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International audience; Human T-cell leukemia/lymphoma virus-1 (HTLV-1), the first human oncoretrovirus, is the etiological agent of a chronic neuromyelopathy named “tropical spastic paraparesis/HTLV-1–associated myelopathy (TSP/HAM). At least 5–10 million individuals are infected worldwide. The areas of highest prevalence are southwestern Japan and parts of the Caribbean, South America, intertropical Africa, and the Middle East. The lifetime risk of developing TSP/HAM in HTLV-1 carriers is estimated at 0.25–4%, depending on the endemic area. The neurological characteristics include spasticity or hyper-reflexia of the lower limbs, urinary bladder disturbances often with lower limb muscle weakness, sensory disturbance, and low back pain. TSP/HAM pathology is characterized by chronic inflammation with perivascular lymphocyte cuffs and mild parenchymal lymphocytic infiltrates, foamy macrophages, astrocyte proliferation, and fibrillary gliosis. Pyramidal tract degenerative lesions with myelin and axonal loss are observed, mainly in the lower thoracic spinal cord; these are linked to inflammatory foci in the CNS. The treatment of TSP/HAM remains largely symptomatic because there is no current disease-modifying therapy with definite long-term clinical benefit.

Human T cell leukemia virus (HTLV-1) is an oncoretrovirus that infects at least 10 million people worldwide. HTLV-1 exhibits a remarkable genetic stability, however, viral strains have been classified in several genotypes and subgroups, which often mirror the geographic origin of the viral strain. The Cosmopolitan genotype HTLV-1a, can be subdivided into geographically related subgroups, e.g. Transcontinental (a-TC), Japanese (a-Jpn), West-African (a-WA), North-African (a-NA), and Senegalese (a-Sen). Within each subgroup, the genetic diversity is low. Genotype HTLV-1b is found in Central Africa; it is the major genotype in Gabon, Cameroon and Democratic Republic of Congo. While strains from the HTLV-1d genotype represent only a few percent of the strains present in Central African countries, genotypes -e, -f, and -g have been only reported sporadically in particular in Cameroon Gabon, and Central African Republic. HTLV-1c genotype, which is found exclusively in Australo-Melanesia, is the most divergent genotype. This reflects an ancient speciation, with a long period of isolation of the infected populations in the different islands of this region (Australia, Papua New Guinea, Solomon Islands and Vanuatu archipelago). Until now, no viral genotype or subgroup is associated with a specific HTLV-1-associated disease. HTLV-1 originates from a simian reservoir (STLV-1); it derives from interspecies zoonotic transmission from non-human primates to humans (ancient or recent). In this review, we describe the genetic diversity of HTLV-1, and analyze the molecular mechanisms that are at play in HTLV-1 evolution. Similar to other retroviruses, HTLV-1 evolves either through accumulation of point mutations or recombination. Molecular studies point to a fairly low evolution rate of HTLV-1 (between 5.6E−7 and 1.5E−6 substitutions/site/year), supposedly because the virus persists within the host via clonal expansion (instead of new infectious cycles that use reverse transcriptase).

HIV-1 can cross the blood-brain barrier (BBB) to penetrate the brain and infect target cells, causing neurocognitive disorders as a result of neuroinflammation and brain damage. The HIV-1 envelope spike gp160 is partially required for viral transcytosis across the BBB endothelium. But do antibodies developing in infected individuals and targeting the HIV-1 gp160 glycoproteins block HIV-1 transcytosis through the BBB? We addressed this issue and discovered that anti-gp160 antibodies do not block HIV-1 transport; instead, free viruses and those in complex with antibodies can transit across BBB endothelial cells. Importantly, we found that only neutralizing antibodies could inhibit posttranscytosis viral infectivity, highlighting their ability to protect susceptible brain cells from HIV-1 infection., HIV-1 can cross the blood-brain barrier (BBB) to penetrate the brain and infect target cells, causing neurocognitive disorders as a result of neuroinflammation and brain damage. Here, we examined whether antibodies targeting the HIV-1 envelope glycoproteins interfere with the transcytosis of virions across the human BBB endothelium. We found that although the viral envelope spike gp160 is required for optimal endothelial cell endocytosis, no anti-gp160 antibodies blocked the BBB transcytosis of HIV-1 in vitro. Instead, both free viruses and those in complex with antibodies transited across endothelial cells in the BBB model, as observed by confocal microscopy. HIV-1 infectious capacity was considerably altered by the transcytosis process but still detectable, even in the presence of nonneutralizing antibodies. Only virions bound by neutralizing antibodies lacked posttranscytosis infectivity. Overall, our data support the role of neutralizing antibodies in protecting susceptible brain cells from HIV-1 infection despite their inability to inhibit viral BBB endocytic transport.

