GWAS reveals determinants of mobilization rate and dynamics of an active endogenous retrovirus of cattle.

Five to ten percent of mammalian genomes is occupied by multiple clades of endogenous retroviruses (ERVs), that may count thousands of members. New ERV clades arise by retroviral infection of the germline followed by expansion by reinfection and/or retrotransposition. ERV mobilization is a source of deleterious variation, driving the emergence of ERV silencing mechanisms, leaving "DNA fossils". Here we show that the ERVK[2-1-LTR] clade is still active in the bovine and a source of disease-causing alleles. We develop a method to measure the rate of ERVK[2-1-LTR] mobilization, finding an average of 1 per ~150 sperm cells, with >10-fold difference between animals. We perform a genome-wide association study and identify eight loci affecting ERVK[2-1-LTR] mobilization. We provide evidence that polymorphic ERVK[2-1-LTR] elements in four of these loci cause the association. We generate a catalogue of full length ERVK[2-1-LTR] elements, and show that it comprises 15% of C-type autonomous elements, and 85% of D-type non-autonomous elements lacking functional genes. We show that >25% of the variance of mobilization rate is determined by the number of C-type elements, yet that de novo insertions are dominated by D-type elements. We propose that D-type elements act as parasite-of-parasite gene drives that may contribute to the observed demise of ERV elements.

Reconstruction of SARS-CoV-2 outbreaks in a primary school using epidemiological and genomic data.

Mathematical modelling studies have shown that repetitive screening can be used to mitigate SARS-CoV-2 transmission in primary schools while keeping schools open. However, not much is known about how transmission progresses within schools and whether there is a risk of importation to households. During the academic year 2020-2021, a prospective surveillance study using repetitive screening was conducted in a primary school and associated households in Liège (Belgium). SARS-CoV-2 screening was performed via throat washing either once or twice a week. We used genomic and epidemiological data to reconstruct the observed school outbreaks using two different models. The outbreaker2 model combines information on the generation time and contact patterns with a model of sequence evolution. For comparison we also used SCOTTI, a phylogenetic model based on the structured coalescent. In addition, we performed a simulation study to investigate how the accuracy of estimated positivity rates in a school depends on the proportion of a school that is sampled in a repetitive screening strategy. We found no difference in SARS-CoV-2 positivity between children and adults and children were not more often asymptomatic compared to adults. Both models for outbreak reconstruction revealed that transmission occurred mainly within the school environment. Uncertainty in outbreak reconstruction was lowest when including genomic as well as epidemiological data. We found that observed weekly positivity rates are a good approximation to the true weekly positivity rate, especially in children, even when only 25% of the school population is sampled. These results indicate that, in addition to reducing infections as shown in modelling studies, repetitive screening in school settings can lead to a better understanding of the extent of transmission in schools during a pandemic and importation risk at the community level.

Evaluation of Screening Program and Phylogenetic Analysis of SARS-CoV-2 Infections among Hospital Healthcare Workers in Liège, Belgium.

Healthcare workers (HCWs) are known to be at higher risk of developing severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections although whether these risks are equal across all occupational roles is uncertain. Identifying these risk factors and understand SARS-CoV-2 transmission pathways in healthcare settings are of high importance to achieve optimal protection measures. We aimed to investigate the implementation of a voluntary screening program for SARS-CoV-2 infections among hospital HCWs and to elucidate potential transmission pathways though phylogenetic analysis before the vaccination era. HCWs of the University Hospital of Liège, Belgium, were invited to participate in voluntary reverse transcriptase-polymerase chain reaction (RT-PCR) assays performed every week from April to December 2020. Phylogenetic analysis of SARS-CoV-2 genomes were performed for a subgroup of 45 HCWs. 5095 samples were collected from 703 HCWs. 212 test results were positive, 15 were indeterminate, and 4868 returned negative. 156 HCWs (22.2%) tested positive at least once during the study period. All SARS-CoV-2 test results returned negative for 547 HCWs (77.8%). Nurses (p < 0.05), paramedics (p < 0.05), and laboratory staff handling respiratory samples (p < 0.01) were at higher risk for being infected compared to the control non-patient facing group. Our phylogenetic analysis revealed that most positive samples corresponded to independent introduction events into the hospital. Our findings add to the growing evidence of differential risks of being infected among HCWs and support the need to implement appropriate protection measures based on each individual's risk profile to guarantee the protection of both HCWs and patients. Furthermore, our phylogenetic investigations highlight that most positive samples correspond to distinct introduction events into the hospital.

