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Rodents are important reservoirs of numerous viruses, some of which have significant impacts on public health. Ecosystem disturbances and decreased host species richness have been associated with the emergence of zoonotic diseases. In this study, we aimed at (a) characterizing the viral diversity in seven neotropical rodent species living in four types of habitats and (b) exploring how the extent of environmental disturbance influences this diversity. Through a metagenomic approach, we identified 77,767 viral sequences from spleen, kidney, and serum samples. These viral sequences were attributed to 27 viral families known to infect vertebrates, invertebrates, plants, and amoeba. Viral diversities were greater in pristine habitats compared with disturbed ones, and lowest in peri-urban areas. High viral richness was observed in savannah areas. Differences in these diversities were explained by rare viruses that were generally more frequent in pristine forest and savannah habitats. Moreover, changes in the ecology and behavior of rodent hosts, in a given habitat, such as modifications to the diet in disturbed vs. pristine forests, are major determinants of viral composition. Lastly, the phylogenetic relationships of four vertebrate-related viral families (Polyomaviridae, Flaviviridae, Togaviridae, and Phenuiviridae) highlighted the wide diversity of these viral families, and in some cases, a potential risk of transmission to humans. All these findings provide significant insights into the diversity of rodent viruses in Amazonia, and emphasize that habitats and the host’s dietary ecology may drive viral diversity. Linking viral richness and abundance to the ecology of their hosts and their responses to habitat disturbance could be the starting point for a better understanding of viral emergence and for future management of ecosystems.

International audience; Yellow fever virus (YFV) is an RNA virus primarily targeting the liver. Severe YF cases are responsible for hemorrhagic fever, plausibly precipitated by excessive proinflammatory cytokine response. Pathogen recognition receptors (PRRs), such as the cytoplasmic retinoic acid inducible gene I (RIG-I)-like receptors (RLRs), and the viral RNA sensor protein kinase R (PKR), are known to initiate a proinflammatory response upon recognition of viral genomes. Here, we sought to reveal the main determinants responsible for the acute cytokine expression occurring in human hepatocytes following YFV infection. Using a RIG-I-defective human hepatoma cell line, we found that RIG-I largely contributes to cytokine secretion upon YFV infection. In infected RIG-I-proficient hepatoma cells, RIG-I was localized in stress granules. These granules are large aggregates of stalled translation preinitiation complexes known to concentrate RLRs and PKR and are so far recognized as hubs orchestrating RNA virus sensing. Stable knockdown of PKR in hepatoma cells revealed that PKR contributes to both stress granule formation and cytokine induction upon YFV infection. However, stress granule disruption did not affect the cytokine response to YFV infection, as assessed by small interfering RNA (siRNA)-knockdown-mediated inhibition of stress granule assembly. Finally, no viral RNA was detected in stress granules using a fluorescence hybridization approach coupled with immunofluorescence. Our findings suggest that both RIG-I and PKR mediate proinflammatory cytokine induction in YFV-infected hepatocytes, in a stress granule-independent manner. Therefore, by showing the uncoupling of the cytokine response from the stress granule formation, our model challenges the current view in which stress granules are required for the mounting of the acute antiviral response. Yellow fever is a mosquito-borne acute hemorrhagic disease caused by yellow fever virus (YFV). The mechanisms responsible for its pathogenesis remain largely unknown, although increased inflammation has been linked to worsened outcome. YFV targets the liver, where it primarily infects hepatocytes. We found that two RNA-sensing proteins, RIG-I and PKR, participate in the induction of proinflammatory mediators in human hepatocytes infected with YFV. We show that YFV infection promotes the formation of cytoplasmic structures, termed stress granules, in a PKR- but not RIG-I-dependent manner. While stress granules were previously postulated to be essential platforms for immune activation, we found that they are not required for the production of proinflammatory mediators upon YFV infection. Collectively, our work uncovered molecular events triggered by the replication of YFV, which could prove instrumental in clarifying the pathogenesis of the disease, with possible repercussions for disease management.

