenesis of HAM/TSP. Especially, the efficiency of an individual's cytotoxic T-cell (CTL) response to HTLV-1 limits the HTLV-1 proviral load and the risk of HAM/TSP. This paper focuses on the recent advances in HAM/TSP research with the aim to identify the precise mechanisms of disease, in order to develop effective treatment and prevention.

ATLL is an aggressive malignancy of T cells that affects about 5% of individuals infected with HTLV-1. The precise mechanism of oncogenesis is not known, but there is evidence that two regulatory viral proteins, Tax and HBZ, are involved. A high set point proviral load is associated with development of ATLL or a chronic inflammatory condition, HAM/TSP. Several lines of evidence, including HLA class 1 association studies and in vitro killing assays, indicate that cytotoxic T lymphocytes are instrumental in determining this proviral load set point. Prior studies have focused chiefly on the CTL response to the immunodominant Tax protein: efficient lysis of Tax-expressing cells inversely correlates with proviral load in nonmalignant infection. However, a recent study showed that strong binding of peptides from HBZ, but not Tax, to HLA class 1 molecules was associated with a low proviral load and a reduced risk of developing HAM/TSP, indicating an important role for HBZ-specific CTL in determining infection outcome. In comparison with nonmalignant infection, HTLV-1-specific CTLs in ATLL patients are reduced in frequency and functionally deficient. Here we discuss the nature of protective CTL responses in nonmalignant HTLV-1 infection and explore the potential of CTLs to protect against ATLL.

Additional file 2. Clonality analysisâ additional data tables.

Additional file 2. Clonality analysisâ additional data tables.

Background Immunogenetic evidence indicates that cytotoxic T lymphocytes (CTLs) specific for the weak CTL antigen HBZ limit HTLV-1 proviral load in vivo, whereas there is no clear relationship between the proviral load and the frequency of CTLs specific for the immunodominant antigen Tax. In vivo, circulating HTLV-1-infected cells express HBZ mRNA in contrast, Tax expression is typically low or undetectable. To elucidate the virus-suppressing potential of CTLs targeting HBZ, we compared the ability of HBZ- and Tax-specific CTLs to lyse naturally-infected cells, by co-incubating HBZ- and Tax-specific CTL clones with primary CD4+ T cells from HLA-matched HTLV-1-infected donors. We quantified lysis of infected cells, and tested whether specific virus-induced host cell surface molecules determine the susceptibility of infected cells to CTL-mediated lysis. Results Primary infected cells upregulated HLA-A*02, ICAM-1, Fas and TRAIL-R1/2 in concert with Tax expression, forming efficient targets for both HTLV-1-specific CTLs and CTLs specific for an unrelated virus. We detected expression of HBZ mRNA (spliced isoform) in both Tax-expressing and non-expressing infected cells, and the HBZ26–34 epitope was processed and presented by cells transfected with an HBZ expression plasmid. However, when coincubated with primary cells, a high-avidity HBZ-specific CTL clone killed significantly fewer infected cells than were killed by a Tax-specific CTL clone. Finally, incubation with Tax- or HBZ-specific CTLs resulted in a significant decrease in the frequency of cells expressing high levels of HLA-A*02. Conclusions HTLV-1 gene expression in primary CD4+ T cells non-specifically increases susceptibility to CTL lysis. Despite the presence of HBZ spliced-isoform mRNA, HBZ epitope presentation by primary cells is significantly less efficient than that of Tax. Electronic supplementary material The online version of this article (doi:10.1186/s12977-014-0116-6) contains supplementary material, which is available to authorized users.