Among all known retroviruses, foamy viruses (FVs) have the most stable virus–host co-speciation history, co-diverging in concert with their vertebrate hosts for hundreds of millions of years. However, detailed molecular analyses indicate that different parts of their genome might have different evolutionary histories. While their polymerase gene displays a robust and straightforward virus–host co-speciation pattern, the evolutionary history of their envelope (env) gene, is much more complicated. Here, we report eleven new FV env sequences in two mandrill populations in Central Africa, geographically separated by the Ogooué River into the North and the South populations. Phylogenetic reconstruction of the polymerase gene shows that the two virus populations are distinct, and each contains two variants of env genes co-existing with one another. The distinction between the two env variants can be mapped to the surface domain, flanked by two recombination hotspots, as previously reported for chimpanzee and gorilla FVs. Our analyses suggest that the two env variants originated during the diversification of Old World monkeys and apes, ∼30 million years ago. We also show that this env gene region forms two phylogenetically distinct clades, each displaying a host co-divergence and geographical separation pattern, while the rest of the genome of the two strains is phylogenetically indistinguishable in each of the host-specific groups. We propose possible evolutionary mechanisms to explain the modular nature of the FV genome.

Japanese encephalitis virus (JEV) is the major cause of viral encephalitis in South East Asia. It has been suggested that JEV gets access to the central nervous system (CNS) as a consequence of a preceding inflammatory process which leads to the blood-brain barrier (BBB) disruption and viral neuroinvasion. However, what happens at early times of JEV contact with the BBB is poorly understood. In the present work, we evaluated the ability of both a virulent and a vaccine strain of JEV (JEV RP9 and SA14-14-2, respectively) to cross anin cellulohuman BBB model consisting of hCMEC/D3 human endothelial cells cultivated on permeable inserts above SK-N-SH human neuroblastoma cells. Using this system, we demonstrated that both JEV RP9 and SA14-14-2 are able to cross the BBB without disrupting it at early times post-addition. Furthermore, this BBB model was able to discriminate between the virulent RP9 and the vaccine SA14-14-2 strains, as demonstrated by the presence of almost 10 times more RP9 infectious particles that crossed the BBB than SA14-14 particles at a high MOI. Besides contributing to the understanding of early events in JEV neuroinvasion, thisin celluloBBB model represents a suitable and useful system to study the viral determinants of JEV neuroinvasiveness and the molecular mechanisms by which this flavivirus crosses the BBB at early times of neuroinvasion.

The blood-brain barrier (BBB), which constitutes the interface between blood and cerebral parenchyma, has been shown to be disrupted during retroviral associated neuromyelopathies. Human T cell leukemia virus (HTLV-1)-associated myelopathy/tropical spastic paraparesis is a slowly progressive neurodegenerative disease, in which evidence of BBB breakdown has been demonstrated by the presence of lymphocytic infiltrates in the CNS and plasma protein leakage through cerebral endothelium. Using an in vitro human BBB model, we investigated the cellular and molecular mechanisms involved in endothelial changes induced by HTLV-1-infected lymphocytes. We demonstrate that coculture with infected lymphocytes induces an increase in paracellular endothelial permeability and transcellular migration, via IL-1α and TNF-α secretion. This disruption is associated with tight junction disorganization between endothelial cells, and alterations in the expression pattern of tight junction proteins such as zonula occludens 1. These changes could be prevented by inhibition of the NF-κB pathway or of myosin light chain kinase activity. Such disorganization was confirmed in histological sections of spinal cord from an HTLV-1-associated myelopathy/tropical spastic paraparesis patient. Based on this BBB model, the present data indicate that HTLV-1-infected lymphocytes can induce BBB breakdown and may be responsible for the CNS infiltration that occurs in the early steps of retroviral-associated neuromyelopathies.

Simian foamy virus (SFV) is a ubiquitous retrovirus in nonhuman primates (NHPs) that can be transmitted to humans, mostly through severe bites. In the past few years, our laboratory has identified more than 50 hunters from central Africa infected with zoonotic SFVs. Analysis of the complete sequences of five SFVs obtained from these individuals revealed that env was the most variable gene. Furthermore, recombinant SFV strains, some of which involve sequences in the env gene, were recently identified. Here, we investigated the variability of the env genes of zoonotic SFV strains and searched for possible recombinants. We sequenced the complete env gene or its surface glycoprotein region (SU) from DNA amplified from the blood of (i) a series of 40 individuals from Cameroon or Gabon infected with a gorilla or chimpanzee foamy virus (FV) strain and (ii) 1 gorilla and 3 infected chimpanzees living in the same areas as these hunters. Phylogenetic analyses revealed the existence of two env variants among both the gorilla and chimpanzee FV strains that were present in zoonotic and NHP strains. These variants differ greatly (>30% variability) in a 753-bp-long region located in the receptor-binding domain of SU, whereas the rest of the gene is very conserved. Although the organizations of the Env protein sequences are similar, the potential glycosylation patterns differ between variants. Analysis of recombination suggests that the variants emerged through recombination between different strains, although all parental strains could not be identified. IMPORTANCE SFV infection in humans is a great example of a zoonotic retroviral infection that has not spread among human populations, in contrast to human immunodeficiency viruses (HIVs) and human T-lymphotropic viruses (HTLVs). Recombination was a major mechanism leading to the emergence of HIV. Here, we show that two SFV molecular envelope gene variants circulate among ape populations in Central Africa and that both can be transmitted to humans. These variants differ greatly in the SU region that corresponds to the part of the Env protein in contact with the environment. These variants may have emerged through recombination between SFV strains infecting different NHP species.