UDP-glucose (UDP-Glc) is synthesized by UGP2-encoded UDP-Glc pyrophosphorylase (UGP) and is required for glycoconjugate biosynthesis and galactose metabolism because it is a uridyl donor for galactose-1-P (Gal1P) uridyltransferase. Chinese hamster lung fibroblasts harboring a hypomrphic UGP(G116D) variant display reduced UDP-Glc levels and cannot grow if galactose is the sole carbon source. Here, these cells were cultivated with glucose in either the absence or presence of galactose in order to investigate glycoconjugate biosynthesis and galactose metabolism. The UGP-deficient cells display &lt, 5% control levels of UDP-Glc/UDP-Gal and &gt, 100-fold reduction of [6-3H]galactose incorporation into UDP-[6-3H]galactose, as well as multiple deficits in glycoconjugate biosynthesis. Cultivation of these cells in the presence of galactose leads to partial restoration of UDP-Glc levels, galactose metabolism and glycoconjugate biosynthesis. The Vmax for recombinant human UGP(G116D) with Glc1P is 2000-fold less than that of the wild-type protein, and UGP(G116D) displayed a mildly elevated Km for Glc1P, but no activity of the mutant enzyme towards Gal1P was detectable. To conclude, although the mechanism behind UDP-Glc/Gal production in the UGP-deficient cells remains to be determined, the capacity of this cell line to change its glycosylation status as a function of extracellular galactose makes it a useful, reversible model with which to study different aspects of galactose metabolism and glycoconjugate biosynthesis.

International audience; Background: We developed a new approach for the treatment of enterovirus infections, the consecutive alternating administration (CAA) of a combination of enterovirus inhibitors. On the model of coxsackievirus B1 (CVB1) in mice, two phenomena were observed: absence of drug resistance and increased susceptibility to the antivirals. This study aims to clarify the genetic basis of these phenomena. Methods: Brain samples from CVB1-infected mice subjected to a CAA course with the combination pleconaril/MDL-860/oxoglaucine were used for viral RNA extraction and next generation sequencing. In parallel, samples from monotherapeutic courses of the three substances included in the combination were studied. Whole genome sequence analysis was carried out on all samples. Results: Samples of pleconaril monotherapy showed mutations in 5 0 untranslated region, VP3, 2C, 3C and 2A regions of viral RNA, translated in amino acid substitution of the 2A protein. The MDL-860 course induced changes in CVB1 RNA in the VP3 and 2C regions. The oxoglaucine monotherapy samples showed RNA mutation and amino acid substitution in the VP1 region and nucleotide substitution in the 3D region. In the specimens taken from mice subjected to the CAA course with pleconaril/MDL-860/oxoglaucine, the following RNA mutations were established: 5 0 untranslated region, 2A, and 2B, and amino acids substitutions in VP3 and 2A, which differ from those mentioned above. These changes could be the reason for the prevention of drug resistance development and also to be considered as the basis for the phenomenon of increased drug susceptibility. Conclusions: The results reveal that the high anti-enteroviral efficacy of the CAA course is substantiated by the appearance of specific changes in the viral genome.

Retroviral integrase (IN) proteins catalyze the permanent integration of the viral genome into host DNA. They can productively recruit cellular proteins, and the human Bromodomain and Extra-Terminal domain (hBET) proteins have been shown to be co-factors for integration of gamma-retroviruses such as Murine Leukemia Virus (MLV) into human cells. By using two-hybrid, co-immunoprecipitation and in vitro interaction assays, we showed that IN of the gamma- Porcine Endogenous Retrovirus-A/C (PERV IN) interacts through its C-terminal domain (CTD) with hBET proteins. We observed that PERV IN interacts with the BRD2, BRD3 and BRD4 proteins in vitro and that the BRD2 protein specifically binds and co-localizes with PERV IN protein in the nucleus of cells. We further mapped the interaction sites to the conserved Extra-Terminal (ET) domain of the hBET proteins and to several amino acids of the of the C-terminal tail of the PERV IN CTD. Finally, we determined the first experimental structure of an IN CTD - BET ET complex from small-angle X-ray scattering data (SAXS). We showed that the two factors assemble as two distinct modules linked by a short loop which confers partial flexibility. The SAXS-restrained model is structurally compatible with the binding of the PERV intasome to BRD2. Altogether, these data confirm the important role of host BET proteins in the gamma-retroviruses' targeting site and efficiency of integration.