Human T-lymphotropic virus type-1 (HTLV-1) persists within hosts via infectious spread (de novoinfection) and mitotic spread (infected cell proliferation), creating a population structure of multiple clones (infected cell populations with identical genomic proviral integration sites). The relative contributions of infectious and mitotic spread to HTLV-1 persistence are unknown, and will determine the efficacy of different approaches to treatment. The prevailing view is that infectious spread is negligible in HTLV-1 persistence beyond early infection. However, in light of recent high-throughput data on the abundance of HTLV-1 clones, and recent estimates of HTLV-1 clonal diversity that are substantially higher than previously thought (typically between 10(4)and 10(5)HTLV-1(+)T cell clones in the body of an asymptomatic carrier or patient with HTLV-1-associated myelopathy/tropical spastic paraparesis), ongoing infectious spread during chronic infection remains possible. We estimate the ratio of infectious to mitotic spread using a hybrid model of deterministic and stochastic processes, fitted to previously published HTLV-1 clonal diversity estimates. We investigate the robustness of our estimates using three alternative estimators. We find that, contrary to previous belief, infectious spread persists during chronic infection, even after HTLV-1 proviral load has reached its set point, and we estimate that between 100 and 200 new HTLV-1 clones are created and killed every day. We find broad agreement between all estimators. The risk of HTLV-1-associated malignancy and inflammatory disease is strongly correlated with proviral load, which in turn is correlated with the number of HTLV-1-infected clones, which are created by de novo infection. Our results therefore imply that suppression of de novo infection may reduce the risk of malignant transformation.

Figure S1. Effect of idelalisib on the frozen and thawed samples of ATL patients. Figure S2. The apoptotic ATL cells by treatment with idelalisib. Figure S3. The viability of non-ATL cells by treatment with idelalisib. Figure S4. The apoptotic non-ATL cells by treatment with idelalisib. (DOCX 38 kb)

Figure S1. Effect of idelalisib on the frozen and thawed samples of ATL patients. Figure S2. The apoptotic ATL cells by treatment with idelalisib. Figure S3. The viability of non-ATL cells by treatment with idelalisib. Figure S4. The apoptotic non-ATL cells by treatment with idelalisib. (DOCX 38 kb)

The lungs are the primary target of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, with severe hypoxia being the cause of death in the most critical cases. Coronavirus disease 2019 (COVID-19) is extremely heterogeneous in terms of severity, clinical phenotype and, importantly, global distribution. Although the majority of affected patients recover from the acute infection, many continue to suffer from late sequelae affecting various organs, including the lungs. The role of the pulmonary vascular system during the acute and chronic stages of COVID-19 has not been adequately studied. A thorough understanding of the origins and dynamic behaviour of the SARS-CoV-2 virus and the potential causes of heterogeneity in COVID-19 is essential for anticipating and treating the disease, in both the acute and the chronic stages, including the development of chronic pulmonary hypertension. Both COVID-19 and chronic pulmonary hypertension have assumed global dimensions, with potential complex interactions. In this Review, we present an update on the origins and behaviour of the SARS-CoV-2 virus and discuss the potential causes of the heterogeneity of COVID-19. In addition, we summarize the pathobiology of COVID-19, with an emphasis on the role of the pulmonary vasculature, both in the acute stage and in terms of the potential for developing chronic pulmonary hypertension. We hope that the information presented in this Review will help in the development of strategies for the prevention and treatment of the continuing COVID-19 pandemic., In this Review, the authors discuss the potential causes of the heterogeneity of COVID-19 and summarize the pathobiology of the disease, with an emphasis on the role of the pulmonary vasculature in the acute stage and the potential for developing chronic pulmonary hypertension., Key points A thorough understanding of the dynamic behaviour of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is essential to understanding its heterogeneous effects on the pulmonary vasculature in patients with coronavirus disease 2019 (COVID-19).The severity and clinical phenotype of COVID-19 are influenced by host factors, including socioeconomic factors and genetics.Silent hypoxia is a major and independent cause of lung damage in COVID-19; the use of modern imaging techniques is proving to be very valuable in identifying silent hypoxia.The pulmonary vascular system has a major role in the pathobiology of COVID-19.Both COVID-19 and chronic pulmonary hypertension are global diseases with a complex interaction.