A 2-month field cohort study of SARS-CoV-2 in saliva of BNT162b2 vaccinated nursing home workers.

Background: Nursing home (NH) residents have been severely affected during the COVID-19 pandemic because of their age and underlying comorbidities. Infection and outbreaks in NHs are most likely triggered by infected workers. Screening for asymptomatic NH workers can prevent risky contact and viral transmission to the residents. This study examined the effect of the BNT162b2 mRNA COVID­19 (Comirnaty®; BioNTech and Pfizer) vaccination on the saliva excretion of SARS-CoV-2 among NH workers, through weekly saliva RT-qPCR testing. Methods: A 2-month cohort study was conducted among 99 NHs in the Walloon region (Belgium), at the start of February 2021. Three groups of workers, i.e., non-vaccinated (n = 1618), one-dosed vaccinated (n = 1454), and two-dosed vaccinated (n = 2379) of BNT162b2 mRNA COVID­19 vaccine, were followed-up weekly. Their saliva samples were used to monitor the shedding of SARS-CoV-2. All positive samples were sequenced and genotyped to identify the circulating wild-type virus or variants of concern. Results: The protection fraction against the excretion of the SARS-CoV-2 in the saliva samples of the workers after the second dose is estimated at 0.90 (95% CI: 0.18; 0.99) at 1 week and 0.83 (95% CI: 0.54; 0.95) at 8 weeks. We observe more circulating SARS-CoV-2 and a greater variability of viral loads in the unvaccinated group compared to those of the vaccinated group. Conclusions: This field cohort study advances our knowledge of the efficacy of the mRNA BNT162b2 COVID-19 vaccine on the viral shedding in the saliva specimens of vaccinated NH workers, contributing to better decision-making in public health interventions and management.

NK cells and monocytes modulate primary HTLV-1 infection.

We investigated the impact of monocytes, NK cells, and CD8+ T-cells in primary HTLV-1 infection by depleting cell subsets and exposing macaques to either HTLV-1 wild type (HTLV-1WT) or to the HTLV-1p12KO mutant unable to infect replete animals due to a single point mutation in orf-I that inhibits its expression. The orf-I encoded p8/p12 proteins counteract cytotoxic NK and CD8+ T-cells and favor viral DNA persistence in monocytes. Double NK and CD8+ T-cells or CD8 depletion alone accelerated seroconversion in all animals exposed to HTLV-1WT. In contrast, HTLV-1p12KO infectivity was fully restored only when NK cells were also depleted, demonstrating a critical role of NK cells in primary infection. Monocyte/macrophage depletion resulted in accelerated seroconversion in all animals exposed to HTLV-1WT, but antibody titers to the virus were low and not sustained. Seroconversion did not occur in most animals exposed to HTLV-1p12KO. In vitro experiments in human primary monocytes or THP-1 cells comparing HTLV-1WT and HTLV-1p12KO demonstrated that orf-I expression is associated with inhibition of inflammasome activation in primary cells, with increased CD47 "don't-eat-me" signal surface expression in virus infected cells and decreased monocyte engulfment of infected cells. Collectively, our data demonstrate a critical role for innate NK cells in primary infection and suggest a dual role of monocytes in primary infection. On one hand, orf-I expression increases the chances of viral transmission by sparing infected cells from efferocytosis, and on the other may protect the engulfed infected cells by modulating inflammasome activation. These data also suggest that, once infection is established, the stoichiometry of orf-I expression may contribute to the chronic inflammation observed in HTLV-1 infection by modulating monocyte efferocytosis.

Transmission of SARS-CoV-2 After COVID-19 Screening and Mitigation Measures for Primary School Children Attending School in Liège, Belgium.