Isolated HTLV-1 viral particles are poorly infectious both in vivo and in vitro. In contrast, the virus is efficiently transmitted through cell-to-cell contact. While the molecular mechanisms of viral cell-to-cell propagation have been extensively studied, the mechanisms facilitating the encounter between an infected cell and a target cell remain unknown. We postulated that the infected cells could behave as a source of chemoattractant, thus recruiting target cells. In 2012, Trindade et al. showed that PBMCs from HTLV-1 infected individuals (HTLV-1 asymptomatic carrier and HAM/TSP patients) could secrete higher levels of leukotriene B4 (LTB4), a potent chemoattractant, than cells from healthy donors. We proposed that, although T cells purportedly secrete no LTB4, LTB4 could be secreted by HTLV-1 infected lymphocytes and facilitate cell-to-cell contacts and viral transmission. We showed by ELISA that HTLV-1 infected lymphocytes (both primary CD4+ T cells and chronically infected cell lines) secrete LTB4. By cell transduction with lentiviral vectors encoding Tax, we suggest that HTLV-1 induced LTB4 secretion is a consequence of Tax-induced cytosolic phospholipase A2 (cPLA2) over expression. Blocking LTB4 secretion (with diverse specific inhibitors of the leukotriene pathway) prevented the recruitment of potential target cells, as well as the formation of cell-to-cell contacts. In conclusion, HTLV-1 diverts the leukotriene pathway and LTB4-mediated chemotaxis facilitates viral propagation.

Four types of primate T-cell lymphotropic viruses (PTLVs) have been discovered. While three of them, i.e., PTLV-1, PTLV-2, and PTLV-3, include human T-cell lymphotropic viruses (HTLV-1, HTLV-2, and HTLV-3) and simian T-cell lymphotropic viruses (STLV-1, STLV-2, and STLV-3), the fourth type (HTLV-4) has so far been found to consist only of a unique human isolate (4, 52). STLV-3 (formerly named PTLV-L) was isolated shortly before STLV-2 (14). The STLV-3 lineage is composed of at least two subtypes that correspond more or less to the geographical sources of the isolates (East Africa and west-central Africa) (7, 14, 24, 25, 27, 46, 47, 49, 51). Although STLV-3 is much more widespread than STLV-2 in African monkeys, STLV-3 infection has not been linked to any pathology so far, yet neither longitudinal follow-up nor clinical studies have been performed. Sequence comparisons of STLV-3 full-length proviruses pointed out that these strains are highly divergent from HTLV-1, HTLV-2, and STLV-2 (40%, 38%, and 38% divergence, respectively) (7, 14, 24, 25, 27, 46, 47, 49, 51). The overall STLV-3 genomic organization is similar to that of HTLV-1 and HTLV-2; the gag, pro, pol, env, tax, and rex genes are present (24, 25, 46, 47, 51). Sequence analyses also revealed that STLV-3 long terminal repeat (LTR) sequences possess only two 21-bp repeats (or Tax-responsive elements [TREs]), while HTLV-1 and HTLV-2 LTRs retain three of these sequences (24, 25, 46). The impact of the lack of a TRE on viral transcription is not known. Interestingly, apart from Tax and Rex, the STLV-3 prototype (STLV-3PH969) pX region was reported to contain only one additional open reading frame (ORF) whose corresponding mRNA can be amplified by reverse transcriptase (RT)-PCR (47). This mRNA could be translated into a putative 84-amino-acid-long protein designated RorfII, which shares some similarities with the HTLV-1 p12 protein (47). Of note, the RorfII mRNA was not detected in the two other STLV-3 strains (PPAF3 and CTO604) that have been analyzed lately by RT-PCR. In fact, sequence analysis of these isolates revealed either a mutation in the splice acceptor sequence and/or a stop codon in the RorfII sequence (24, 25). At this point, it is therefore not clear whether, apart from Tax and Rex, additional ORFs are commonly present in the STLV-3 pX sequence. We and others have uncovered the existence of HTLV-3, a third member of the HTLV family, in two Cameroonian individuals (4, 52). We therefore ought to obtain the complete sequence of HTLV-3Pyl43 in order to determine its genomic organization and to characterize its Tax protein. Full-length genome comparisons and phylogenetic analyses allow us to demonstrate that the HTLV-3 genome organization is similar to that of HTLV-1 and HTLV-2 and is related to some central African STLV-3 strains. Like STLV-3, HTLV-3 LTRs contain only two Tax-responsive elements. Unexpectedly, a 366-bp sequence is lacking in the HTLV-3 proximal pX region. This sequence corresponds in part to the RorfII sequence that was previously described for STLV-3PH969 and also to the 3′ end of a putative antisense transcript. We have lately demonstrated that HTLV-3 Tax protein is expressed in vivo (6). Our results now reveal that Tax3Pyl43 is functionally more closely related to the transforming HTLV-1 Tax protein than to HTLV-2 Tax, suggesting that HTLV-3, like HTLV-1, might be pathogenic in vivo.