Restriction factors are cellular proteins that constitute one of the defence mechanisms developed by the host to fight viral infections. Friend virus susceptibility-1 (Fv1) is the prototypic restriction factor known for blocking murine leukemia virus (MLV) replication by targeting the viral capsid protein. This gene was found to be highly polymorphic between murine species as well as being under strong positive selection and having different restriction specificities. The origin of Fv1 remains unclear, however, its acquisition is known to have happened before the emergence of MLVs. It is, therefore, reasonable to speculate that non-MLV retroviruses have been involved in shaping Fv1 evolution. The aim of this PhD project was to further understand how Fv1 evolves and interacts with retroviruses. A number of retroviruses from different genera were tested against a panel of Fv1s from different wild mice. Novel restriction activities were found for the gammaretroviruses Gibbon Ape Leukemia Virus and Feline Leukemia Virus as well as for two newly discovered endogenous retroviruses. More interestingly, the alpharetrovirus Rous Sarcoma Virus and the betaretrovirus Mouse Mammary Tumour Virus could be restricted by two Fv1s. These Fv1s showed no sign of gammaretrovirus restriction. Mapping studies pointed to the Fv1 C-terminal domain variable regions VA and VD as the interacting domains involved in these new specificities. Fine mapping revealed that single residue determinants were different for each virus. It was also shown that loss of activity is possible with very limited changes. To investigate the stage of the life cycle blocked for these viruses, qPCR assays were performed in Fv1 transduced cells to detect late reverse transcription and nuclear entry. The results confirmed that infection is blocked at the post-entry level after reverse transcription and before integration, as previously observed with MLV. Taken together with previous published data, these findings support the hypothesis that Fv1 is a very plastic system and that it might have evolved in response to non-gamma retroviruses from different genera that would have predated MLVs. Open Access

The human leukaemia virus HTLV-1 expresses essential accessory genes that manipulate the expression, splicing and transport of viral mRNAs. Two of these genes, tax and hbz, also promote proliferation of the infected cell, and both genes are thought to contribute to oncogenesis in adult T-cell leukaemia/lymphoma. The regulation of HTLV-1 proviral latency is not understood. tax, on the proviral plus strand, is usually silent in freshly-isolated cells, whereas the minus-strand-encoded hbz gene is persistently expressed at a low level. However, the persistently activated host immune response to Tax indicates frequent expression of tax in vivo. By using single-molecule RNA-FISH, I was able to quantify the expression of HTLV-1 plus-strand and minus-strand transcripts at the single-cell level in naturally-infected T-cell clones isolated from peripheral blood. While hbz is the lone transcript located on the HTLV-1 minus-strand, it is not possible to definitively separate tax transcripts from its neighbours on the plus-strand by means of single-molecule RNA-FISH. Detected plus-strand transcripts are referred to as tax transcripts throughout the thesis, but it is important to remember that the probe which targets the tax sequence will also detect the other plus-strand products, even if they are not thought to be expressed in the same volume as tax. Measuring viral transcription at the single-cell level revealed strong within-clone and between-clone heterogeneity in the expression of tax and hbz. Both tax and hbz are transcribed in bursts; tax expression is enhanced in the absence of hbz, while hbz expression increased in cells with high tax expression. Unexpectedly, hbz expression was found to be strongly associated with the G2/M phase of the cell cycle, independent of tax expression. There is also a link between a cell’s level of hbz expression and its volume. These results offer an explanation for the paradoxical correlations observed between the host immune response and HTLV-1 transcription. While hbz is the lone transcript located on the HTLV-1 minus-strand, it is not possible to definitively separate tax transcripts from its neighbours on the plus-strand by means of single-molecule RNA-FISH. Detected plus-strand transcripts are referred to as tax transcripts throughout the thesis, but it is important to remember that the probe which target the tax sequence will also detect the other plus-strand products, even if they are not thought to be expressed in the same volume as tax. Measuring viral transcription at the single-cell level revealed strong within-clone and between-clone heterogeneity in the expression of tax and hbz. Both tax and hbz are transcribed in bursts; tax expression is enhanced in the absence of hbz, while hbz expression increased in cells with high tax expression. Unexpectedly, hbz expression was found to be strongly associated with the G2/M phase of the cell cycle, independent of tax expression. There is also a link between a cell’s level of hbz expression and its volume. These results offer an explanation for the paradoxical correlations observed between the host immune response and HTLV-1 transcription. Open Access