Importance: Recent data suggest a relatively low incidence of COVID-19 among children. The possible role that children attending primary school may play in the transmission of SARS-CoV-2 remains poorly understood. Objective: To gain a better understanding of the possible role of children in the transmission of SARS-CoV-2. Design, Setting, and Participants: This prospective cohort study was conducted from September 21 to December 31, 2020, in a primary school in Liège, Belgium, among a volunteer sample of 181 children, parents, and school employees. Exposures: Participants were tested for SARS-CoV-2 infection once a week for 15 weeks through throat washing, performed with 5 mL of saline and collected in a sterile tube after approximately 30 seconds of gargling. Quantitative reverse transcription-polymerase chain reaction was performed to detect SARS-CoV-2 infection. Main Outcomes and Measures: In case of test positivity, participants were asked to complete a questionnaire aimed at determining the timing of symptom onset and symptom duration. SARS-CoV-2 genetic sequencing was also performed. Confirmed cases were linked based on available information on known contacts and viral sequences. Results: A total of 181 individuals participated in this study, including 63 children (34 girls [54.0%]; mean [SD] age, 8.6 [1.9] years [range, 5-13 years]) and 118 adults (75 women [63.6%]; mean [SD] age, 42.5 [5.7] years [range, 30-59 years]). Forty-five individuals (24.9%) tested positive: 13 children (20.6%; 95% CI, 10.6%-30.6%) and 32 adults (27.1%; 95% CI, 19.1%-35.7%) (P = .34). Children were more often asymptomatic compared with adults (6 [46.2%; 95% CI, 19.1%-73.3%] vs 4 of 31 [12.9%; 95% CI, 1.3%-24.5%]; P = .04). The median duration of symptoms was shorter in children than in adults (0.00 days [IQR, 0.00-1.00 days] vs 15.00 days [IQR, 7.00-22.00 days]). A reconstruction of the outbreak revealed that most transmission events occurred between teachers and between children within the school. Of the observed household transmission events, most seemed to have originated from a child or teacher who acquired the infection at school. Conclusions and Relevance: Despite the implementation of several mitigation measures, the incidence of COVID-19 among children attending primary school in this study was comparable to that observed among teachers and parents. Transmission tree reconstruction suggests that most transmission events originated from within the school. Additional measures should be considered to reduce the transmission of SARS-CoV-2 at school, including intensified testing.

Genomic sequencing of SARS-CoV-2 in Rwanda reveals the importance of incoming travelers on lineage diversity.

COVID-19 transmission rates are often linked to locally circulating strains of SARS-CoV-2. Here we describe 203 SARS-CoV-2 whole genome sequences analyzed from strains circulating in Rwanda from May 2020 to February 2021. In particular, we report a shift in variant distribution towards the emerging sub-lineage A.23.1 that is currently dominating. Furthermore, we report the detection of the first Rwandan cases of the B.1.1.7 and B.1.351 variants of concern among incoming travelers tested at Kigali International Airport. To assess the importance of viral introductions from neighboring countries and local transmission, we exploit available individual travel history metadata to inform spatio-temporal phylogeographic inference, enabling us to take into account infections from unsampled locations. We uncover an important role of neighboring countries in seeding introductions into Rwanda, including those from which no genomic sequences were available. Our results highlight the importance of systematic genomic surveillance and regional collaborations for a durable response towards combating COVID-19.

Exploiting genomic surveillance to map the spatio-temporal dispersal of SARS-CoV-2 spike mutations in Belgium across 2020.

At the end of 2020, several new variants of SARS-CoV-2-designated variants of concern-were detected and quickly suspected to be associated with a higher transmissibility and possible escape of vaccine-induced immunity. In Belgium, this discovery has motivated the initiation of a more ambitious genomic surveillance program, which is drastically increasing the number of SARS-CoV-2 genomes to analyse for monitoring the circulation of viral lineages and variants of concern. In order to efficiently analyse the massive collection of genomic data that are the result of such increased sequencing efforts, streamlined analytical strategies are crucial. In this study, we illustrate how to efficiently map the spatio-temporal dispersal of target mutations at a regional level. As a proof of concept, we focus on the Belgian province of Liège that has been consistently sampled throughout 2020, but was also one of the main epicenters of the second European epidemic wave. Specifically, we employ a recently developed phylogeographic workflow to infer the regional dispersal history of viral lineages associated with three specific mutations on the spike protein (S98F, A222V and S477N) and to quantify their relative importance through time. Our analytical pipeline enables analysing large data sets and has the potential to be quickly applied and updated to track target mutations in space and time throughout the course of an epidemic.