Although recombination is a major source of genetic variability in retroviruses, no recombinant strain had been observed for HTLV-1, the first isolated human-pathogenic retrovirus. Different genotypes exist for HTLV-1: Genotypes b and d to g are restricted to central Africa, while genotype c is only endemic in Australo-Melanesia. In contrast, the cosmopolitan genotype A is widely distributed. We applied a combination of phylogenetics and recombination analysis approaches to a set of new HTLV-1 sequences, including the complete LTR and a 522-bp fragment of the env gene, which we collected from 19 countries throughout North, West and Central Africa, the continent where the virus has the largest endemic presence. The samples were obtained from 41 HTLV-1 infected individuals with different clinical statuses including ATL, TSP/HAM and asymptomatic carriers. The recombinant search and breakpoint detection were performed by boot scanning in Simplot to compare inferred clusters of sequences to each other. Finally, molecular clock analyses were performed to date the recombinant event observed. This led us to demonstrate the presence of recombinants in HTLV-1. Indeed, the HTLV-1 strains currently present in North Africa have originated from a recombinant event between strains from Senegal and West Africa. This recombination is estimated to have occurred around 4,0 years ago. This recombination seems to have been generated during reverse transcription. In conclusion, we demonstrate that, albeit rare, recombination can occur in HTLV-1 and may play a role in the evolution of this retrovirus (Desrames et al., J. Virology, 88, 2014). In order to precise the geographical distribution of this recombinant within the African Continent, we studied a new set of samples from HTLV-1 infected patients of diverse African origin. The data on this on-going study will be presented.

Merkel cell polyomavirus (MCPyV) is linked to a cutaneous cancer mainly occurring in Caucasians. DNA from skin swabs of 255 adults, originating from the 5 continents, were subjected to MCPyV PCRs. Phylogenetic analyses demonstrate the existence of 5 major geographically related MCPyV genotypes (Europe/North America, Africa [sub-Saharan], Oceania, South America, and Asia/Japan).

Efficient HTLV-1 viral transmission occurs through cell-to-cell contacts. The Tax viral transcriptional activator protein facilitates this process. Using a comparative transcriptomic analysis, we recently identified a series of genes up-regulated in HTLV-1 Tax expressing T-lymphocytes. We focused our attention towards genes that are important for cytoskeleton dynamic and thus may possibly modulate cell-to-cell contacts. We first demonstrate that Gem, a member of the small GTP-binding proteins within the Ras superfamily, is expressed both at the RNA and protein levels in Tax-expressing cells and in HTLV-1-infected cell lines. Using a series of ChIP assays, we show that Tax recruits CREB and CREB Binding Protein (CBP) onto a c-AMP Responsive Element (CRE) present in the gem promoter. This CRE sequence is required to drive Tax-activated gem transcription. Since Gem is involved in cytoskeleton remodeling, we investigated its role in infected cells motility. We show that Gem co-localizes with F-actin and is involved both in T-cell spontaneous cell migration as well as chemotaxis in the presence of SDF-1/CXCL12. Importantly, gem knock-down in HTLV-1-infected cells decreases cell migration and conjugate formation. Finally, we demonstrate that Gem plays an important role in cell-to-cell viral transmission., Author Summary HTLV-1 was the first human oncoretrovirus to be discovered. Five to ten million people are infected, and 1–6% will develop either Adult T-cell Leukemia, or Tropical Spastic Paraparesis/HTLV-1 Associated Myelopathy (TSP/HAM). HTLV-1 infects primarily T-cells, but dendritic cells were also found to carry proviruses. Contrary to HIV-1, cell-free HTLV-1 viral particles are poorly infectious. Thus, efficient viral transmission relies on formation of virological synapses or formation and transfer of viral biofilm-like structures. The Tax viral transactivator plays a key role in both modes of transmission. Using transcriptomic analyses, we recently identified cellular genes that are deregulated following Tax expression in T-cells. We focused our attention on genes that are important for cell architecture and are thus likely to modulate cell-to-cell contacts and motility. We found that Gem was highly upregulated both at the RNA and protein levels in Tax-expressing cells and HTLV-1-infected cell lines. We further show that Tax binds cellular co-activators and transcription factor and activates transcription from the gem promoter. We demonstrated that Gem is involved in cellular migration of HTLV-1-infected cells. Importantly, gem knockdown decreases the rate of HTLV-1-infected cell migration and cell-to-cell conjugate formation. We also show that Gem plays an important role in HTLV-1 transmission through cell-to-cell contacts, the most efficient mode of viral infection.

Approximately 3% of all human T-lymphotropic virus type 1 (HTLV-1)–infected persons will develop a disabling inflammatory disease of the central nervous system known as HTLV-1–associated myelopathy/tropical spastic paraparesis, against which there is currently no efficient treatment. As correlation exists between the proviral load (PVL) and the clinical status of the carrier, it is thought that diminishing the PVL could prevent later occurrence of the disease. We have conducted a study combining valproate, an inhibitor of histone deacetylases, and azidothymidine, an inhibitor of reverse transcriptase, in a series of baboons naturally infected with simian T-lymphotropic virus type 1 (STLV-1), whose PVL was equivalent to that of HTLV-1 asymptomatic carriers. We show that the combination of drugs caused a strong decrease in the PVL and prevented the transient rise in PVL that is seen after treatment with histone deacetylases alone. We then demonstrate that the PVL decline was associated with an increase in the STLV-1–specific cytotoxic T-cell population. We conclude that combined treatment with valproate to induce viral expression and azidothymidine to prevent viral propagation is a safe and effective means to decrease PVL in vivo. Such treatments may be useful to reduce the risk of HAM/TSP in asymptomatic carriers with a high PVL.