Retroviruses must integrate a DNA copy of their genome into host cell chromatin to establish infection. The pre-integration complex of the retroviruses, HIV-1 and murine leukemia virus interact with a host co-factor through their integrase enzyme to preferentially integrate into particular sites of host cell chromatin. This results in a unique pattern of integration site distribution in the host genome for each retroviral genus. HTLV-1, a delta-retrovirus, has a well-defined preferential integration pattern that is important for supporting viral gene expression during chronic infection. So far, a deltaretroviral integrase host co-factor has not yet been identified, however, biochemical evidence suggested that the protein phosphatase 2A (PP2A) complex could fulfil this function. Here, I report the results of a Y2H screen which independently confirms that PP2A B’ regulatory subunits interact directly with HTLV-1 IN. Second, I show, using three different experimental approaches, that HTLV-1 integration is redistributed in cells that lack functional PP2A. This is the first evidence that a loss of PP2A activity in live cells leads to an altered integration site preference and strongly suggests that PP2A acts as an HTLV-1 IN co-factor. ShRNA-mediated knockdown of either of the two B’ isoforms most highly expressed in Jurkat T cells, B’ γ and B’ δ, indicates that B’ γ mediates the majority of IN co-factor function, since B’ γ depletion resulted in the most extensive redistribution of integration sites compared to cells treated with control shRNA. However, it is likely that other PP2A holoenzymes can also act as co-factors for HTLV-1 IN. PP2A is ubiquitously and highly expressed, making it an attractive target for co-factor function. Future analysis of PP2A’s co-factor function should aim to use HTLV-1 IN mutants to specifically disrupt the interaction between PP2A and HTLV-1 IN. Open Access

Human T-lymphotropic virus type 1 (HTLV-1) causes the debilitating neuroinflammatory disease HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) in a minority of infected individuals, while the majority are lifelong asymptomatic carriers (ACs). The effectiveness of the CD8+ T-cell response to HTLV-1 is a major determinant of the HTLV-1 proviral load (PVL) and the associated HAM/TSP risk. Host genotype, particularly of HLA class I, strongly influences CD8+ T-cell effectiveness against HTLV-1. Furthermore, possession of the KIR gene 2DL2 was recently shown to enhance protective and detrimental HLA class I-associated immunity to HTLV-1 and HCV (Seich al Basatena et al., 2011). The proposed mechanism explaining this effect is a KIR expression-induced increase in activation thresholds, thus promoting survival. I investigated KIR expression and function on T-cells and NK cells in HTLV-1 infection, focussing on CD8+ T-cells and using new anti-KIR mAbs to analyse 2DL2 expression alongside four other KIRs. This thesis presents evidence supporting the genetic findings and the KIR expression-induced CD8+ T-cell survival hypothesis. AC status was associated with expanded 2DL2+CD8+ T EM cell populations and 2DL2+ and 2DL3+CD8+ T EM cell frequencies were inversely correlated with PVL, consistent with a protective role for 2DL2 (and potentially 2DL3) expression on CD8+ T-cells in HTLV-1 infection. Rather than a generalised reduction in CD8+ T-cell function, specific KIR expression was associated with a shift in effector function profile towards a cytotoxic phenotype without cytokine production. This is consistent with raised activation thresholds and may suggest fine-tuning of CD8+ T-cell effector functions by KIRs, preserving killing without causing inflammation. I further speculate that 2DL2/3 could be a context-dependent modulator of CD8+ T-cell function, optimally tuning responses and contributing to CD8+ T-cell effectiveness. Together with the longevity of 2DL2/3+CD8+ T-cells in vivo, this hypothesis requires further testing, since it may be relevant to other persistent viral infections. Open Access