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) Genome Sequencing from Post-Mortem Formalin-Fixed, Paraffin-Embedded Lung Tissues.

Implementation of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) testing in the daily practice of pathology laboratories requires procedure adaptation to formalin-fixed, paraffin-embedded (FFPE) samples. So far, one study reported the feasibility of SARS-CoV-2 genome sequencing on FFPE tissues with only one contributory case of two. This study optimized SARS-CoV-2 genome sequencing using the Ion AmpliSeq SARS-CoV-2 Panel on 22 FFPE lung tissues from 16 deceased coronavirus disease 2019 (COVID-19) patients. SARS-CoV-2 was detected in all FFPE blocks using a real-time RT-qPCR targeting the E gene with crossing point (Cp) values ranging from 16.02 to 34.16. Sequencing was considered as contributory (i.e. with a uniformity >55%) for 17 FFPE blocks. Adapting the number of target amplification PCR cycles according to the RT-qPCR Cp values allowed optimization of the sequencing quality for the contributory blocks (i.e. 20 PCR cycles for blocks with a Cp value <28 and 25 PCR cycles for blocks with a Cp value between 28 and 30). Most blocks with a Cp value >30 were non-contributory. Comparison of matched frozen and FFPE tissues revealed discordance for only three FFPE blocks, all with a Cp value >28. Variant identification and clade classification was possible for 13 patients. This study validates SARS-CoV-2 genome sequencing on FFPE blocks and opens the possibility to explore correlation between virus genotype and histopathologic lesions.

PCIP-seq: simultaneous sequencing of integrated viral genomes and their insertion sites with long reads.

The integration of a viral genome into the host genome has a major impact on the trajectory of the infected cell. Integration location and variation within the associated viral genome can influence both clonal expansion and persistence of infected cells. Methods based on short-read sequencing can identify viral insertion sites, but the sequence of the viral genomes within remains unobserved. We develop PCIP-seq, a method that leverages long reads to identify insertion sites and sequence their associated viral genome. We apply the technique to exogenous retroviruses HTLV-1, BLV, and HIV-1, endogenous retroviruses, and human papillomavirus.

A Phylodynamic Workflow to Rapidly Gain Insights into the Dispersal History and Dynamics of SARS-CoV-2 Lineages.

Since the start of the COVID-19 pandemic, an unprecedented number of genomic sequences of SARS-CoV-2 have been generated and shared with the scientific community. The unparalleled volume of available genetic data presents a unique opportunity to gain real-time insights into the virus transmission during the pandemic, but also a daunting computational hurdle if analyzed with gold-standard phylogeographic approaches. To tackle this practical limitation, we here describe and apply a rapid analytical pipeline to analyze the spatiotemporal dispersal history and dynamics of SARS-CoV-2 lineages. As a proof of concept, we focus on the Belgian epidemic, which has had one of the highest spatial densities of available SARS-CoV-2 genomes. Our pipeline has the potential to be quickly applied to other countries or regions, with key benefits in complementing epidemiological analyses in assessing the impact of intervention measures or their progressive easement.

Targeted deep sequencing reveals clonal and subclonal mutational signatures in Adult T-cell leukemia/lymphoma and defines an unfavorable indolent subtype.