Little is known about the transmission or tropism of the newly discovered human retrovirus, human T-cell lymphotropic virus type 3 (HTLV-3). Here, we examine the entry requirements of HTLV-3 using independently expressed Env proteins. We observed that HTLV-3 surface glycoprotein (SU) binds efficiently to both activated CD4 + and CD8 + T cells. This contrasts with both HTLV-1 SU, which primarily binds to activated CD4 + T cells, and HTLV-2 SU, which primarily binds to activated CD8 + T cells. Binding studies with heparan sulfate proteoglycans (HSPGs) and neuropilin-1 (NRP-1), two molecules important for HTLV-1 entry, revealed that these molecules also enhance HTLV-3 SU binding. However, unlike HTLV-1 SU, HTLV-3 SU can bind efficiently in the absence of both HSPGs and NRP-1. Studies of entry performed with HTLV-3 Env-pseudotyped viruses together with SU binding studies revealed that, for HTLV-1, glucose transporter 1 (GLUT-1) functions at a postbinding step during HTLV-3 Env-mediated entry. Further studies revealed that HTLV-3 SU binds efficiently to naïve CD4 + T cells, which do not bind either HTLV-1 or HTLV-2 SU and do not express detectable levels of HSPGs, NRP-1, and GLUT-1. These results indicate that the complex of receptor molecules used by HTLV-3 to bind to primary T lymphocytes differs from that of both HTLV-1 and HTLV-2.

Nuclear factor (NF)-κB is a major survival pathway engaged by the Human T-Lymphotropic Virus type 1 (HTLV-1) Tax protein. Tax1 activation of NF-κB occurs predominantly in the cytoplasm, where Tax1 binds NF-κB Essential Modulator (NEMO/IKKγ) and triggers the activation of IκB kinases. Several independent studies have shown that Tax1-mediated NF-κB activation is dependent on Tax1 ubiquitination. Here, we identify by co-immunoprecipitation assays NEMO-Related Protein (NRP/Optineurin) as a binding partner for Tax1 in HTLV-1 infected and Tax1/NRP co-expressing cells. Immunofluorescence studies reveal that Tax1, NRP and NEMO colocalize in Golgi-associated structures. The interaction between Tax1 and NRP requires the ubiquitin-binding activity of NRP and the ubiquitination sites of Tax1. In addition, we observe that NRP increases the ubiquitination of Tax1 along with Tax1-dependent NF-κB signaling. Surprisingly, we find that in addition to Tax1, NRP interacts cooperatively with the Tax1 binding protein TAX1BP1, and that NRP and TAX1BP1 cooperate to modulate Tax1 ubiquitination and NF-κB activation. Our data strongly suggest for the first time that NRP is a critical adaptor that regulates the assembly of TAX1BP1 and post-translationally modified forms of Tax1, leading to sustained NF-κB activation., Author Summary Oncogenic viruses (i.e., viruses that can induce cancer) have usually been found to deregulate several cellular signaling pathways controlling cell survival and proliferation. Among those, the NF-κB pathway is particularly important. In this study, we focus on the Human T-Lymphotropic Virus type 1 (HTLV-1), which infects immune T cells, and is associated with the development of a severe hematological disease, termed adult T cell leukemia. The viral Tax oncoprotein is known to activate the NF-κB pathway, but the precise mechanism is still under investigation. In cells, proteins can undergo modifications that can modulate their function. In the case of Tax, a modified form of the protein (ubiquitinated Tax) is able to activate the NF-κB pathway. Our aim was to identify cellular proteins that participate in the modification of Tax, and in turn in the regulation of its function. We show for the first time that the cellular protein NRP/Optineurin interacts with Tax and increases its ubiquitination, thus leading to an enhanced NF-κB activation. We further demonstrate that TAX1BP1, another cellular protein that had been previously identified as a partner of Tax, also participates in this regulation. Thus, this study uncovers new actors of the virally induced cell signaling.