Infection with HIV-1 results in the progressive dysfunction of the immune system eventually leading to AIDS, characterised by low CD4+ T lymphocyte counts and increased susceptibility to opportunistic infections. In contrast to HIV-1, individuals infected with HIV-2 often remain asymptomatic, with lower viral loads and higher CD4+ T cell counts throughout the course of disease. Furthermore, HIV-2+ individuals display enhanced HIV-specific T cell responses. In addition, HIV-1 disease progression is slower in patients with pre-existing HIV-2 infection. Plasmacytoid dendritic cells (pDCs) are key mediators of the early innate immune response. Upon viral infection, pDCs secrete high levels of type I IFN (IFN-α/β) which limit viral replication and prime the adaptive immune response. Activated pDCs, in particular excessive IFN-α/β, have been implicated in HIV-1 immunopathogenesis. Specifically pDCs may contribute to the recruitment of target cells to the site of HIV-1 infection, increased apoptosis of immune cells and suppression of memory T cell responses. The aim of this study was to compare the abilities of HIV-1 and HIV-2 to activate pDCs in vitro. HIV-1 was a more potent inducer of type I IFN responses in PBMCs compared to HIV-2, measured at both the transcriptional level, and by measuring IFN-α/β secretion into cell culture supernatants. Furthermore, HIV-2, but not HIV-1, inhibited IFN-α production in response to synthetic stimuli. Phenotypic analysis of pDCs by flow cytometry revealed that both HIV-1 and HIV-2 were equally able to induce an up-regulation of co-stimulatory marker expression. Measurement of co-stimulatory molecule expression in conjunction with IFN-α secretion showed that HIV-1 favoured an IFN response, whereas HIV-2 preferentially maturated pDCs towards an antigen-presenting cell (APC) phenotype. The ability of HIV-2 to mature pDCs into APCs while reducing IFN-α secretion may be an important contributor to more robust T cell responses and therefore slower progressing HIV disease. Open Access

Human T-lymphotropic virus type-1 (HTLV-1) infects approximately 10-20 million people worldwide. The virus persists within hosts via de novo infection and infected cell proliferation, creating a population structure of multiple clones (infected cell populations with identical genomic proviral integration sites). The number of clones in one host is unknown, and is determined by the rate of de novo infection. Our primary objectives are: i) to estimate HTLV-1 clonal diversity; and ii) to develop a model of HTLV-1 dynamics that can estimate the relative contributions of de novo infection and mitotic replication. We use a combination of mathematical modelling, computer simulation and statistical methods to interpret experimental observation. We develop an estimator (named DivE) to estimate the number of HTLV-1 clones. DivE uses the ecological method of rarefaction, and includes novel model selection criteria. We show that DivE is more accurate than widely-used estimators from population ecology, and we demonstrated that this holds across many systems. Differences between these systems and ecological populations are investigated, and DivE is applied to patients with a range of HTLV-associated diseases. HTLV-1 clonal abundance varies by several orders of magnitude: quantifying within-host HTLV-1 dynamics requires mathematical modelling at multiple scales. Stochastic processes, important for modelling small populations, are introduced, and we explore properties and approximations of a mass-action birth-death process for biologically realistic species extinction scenarios. We combine ordinary differential equations with stochastic processes in a hybrid model and explore its consequences. The estimated HTLV-1 clonal diversity is substantially higher than previously thought, which strongly implies higher rates of de novo infection. The hybrid model captures known behaviour of HTLV-1, and can be used to infer rates of viral persistence. DivE and the hybrid model are applicable to other biological systems, in particular the study of T and B cell receptor repertoires. Open Access

Human T-lymphotropic virus type 1 (HTLV-1) is a retrovirus that persists lifelong within the infected host by driving expansion of infected CD4+T-cells. It is the cause of adult T-cell leukaemia/lymphoma (ATL), an aggressive CD4+ T-cell malignancy, which arises in approximately 5% of individuals typically following decades of asymptomatic infection. The reasons why some individuals develop ATL remain unknown. In this laboratory a novel customised high throughput sequencing and bioinformatic method has been developed in order to map and accurately quantify the proviral integration sites within each host genome in order to identify clonal populations within each host. In this study I aimed first to test the hypothesis that there is a single provirus integrated into each host genome, and secondly to test the hypothesis that the site of retroviral integration determines the risk of leukaemia. In order to quantify the average number of proviral integration sites in each host cell, we isolated infected T-cells from the peripheral blood of infected individuals by limiting dilution cloning. Integration site analysis of these clones revealed that in natural infection each T-cell clone carries a single integrated provirus. This work formed the basis of a publication in the journal Blood (Cook et al 2012). I describe the systematic analysis of the clonality, structure and the integrity of the proviral tax gene in a large cohort of ATL patients (n=197). I correlate these findings with the clinical subtype of ATL and the landscape of the host genome flanking the proviral integration site. Based upon our findings we conclude that the integration site in cis does not directly cause leukaemogenesis and hypothesise that the absolute number of infected clones within an individual, and not oligoclonal proliferation, predisposes to malignant transformation. Open Access