Adult T-cell leukemia/lymphoma (ATL) carries a poor prognosis even in indolent subtypes. We performed targeted deep sequencing combined with mapping of HTLV-1 proviral integration sites of 61 ATL patients of African and Caribbean origin. This revealed mutations mainly affecting TCR/NF-kB (74%), T-cell trafficking (46%), immune escape (29%), and cell cycle (26%) related pathways, consistent with the genomic landscape previously reported in a large Japanese cohort. To examine the evolution of mutational signatures upon disease progression while tracking the viral integration architecture of the malignant clone, we carried out a longitudinal study of patients who either relapsed or progressed from an indolent to an aggressive subtype. Serial analysis of relapsing patients identified several patterns of clonal evolution. In progressing patients, the longitudinal study revealed NF-kB/NFAT mutations at progression that were present at a subclonal level at diagnosis (allelic frequency < 5%). Moreover, the presence in indolent subtypes of mutations affecting the TCR/NF-kB pathway, whether clonal or subclonal, was associated with significantly shorter time to progression and overall survival. Our observations reveal the clonal dynamics of ATL mutational signatures at relapse and during progression. Our study defines a new subgroup of indolent ATLs characterized by a mutational signature at high risk of transformation.

An Improved Sequencing-Based Bioinformatics Pipeline to Track the Distribution and Clonal Architecture of Proviral Integration Sites.

The combined application of linear amplification-mediated PCR (LAM-PCR) protocols with next-generation sequencing (NGS) has had a large impact on our understanding of retroviral pathogenesis. Previously, considerable effort has been expended to optimize NGS methods to explore the genome-wide distribution of proviral integration sites and the clonal architecture of clinically important retroviruses like human T-cell leukemia virus type-1 (HTLV-1). Once sequencing data are generated, the application of rigorous bioinformatics analysis is central to the biological interpretation of the data. To better exploit the potential information available through these methods, we developed an optimized bioinformatics pipeline to analyze NGS clonality datasets. We found that short-read aligners, specifically designed to manage NGS datasets, provide increased speed, significantly reducing processing time and decreasing the computational burden. This is achieved while also accounting for sequencing base quality. We demonstrate the utility of an additional trimming step in the workflow, which adjusts for the number of reads supporting each insertion site. In addition, we developed a recall procedure to reduce bias associated with proviral integration within low complexity regions of the genome, providing a more accurate estimation of clone abundance. Finally, we recommend the application of a "clean-and-recover" step to clonality datasets generated from large cohorts and longitudinal studies. In summary, we report an optimized bioinformatics workflow for NGS clonality analysis and describe a new set of steps to guide the computational process. We demonstrate that the application of this protocol to the analysis of HTLV-1 and bovine leukemia virus (BLV) clonality datasets improves the quality of data processing and provides a more accurate definition of the clonal landscape in infected individuals. The optimized workflow and analysis recommendations can be implemented in the majority of bioinformatics pipelines developed to analyze LAM-PCR-based NGS clonality datasets.

A Recurrent Mutation at Position 26340 of SARS-CoV-2 Is Associated with Failure of the E Gene Quantitative Reverse Transcription-PCR Utilized in a Commercial Dual-Target Diagnostic Assay.

Control of the ongoing severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic requires accurate laboratory testing to identify infected individuals while also clearing essential staff to continue to work. At the current time, a number of quantitative real-time PCR (qRT-PCR) assays have been developed to identify SARS-CoV-2, targeting multiple positions in the viral genome. While the mutation rate of SARS-CoV-2 is moderate, given the large number of transmission chains, it is prudent to monitor circulating viruses for variants that might compromise these assays. Here, we report the identification of a C-to-U transition at position 26340 of the SARS-CoV-2 genome that is associated with failure of the cobas SARS-CoV-2 E gene qRT-PCR in eight patients. As the cobas SARS-CoV-2 assay targets two positions in the genome, the individuals carrying this variant were still called SARS-CoV-2 positive. Whole-genome sequencing of SARS-CoV-2 showed all to carry closely related viruses. Examination of viral genomes deposited on GISAID showed this mutation has arisen independently at least four times. This work highlights the necessity of monitoring SARS-CoV-2 for the emergence of single-nucleotide polymorphisms that might adversely affect RT-PCRs used in diagnostics. Additionally, it argues that two regions in SARS-CoV-2 should be targeted to avoid false negatives.

Essential Role of Human T Cell Leukemia Virus Type 1 orf-I in Lethal Proliferation of CD4+ Cells in Humanized Mice.

