Search

The human T cell leukemia virus type 1 (HTVL-1), first reported in 1980 by Robert Gallo’s group, is the etiologic agent of both cancer and inflammatory diseases. Despite approximately 40 years of investigation, the prognosis for afflicted patients remains poor with no effective treatments. The virus persists in the infected host by evading the host immune response and inducing proliferation of infected CD4+T-cells. Here, we will review the role that viralorf-Iprotein products play in altering intracellular signaling, protein expression and cell–cell communication in order to escape immune recognition and promote T-cell proliferation. We will also review studies oforf-Imutations found in infected patients and their potential impact on viral load, transmission and persistence. Finally, we will compare theorf-Igene in HTLV-1 subtypes as well as related STLV-1.

The extraordinarily high prevalence of HTLV-1 subtype C (HTLV-1C) in some isolated indigenous communities in Oceania and the severity of the health conditions associated with the virus impress the great need for basic and translational research to prevent and treat HTLV-1 infection. The genome of the virus’s most common subtype, HTLV-1A, encodes structural, enzymatic, and regulatory proteins that contribute to viral persistence and pathogenesis. Among these is the p30 protein encoded by the doubly splicedTax-orf IImRNA, a nuclear/nucleolar protein with both transcriptional and post-transcriptional activity. The p30 protein inhibits the productive replication cycle via nuclear retention of the mRNA that encodes for both the viral transcriptional trans-activator Tax, and the Rex proteins that regulate the transport of incompletely spliced viral mRNA to the cytoplasm. In myeloid cells, p30 inhibits the PU-1 transcription factor that regulates interferon expression and is a critical mediator of innate and adaptive immunity. Furthermore, p30 alters gene expression, cell cycle progression, and DNA damage responses in T-cells, raising the hypothesis that p30 may directly contribute to T cell transformation. By fine-tuning viral expression while also inhibiting host innate responses, p30 is likely essential for viral infection and persistence. This concept is supported by the finding that macaques, a natural host for the closely genetically related simian T-cell leukemia virus 1 (STLV-1), exposed to an HTLV-1 knockout for p30 expression by a single point mutation do not became infected unless reversion and selection of the wild type HTLV-1 genotype occurs. All together, these data suggest that inhibition of p30 may help to curb and eventually eradicate viral infection by exposing infected cells to an effective host immune response.

During human T-cell leukemia virus type 1 (HTLV-1) infection, the frequency of cells harboring an integrated copy of viral cDNA, the proviral load (PVL), is the main risk factor for progression of HTLV-1-associated diseases. Accurate quantification of provirus by droplet digital PCR (ddPCR) is a powerful diagnostic tool with emerging uses for monitoring viral expression. Current ddPCR techniques quantify HTLV-1 PVL in terms of whole genomic cellular material, while the main targets of HTLV-1 infection are CD4(+) and CD8(+) T cells. Our understanding of HTLV-1 proliferation and the amount of viral burden present in different compartments is limited. Recently a sensitive ddPCR assay was applied to quantifying T cells by measuring loss of germ line T-cell receptor genes as method of distinguishing non-T-cell from recombined T-cell DNA. In this study, we demonstrated and validated novel applications of the duplex ddPCR assay to quantify T cells from various sources of human genomic DNA (gDNA) extracted from frozen material (peripheral blood mononuclear cells [PBMCs], bronchoalveolar lavage fluid, and induced sputum) from a cohort of remote Indigenous Australians and then compared the T-cell measurements by ddPCR to the prevailing standard method of flow cytometry. The HTLV-1 subtype c (HTLV-1c) PVL was then calculated in terms of extracted T-cell gDNA from various compartments. Because HTLV-1c preferentially infects CD4(+) T cells, and the amount of viral burden correlates with HTLV-1c disease pathogenesis, application of this ddPCR assay to accurately measure HTLV-1c-infected T cells can be of greater importance for clinical diagnostics and prognostics as well as monitoring therapeutic applications.

Background Human T-cell Lymphotropic Virus type-1 (HTLV-1) infects an estimated 20 million people worldwide causing a fatal T- cell leukaemia and inflammatory conditions, such as HTLV-1-associated myelopathy, resulting from a dysregulated inflammatory response to the virus. HTLV-1 is highly endemic to remote indigenous communities in Central Australia where infection contributes to racial disparities in morbidity and mortality. HTLV-1 related complications are associated with elevated numbers of T-cells in which the HTLV-1 provirus integrates (HTLV-1 proviral load, PVL). Digital-droplet PCR (ddPCR) is capable of absolute quantification of PVL, offering low variability between assays with few cell numbers. We measured HTLV-1 PVLs from buffy coat cells (BCCs) of infected individuals, and bronchoalveolar lavage (BALs) infiltrates from a bronchiectasis patient to determine the range of HTLV-1 copies/cell relative to illness. Methods All samples were from Alice Springs Hospital HTLV-1 cohort (8 BCCs, 1 BAL) and obtained following ethics approval and patient consent in first language. Genomic DNA was extracted from BCCs for ddPCR PVL quantification. HTLV-1 gag/tax primers and FAM/MGB probes were optimised using temperature gradient amplification. Samples were tested in duplicate relative to an internal reference gene standard, RPP30, or negative threshold controls that used VIC/MGB probe. QuantaSoft software was used for data analysis. Results Of the 8 samples from BCCs, the PVL fell between 320 – 8,040 proviral copies per 10 6 cells. The BAL sample PVL was 1,110 DNA copies per 10 6 cells. The sensitivity (or limit of detection) of our ddPCR assay is 2 copies of proviral DNA per 10 4 cells. Conclusion The ddPCR assay demonstrates extremely high sensitivity, low assay-variability and the capability to reliably quantify HTLV-1 PVL. This technique offers logistic advantages in studying relationships between PVL in HTLV-1 patient’s BCCs and BAL. Disclosure of interest statement No Conflicts of interest. ISSTDR and IUSTI recognise the considerable contribution that industry partners make to professional and research activities. We also recognise the need for transparency of disclosure of potential conflicts of interest by acknowledging these relationships in publications and presentations. No commercial contributions have been received for this research project.