Human T-Lymphotropic virus type 1 (HTLV-1) maintains persistent infection in the host by driving expansion of HTLV-1-infected T cells. However, the wide variation observed in clonal abundance in vivo remains unexplained. We hypothesize that the site of proviral integration in the host cell genome determines the rate of expression of HTLV-1 genes such as the viral transactivator Tax, which in turn determine the level of clonal expansion in vivo. Utilising Molecular biology techniques, High-throughput sequencing and computing tools, we have developed a high-throughput method for the amplification, mapping and quantification of proviral integration sites. We have used this technique, in combination with bioinformatics analysis, to identify factors associated with clonal expansion. Using PBMCs sorted for CD4+ and CD8+ T cell subsets, we observed differences in the form of the clonal distribution between the cell types. In addition, using flow-cytometric sorting of Tax expressing CD4+ cells, we identified genomic patterns associated with the presence or absence of spontaneous Tax expression. We have also identified a correlation between the expression of Tax and the size of the infected clones. Finally, we identified genomic factors associated with HTLV-1 integration in vitro, suggesting a possible role of these factors in targeting of the pre-integration complex to particular sites in the host genome. The thesis will detail and discuss the findings from these analyses, as well as their impact on the current knowledge. These results would play a role in improving our understanding of HTLV-1 persistence in the face of a strong immune response, and may provide excellent basis from a population level to test mechanisms of proviral latency in natural infection. Open Access

associated myelopathy (HAM). The pathogenesis of HAM is not fully understood. However, considerable evidence points to the importance of the regulatory protein Tax. Tax promotes the proliferation of HTLV-1 infected cells but also induces a strong cytotoxic-T-lymphocyte response. The contribution of the HTLV-1 basic leucine zipper protein (HBZ), which promotes T cell proliferation but suppresses Tax mediated transactivation, remains to be elucidated. This thesis explores the following hypotheses:- 1. HBZ is expressed in vivo and will elicit an immune response in HTLV-1 infected subjects. 2. HBZ mRNA will be detected in peripheral blood mononuclear cells (PBMCs) from HTLV-1 infected subjects. To test these hypotheses 48 subjects: 14 patients with HAM, 28 asymptomatic carriers (ACs) (14 with high viral load >1% and 14 with low viral load

Human T-lymphotropic virus Type 1 (HTLV-1) is a retrovirus that persists lifelong in the host. In ~4% of infected people, HTLV-1 causes a chronic disabling neuroinflammatory disease known as HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). The pathogenesis of HAM/TSP is unknown and treatment remains ineffective. In this study we aimed to identify patterns in frequencies of peripheral leukocyte populations and blood gene expression profiles of HTLV-1 carriers that suggest new hypotheses as to the mechanisms of HTLV-1 persistence and HAM/TSP pathology. Flow cytometric immunophenotyping of peripheral blood leukocytes revealed abnormal activation and maturation profiles of effector T cells but not antigen-presenting cells. High frequencies of circulating granzyme and perforin-rich CD8+ T cells were associated with an increased probability of HAM/TSP. However, the cytolytic capacity of these T cells is not known as although they accumulated cytolytic proteins, granzyme mRNA levels were down-regulated in patients with HAM/TSP. Furthermore, presence of HAM/TSP was associated with an expansion of CD56-negative NK cells, which are thought to have decreased cytolytic functions. Blood gene expression profiles identified perturbations of the p53 signalling pathway as a hallmark of HTLV-1 infection. In contrast, a subset of interferon (IFN)-stimulated genes was over-expressed in patients with HAM/TSP but not in asymptomatic HTLV-1 carriers or patients with the clinically similar disease multiple sclerosis. The IFN-inducible signature was present in all circulating leukocytes and its intensity correlated with the clinical severity of HAM/TSP. Leukocytes from patients with HAM/TSP were primed to respond strongly to stimulation with exogenous IFN. However, while type I IFN suppressed expression of the HTLV-1 structural protein Gag it failed to suppress the highly immunogenic viral transcriptional transactivator Tax. Based on our findings we hypothesise that impaired NK cell and T cell-mediated immune responses result in high HTLV-1 proviral loads but that the over-expression of a subset of IFN-stimulated genes contributes to the development of HAM/TSP.