The blood–brain barrier (BBB), which forms the interface between the blood and the cerebral parenchyma, has been shown to be disrupted during retroviral-associated neuromyelopathies. Human T Lymphotropic Virus (HTLV-1) Associated Myelopathy/Tropical Spastic Paraparesis (HAM/TSP) is a slowly progressive neurodegenerative disease associated with BBB breakdown. The BBB is composed of three cell types: endothelial cells, pericytes and astrocytes. Although astrocytes have been shown to be infected by HTLV-1, until now, little was known about the susceptibility of BBB endothelial cells to HTLV-1 infection and the impact of such an infection on BBB function. We first demonstrated that human cerebral endothelial cells express the receptors for HTLV-1 (GLUT-1, Neuropilin-1 and heparan sulfate proteoglycans), both in vitro, in a human cerebral endothelial cell line, and ex vivo, on spinal cord autopsy sections from HAM/TSP and non-infected control cases. In situ hybridization revealed HTLV-1 transcripts associated with the vasculature in HAM/TSP. We were able to confirm that the endothelial cells could be productively infected in vitro by HTLV-1 and that blocking of either HSPGs, Neuropilin 1 or Glut1 inhibits this process. The expression of the tight-junction proteins within the HTLV-1 infected endothelial cells was altered. These cells were no longer able to form a functional barrier, since BBB permeability and lymphocyte passage through the monolayer of endothelial cells were increased. This work constitutes the first report of susceptibility of human cerebral endothelial cells to HTLV-1 infection, with implications for HTLV-1 passage through the BBB and subsequent deregulation of the central nervous system homeostasis. We propose that the susceptibility of cerebral endothelial cells to retroviral infection and subsequent BBB dysfunction is an important aspect of HAM/TSP pathogenesis and should be considered in the design of future therapeutics strategies., Author Summary The blood–brain barrier (BBB) forms the interface between the blood and the central nervous system (CNS). BBB disruption is considered to be a key event in the pathogenesis of retroviral-associated neurological diseases. The present paper deals with the susceptibility of the endothelial cells (i.e., one of the main cellular components of BBB) to retroviral infection, and with the impact of infection in BBB function. This study focuses on the Human T-Lymphotropic Virus (HTLV-1), which infects 20 million people worldwide, and is the etiological agent of a neurodegenerative disease called HTLV-1 Associated Myelopathy/Tropical Spastic Paraparesis (HAM/TSP). We first demonstrated that the cerebral endothelial cells express the receptors for the retrovirus in vitro, and on spinal cord autopsy sections from non-infected and HAM/TSP patients. We found on these latter that vascular-like structures were infected and confirmed in vitro that the endothelial cells could be productively infected by HTLV-1. We demonstrated that such an infection impairs BBB properties in vitro, as well as tight junctions, that are cell adhesion structures. This study is the first to demonstrate the impact of HTLV-1 infection on human BBB integrity; such a susceptibility has to be considered in the design of future therapeutics strategies.

International audience; We show human herpesvirus 8 with diverse molecular subtype D variants to be highly endemic among the Ni-Vanuatu population. Most K1 genes were nearly identical to Polynesian strains, although a few clustered with Australian or Taiwanese strains. These results suggest diverse origins of the Ni-Vanuatu population and raise questions about the ancient human population movements in Melanesia.

International audience; HHV-8-associated solid lymphomas which develop in extracavitary sites during the course of primary effusion lymphoma (PEL) could represent the relapse of original PEL tumors in different anatomical sites, or newly occurring distinct HHV-8-associated lymphomas, such as multicentric Castleman disease-related microlymphomas. HHV-8 episome clonality might help identify which event takes place.

An unprecedented epidemic of chikungunya virus (CHIKV) infection recently started in countries of the Indian Ocean area, causing an acute and painful syndrome with strong fever, asthenia, skin rash, polyarthritis, and lethal cases of encephalitis. The basis for chikungunya disease and the tropism of CHIKV remain unknown. Here, we describe the replication characteristics of recent clinical CHIKV strains. Human epithelial and endothelial cells, primary fibroblasts and, to a lesser extent, monocyte-derived macrophages, were susceptible to infection and allowed viral production. In contrast, CHIKV did not replicate in lymphoid and monocytoid cell lines, primary lymphocytes and monocytes, or monocyte-derived dendritic cells. CHIKV replication was cytopathic and associated with an induction of apoptosis in infected cells. Chloroquine, bafilomycin-A1, and short hairpin RNAs against dynamin-2 inhibited viral production, indicating that viral entry occurs through pH-dependent endocytosis. CHIKV was highly sensitive to the antiviral activity of type I and II interferons. These results provide a general insight into the interaction between CHIKV and its mammalian host., Author Summary Chikungunya virus (CHIKV) is a reemerging alphavirus responsible for an unprecedented epidemic in countries of the Indian Ocean region, causing an acute and painful syndrome with strong fever, asthenia, skin rash, polyarthritis, and lethal cases of encephalitis. The most recent epidemic reemergences were documented in Kinshasa, (50,000 estimated cases in 1999–2000), in Indonesia (2001–2003), the Indian Ocean islands of Mayotte, Mauritius, Réunion, and the Seychelles (270,000 cases in 2005–2006 in La Réunion island), and in India (1.4 to 6.5 million estimated cases in 2006–2007). There is a critical lack of knowledge on the biology of CHIKV. In particular, virtually nothing is known about the interaction of CHIKV (and of most alpahaviruses) with human primary cells. We have studied the replication characteristics and the tropism of clinical CHIKV strains from La Réunion. We designed various assays and reagents to follow viral replication, and we report here that adherent cells (epithelial and endothelial cells, primary fibroblasts), as well as macrophages, are sensitive to infection. In contrast, blood cells did not allow viral replication. We also characterized viral entry pathways and sensitivity to interferons. These results provide a general insight into the interaction between CHIKV and its mammalian host. This paper is the result of a collaborative effort between numerous teams from Institut Pasteur, the Groupe Hospitalier Sud Réunion, and other institutions. Our aim was to establish a task force with multiple and complementary expertise on virology, immunology, and cell biology in order to characterize this enigmatic virus.