Human T cell leukemia virus type 1 (HTLV-1) is the ethological agent of adult T cell leukemia/lymphoma (ATLL) and a number of lymphocyte-mediated inflammatory conditions, including HTLV-1-associated myelopathy/tropical spastic paraparesis. HTLV-1 orf-I encodes two proteins, p8 and p12, whose functions in humans are to counteract innate and adaptive responses and to support viral transmission. However, the in vivo requirements for orf-I expression vary in different animal models. In macaques, the ablation of orf-I expression by mutation of its ATG initiation codon abolishes the infectivity of the molecular clone HTLV-1p12KO In rabbits, HTLV-1p12KO is infective and persists efficiently. We used humanized mouse models to assess the infectivity of both wild-type HTLV-1 (HTLV-1WT) and HTLV-1p12KO We found that NOD/SCID/γC-/- c-kit+ mice engrafted with human tissues 1 day after birth (designated NSG-1d mice) were highly susceptible to infection by HTLV-1WT, with a syndrome characterized by the rapid polyclonal proliferation and infiltration of CD4+ CD25+ T cells into vital organs, weight loss, and death. HTLV-1 clonality studies revealed the presence of multiple clones of low abundance, confirming the polyclonal expansion of HTLV-1-infected cells in vivo HTLV-1p12KO infection in a bone marrow-liver-thymus (BLT) mouse model prone to graft-versus-host disease occurred only following reversion of the orf-I initiation codon mutation within weeks after exposure and was associated with high levels of HTLV-1 DNA in blood and the expansion of CD4+ CD25+ T cells. Thus, the incomplete reconstitution of the human immune system in BLT mice may provide a window of opportunity for HTLV-1 replication and the selection of viral variants with greater fitness.IMPORTANCE Humanized mice constitute a useful model for studying the HTLV-1-associated polyclonal proliferation of CD4+ T cells and viral integration sites in the human genome. The rapid death of infected animals, however, appears to preclude the clonal selection typically observed in human ATLL, which normally develops in 2 to 5% of individuals infected with HTLV-1. Nevertheless, the expansion of multiple clones of low abundance in these humanized mice mirrors the early phase of HTLV-1 infection in humans, providing a useful model to investigate approaches to inhibit virus-induced CD4+ T cell proliferation.

PDZ domain-binding motif of Tax sustains T-cell proliferation in HTLV-1-infected humanized mice.

Human T-cell leukemia virus type 1 (HTLV-1) is the etiological agent of adult T-cell leukemia/lymphoma (ATLL), an aggressive malignant proliferation of activated CD4+ T lymphocytes. The viral Tax oncoprotein is critically involved in both HTLV-1-replication and T-cell proliferation, a prerequisite to the development of ATLL. In this study, we investigated the in vivo contribution of the Tax PDZ domain-binding motif (PBM) to the lymphoproliferative process. To that aim, we examined T-cell proliferation in humanized mice (hu-mice) carrying a human hemato-lymphoid system infected with either a wild type (WT) or a Tax PBM-deleted (ΔPBM) provirus. We observed that the frequency of CD4+ activated T-cells in the peripheral blood and in the spleen was significantly higher in WT than in ΔPBM hu-mice. Likewise, human T-cells collected from WT hu-mice and cultivated in vitro in presence of interleukin-2 were proliferating at a higher level than those from ΔPBM animals. We next examined the association of Tax with the Scribble PDZ protein, a prominent regulator of T-cell polarity, in human T-cells analyzed either after ex vivo isolation or after in vitro culture. We confirmed the interaction of Tax with Scribble only in T-cells from the WT hu-mice. This association correlated with the presence of both proteins in aggregates at the leading edge of the cells and with the formation of long actin filopods. Finally, data from a comparative genome-wide transcriptomic analysis suggested that the PBM-PDZ association is implicated in the expression of genes regulating proliferation, apoptosis and cytoskeletal organization. Collectively, our findings suggest that the Tax PBM is an auxiliary motif that contributes to the sustained growth of HTLV-1 infected T-cells in vivo and in vitro and is essential to T-cell immortalization.

HTLV-1-induced leukotriene B4 secretion by T cells promotes T cell recruitment and virus propagation.