Centrosomes are the major microtubule organizing structures in vertebrate cells. They localize in close proximity to the nucleus for the duration of interphase and play major roles in numerous cell functions. Consequently, any deficiency in centrosome function or number may lead to genetic instability. Several viruses including retroviruses such as, Foamy Virus, HIV-1, JSRV, M-PMV and HTLV-1 have been shown to hamper centrosome functions for their own profit, but the outcomes are very different. Foamy viruses, HIV-1, JSRV, M-PMV and HTLV-1 use the cellular machinery to traffic towards the centrosome during early and/or late stages of the infection. In addition HIV-1 Vpr protein alters the cell-cycle regulation by hijacking centrosome functions. Enthrallingly, HTLV-1 Tax expression also targets the functions of the centrosome, and this event is correlated with centrosome amplification, aneuploidy and transformation.

In several areas of high endemicity, HTLV-1 can be transmitted from mother to child, through prolonged breast-feeding. This way of transmission is particularly linked to the development of Adult T Cell Leukemia (ATL). In this context, we studied the mechanisms of HTLV-1 transmission through the digestive tract. We previously demonstrated in an in vitro model of compartimentalized co-culture device of human enterocytes, lymphocytes and dendritic cells, that HTLV-1 was unable to infect enterocytes, or disrupt tight cell junctions, but could rather be transported by transcytosis to infect sub-epithelial dendritic cells (Martin-Latil et al., Blood, 2012). Since HTLV-1 infection has been shown to be more efficient through cell-associated virions, we also focused on the crossing of HTLV-1 infected lymphocytes through the intestinal barrier. We thus studied the migration of HTLV-1 infected lymphocytes across the intestinal epithelium, both ex vivo (on intestinal explant model) and in vitro (on a monolayer of human enterocytic cell line cultured on Transwell devices). We demonstrate that, in both systems, infected lymphocytes are able to cross the epithelial barrier, with increased capacities compared to uninfected lymphocytes. Our work suggests that HTLV-1 infected lymphocytes efficiently cross the intestinal barrier, which could constitute an additional way for mother to child HTLV-1 transmission during breast-feeding.

Simian foamy virus (SFV) is a retrovirus ubiquitous in non-human primates (NHPs) that can be transmitted to humans, mostly through severe bites. In the past few years, our laboratory identified more than 50 hunters from Central Africa infected with zoonotic SFVs. Analysis of SFV complete sequences obtained from 5 of these individuals had revealed that the env gene was the most variable one. Furthermore, recombinant SFV strains have been recently shown; some of them involved the env gene. This led us to investigate the env gene variability of zoonotic SFV strains, looking especially for possible recombinants. We sequenced the complete or the surface glycoprotein region (SU) of SFV env gene amplified from blood DNA of: 1) a series of 40 individuals from Cameroon or Gabon infected with a gorilla or chimpanzee FV strain; 2) one gorilla and 3 infected chimpanzees living in the same areas than the hunters. All sequences were aligned and analysed by phylogenetic (neighbour-joining and maximum likelihood) and recombinant detection methods (similarity plot and bootscan analysis, Recombination Detection Program). Phylogenetic analyses revealed the existence of two different env variants among both gorilla FV and chimpanzee FV strains. These were present in zoonotic as well as in NHP strains. These variants differ greatly (more than 30% variability) in a 750 bp long region located in the receptor binding domain of the SU; the rest of the gene is very conserved (less than 5% variability among strains from the same species). Within a given variant, protein sequences of the SU are very conserved and stable (non-synonymous/synonymous mutations ratio

International audience; BACKGROUND: Melanesia is endemic for human T lymphotropic virus type 1 (HTLV-1) subtype C. In 2005, we identified 4 infected women from Ambae Island, Vanuatu. Subsequently, 4247 Ni-Vanuatu originating from 18 islands were enrolled to define HTLV-1 epidemiological determinants and to characterize the viral strains molecularly. METHODS: Plasma from 1074 males and 3173 females were screened for HTLV-1/2 antibodies by particle agglutination (PA) and an immunofluorescence assay (IFA). Positive and/or borderline samples were then tested by a Western blot (WB) confirmatory assay. DNAs were amplified to obtain a 522-bp env gene fragment. Phylogenetic and molecular-clock analyses were performed. RESULTS: Of 4247 samples, 762 were positive and/or borderline by IFA/PA, and 26 of them were confirmed to be HTLV-1 positive by WB. The overall HTLV-1 seroprevalence was 0.62%. Viral transmission was found within families of infected index case patients. A geographic heterogeneity of HTLV-1 seroprevalence was observed among the islands. All 41 of the new env sequences belonged to HTLV-1 subtype C. Phylogenetic and molecular-clock analyses suggested that Ni-Vanuatu and Solomon Islander strains emerged from a common ancestor ~10,000 years ago. CONCLUSION: The Vanuatu archipelago is endemic for HTLV-1 with a diversity of subtype C variants. These strains were probably introduced into Vanuatu during ancient migration of the original settlers a few thousand years ago.

Background HTLV-1 infection is endemic among people of Melanesian descent in Papua New Guinea, the Solomon Islands and Vanuatu. Molecular studies reveal that these Melanesian strains belong to the highly divergent HTLV-1c subtype. In Australia, HTLV-1 is also endemic among the Indigenous people of central Australia; however, the molecular epidemiology of HTLV-1 infection in this population remains poorly documented. Findings Studying a series of 23 HTLV-1 strains from Indigenous residents of central Australia, we analyzed coding (gag, pol, env, tax) and non-coding (LTR) genomic proviral regions. Four complete HTLV-1 proviral sequences were also characterized. Phylogenetic analyses implemented with both Neighbor-Joining and Maximum Likelihood methods revealed that all proviral strains belong to the HTLV-1c subtype with a high genetic diversity, which varied with the geographic origin of the infected individuals. Two distinct Australians clades were found, the first including strains derived from most patients whose origins are in the North, and the second comprising a majority of those from the South of central Australia. Time divergence estimation suggests that the speciation of these two Australian clades probably occurred 9,120 years ago (38,000–4,500). Conclusions The HTLV-1c subtype is endemic to central Australia where the Indigenous population is infected with diverse subtype c variants. At least two Australian clades exist, which cluster according to the geographic origin of the human hosts. These molecular variants are probably of very ancient origin. Further studies could provide new insights into the evolution and modes of dissemination of these retrovirus variants and the associated ancient migration events through which early human settlement of Australia and Melanesia was achieved., Author Summary The Human T-lymphotropic virus type 1 (HTLV-1) infects at least 5–10 million persons worldwide. In Oceania, previous studies have shown that HTLV-1 is present in a few ancient populations from remote areas of Papua New Guinea, the Solomon Islands, the Vanuatu archipelago and central Australia. The latter comprise one of the most socio-economically disadvantaged groups within any developed country. Characterization of the few available HTLV-1 viruses from Oceania indicates that these belong to a specific HTLV-1 genotype, the Australo-Melanesian c-subtype. In this study, we provide details for 23 HTLV-1 viruses derived from the Indigenous population of central Australia, a vast remote area of 1,000,000 km2. We reveal considerable genetic diversity of HTLV-1c subtype viruses and the existence of two HTLV-1c clades within which a high degree of genetic diversity was also apparent. These newly described HTLV-1c clades clustered according to the geographic origin of their human hosts. Indigenous Australians from the North of central Australia harbor HTLV-1c subtype viruses that are distinct from those of individuals from regions to the South. These data suggest that HTLV-1 was probably introduced to Australia during ancient migration events and was then confined to isolated Indigenous communities in central Australia.

The genetic variability of the human herpesvirus 8 (HHV-8) strains circulating in the populations living in the Maghreb region, an endemic area for HHV-8 and associated Kaposi's sarcoma, remains largely unknown. We have thus analyzed the genetic variation of the complete K1 gene of HHV-8 in a series of 35 viral strains, originating from 28 Moroccan patients with classic, AIDS-associated or iatrogenic Kaposi's sarcoma lesions. All but one of the 35 strains belonged to the large C molecular subtype. Furthermore, high genetic diversity within the C subtype was observed in the 35 sequenced HHV-8 K1 genes, with strains belonging to several and distinct subgroups highly supported from a phylogenetically viewpoint (e.g., C3, C7, C″ and C5). Considering these newly identified Moroccan viral strains in the context of 189 complete K1 genes, we were able to characterized, using the Simplot program, two main groups of recombinant chimeric K1 genes, either intertypic (C5) or intratypic (C7). In addition, the genetic characterization of the host maternal gene pool, through the analyses of mtDNA variation, did not provide evidence for any association between a particular human ethno-geographic background (i.e., North African vs. sub-Saharan African vs. West Eurasian linages) and any HHV-8 strain because both C′ and C″ strains were randomly distributed among the different patients' population backgrounds.

International audience; Background:Presence and origin of endemic foci of HTLV-2 infection in Africa remain a matter of debate.Material and methods:To better appreciate the epidemiological and molecular determinants of HTLV-2 infection in Central Africa, we performed a survey in 3903 inhabitants of a South Cameroon forest area, including 1051 Bakola Pygmies, 815 Baka Pygmies and 2037 Bantus living in their neighboring. HTLV-1 and HTLV-2 infection was determined by both specific serological (IFA and WB) and molecular (different generic and specific PCR) methods.Results:HTLV-1/2 prevalence was of 3% (117/3903) with 90 HTLV-1 (2.3%) and 27 HTLV-2 (0.7%). Surprisingly, HTLV-2 infection was restricted to Bakola Pygmies (27/1051 2.5%) with no HTLV-2 infection in any of the 2852 Baka or Bantus individuals. In Bakola Pygmies, HTLV-2 seroprevalence increased with age, reaching 6.5% in the elder persons. Ongoing intrafamilial HTLV-2 transmission was evidenced. Lymphoid T cell lines (CD8+ or CD4+, CD25 +) producing HTLV-2 antigens, were established from PBMCs cultures of HTLV-2 infected individuals. Sequences of a 672 nucleotide LTR fragment, obtained from 7 HTLV-2 samples, showed a very high degree of homologies among samples (< 1% nucleotide divergence) but also surprisingly with Amerindian HTLV-2 B strains. Complete sequence (8954 bp) of one isolate confirmed a typical HTLV-2 B strain.Conclusion:This study demonstrates clearly a HTLV-2 endemic population, with ongoing transmission, in Central Africa. Furthermore, it gives insights into several central questions regarding the origin and evolution rate of HTLV-2 and the migrations of infected populations.