The human T-lymphotropic virus type 1 (HTLV-1) is efficiently transmitted through cellular contacts. While the molecular mechanisms of viral cell-to-cell propagation have been extensively studied in vitro, those facilitating the encounter between infected and target cells remain unknown. In this study, we demonstrate that HTLV-1-infected CD4 T cells secrete a potent chemoattractant, leukotriene B4 (LTB4). LTB4 secretion is dependent on Tax-induced transactivation of the pla2g4c gene, which encodes the cytosolic phospholipase A2 gamma. Inhibition of LTB4 secretion or LTB4 receptor knockdown on target cells reduces T-cell recruitment, cellular contact formation and virus propagation in vitro. Finally, blocking the synthesis of LTB4 in a humanized mouse model of HTLV-1 infection significantly reduces proviral load. This results from a decrease in the number of infected clones while their expansion is not impaired. This study shows the critical role of LTB4 secretion in HTLV-1 transmission both in vitro and in vivo.

Cis-perturbation of cancer drivers by the HTLV-1/BLV proviruses is an early determinant of leukemogenesis.

Human T-cell leukaemia virus type-1 (HTLV-1) and bovine leukaemia virus (BLV) infect T- and B-lymphocytes, respectively, provoking a polyclonal expansion that will evolve into an aggressive monoclonal leukaemia in ∼5% of individuals following a protracted latency period. It is generally assumed that early oncogenic changes are largely dependent on virus-encoded products, especially TAX and HBZ, while progression to acute leukaemia/lymphoma involves somatic mutations, yet that both are independent of proviral integration site that has been found to be very variable between tumours. Here, we show that HTLV-1/BLV proviruses are integrated near cancer drivers which they affect either by provirus-dependent transcription termination or as a result of viral antisense RNA-dependent cis-perturbation. The same pattern is observed at polyclonal non-malignant stages, indicating that provirus-dependent host gene perturbation contributes to the initial selection of the multiple clones characterizing the asymptomatic stage, requiring additional alterations in the clone that will evolve into full-blown leukaemia/lymphoma.

Characterization of novel Bovine Leukemia Virus (BLV) antisense transcripts by deep sequencing reveals constitutive expression in tumors and transcriptional interaction with viral microRNAs.

BACKGROUND: Bovine Leukemia Virus (BLV) is a deltaretrovirus closely related to the Human T cell leukemia virus-1 (HTLV-1). Cattle are the natural host of BLV where it integrates into B-cells, producing a lifelong infection. Most infected animals remain asymptomatic but following a protracted latency period about 5 % develop an aggressive leukemia/lymphoma, mirroring the disease trajectory of HTLV-1. The mechanisms by which these viruses provoke cellular transformation remain opaque. In both viruses little or no transcription is observed from the 5'LTR in tumors, however the proviruses are not transcriptionally silent. In the case of BLV a cluster of RNA polymerase III transcribed microRNAs are highly expressed, while the HTLV-1 antisense transcript HBZ is consistently found in all tumors examined. RESULTS: Here, using RNA-seq, we demonstrate that the BLV provirus also constitutively expresses antisense transcripts in all leukemic and asymptomatic samples examined. The first transcript (AS1) can be alternately polyadenylated, generating a transcript of ~600 bp (AS1-S) and a less abundant transcript of ~2200 bp (AS1-L). Alternative splicing creates a second transcript of ~400 bp (AS2). The coding potential of AS1-S/L is ambiguous, with a small open reading frame of 264 bp, however the transcripts are primarily retained in the nucleus, hinting at a lncRNA-like role. The AS1-L transcript overlaps the BLV microRNAs and using high throughput sequencing of RNA-ligase-mediated (RLM) 5'RACE, we show that the RNA-induced silencing complex (RISC) cleaves AS1-L. Furthermore, experiments using altered BLV proviruses with the microRNAs either deleted or inverted point to additional transcriptional interference between the two viral RNA species. CONCLUSIONS: The identification of novel viral antisense transcripts shows the BLV provirus to be far from silent in tumors. Furthermore, the consistent expression of these transcripts in both leukemic and nonmalignant clones points to a vital role in the life cycle of the virus and its tumorigenic potential. Additionally, the cleavage of the AS1-L transcript by the BLV encoded microRNAs and the transcriptional interference between the two viral RNA species suggest a shared role in the regulation of BLV.