High-throughput sequencing furnishes a large number of short sequence reads from uncloned DNA and has rapidly become a major tool for identifying viruses in biological samples, and in particular when the target sequence is undefined. In this study, we assessed the analytical sensitivity of a pipeline for detection of viruses in biological samples based on either the Roche-454 genome sequencer or Illumina genome analyzer platforms. We sequenced biological samples artificially spiked with a wide range of viruses with genomes composed of single or double-stranded DNA or RNA, including linear or circular single-stranded DNA. Viruses were added at a very low concentration most often corresponding to 3 or 0.8 times the validated level of detection of quantitative reverse transcriptase PCRs (RT-PCRs). For the viruses represented, or resembling those represented, in public nucleotide sequence databases, we show that the higher output of Illumina is associated with a much greater sensitivity, approaching that of optimized quantitative (RT-)PCRs. In this blind study, identification of viruses was achieved without incorrect identification. Nevertheless, at these low concentrations, the number of reads generated by the Illumina platform was too small to facilitate assembly of contigs without the use of a reference sequence, thus precluding detection of unknown viruses. When the virus load was sufficiently high, de novo assembly permitted the generation of long contigs corresponding to nearly full-length genomes and thus should facilitate the identification of novel viruses.

Most studies on the ability of insect populations to transmit pathogens consider only constant temperatures and do not account for realistic daily temperature fluctuations that can impact vector–pathogen interactions. Here, we show that diurnal temperature range (DTR) affects two important parameters underlying dengue virus (DENV) transmission by Aedes aegypti . In two independent experiments using different DENV serotypes, mosquitoes were less susceptible to virus infection and died faster under larger DTR around the same mean temperature. Large DTR (20 °C) decreased the probability of midgut infection, but not duration of the virus extrinsic incubation period (EIP), compared with moderate DTR (10 °C) or constant temperature. A thermodynamic model predicted that at mean temperatures 18 °C, larger DTR reduces DENV transmission. The negative impact of DTR on Ae. aegypti survival indicates that large temperature fluctuations will reduce the probability of vector survival through EIP and expectation of infectious life. Seasonal variation in the amplitude of daily temperature fluctuations helps to explain seasonal forcing of DENV transmission at locations where average temperature does not vary seasonally and mosquito abundance is not associated with dengue incidence. Mosquitoes lived longer and were more likely to become infected under moderate temperature fluctuations, which is typical of the high DENV transmission season than under large temperature fluctuations, which is typical of the low DENV transmission season. Our findings reveal the importance of considering short-term temperature variations when studying DENV transmission dynamics.

© the American Society for Biochemistry and Molecular Biology"; International audience; Obtaining accurate protein profiles from homogeneous cell populations in heterogeneous tissues can enhance the capability to discover protein biomarkers. In this context, methodologies to access specific cellular populations and analyze their proteome with exquisite sensitivity have to be selected. We report here the results of an investigation using a combination of laser microdissection and accurate mass and time tag proteomics. The study was aimed at the precise determination of proteome alterations in intrahepatic cholangiocarcinoma ICC, a markedly heterogeneous tumor. This cancer, which is difficult to diagnose and carries a very poor prognosis, has shown an unexplained increase in incidence over the last few years. Among a pool of 574 identified proteins, we were able to report on altered abundance patterns affecting 39 proteins conforming to a variety of potential tumorigenic pathways. The reliability of the proteomics results was confirmed by Western blot and immunohistochemistry on matched samples. Most of the proteins displaying perturbed abundances had not yet been described in the setting of ICC. These include proteins involved in cell mobility and actin cytoskeleton remodeling, which may participate in the epithelial to mesenchymal transition, a process invoked in migration and invasion of cancer cells. The biological relevance of these findings was explored using a tissue microarray. An increased abundance of vimentin was thus detected in 70% of ICC and none of the controls. These results suggest that vimentin could play a role in the aggressiveness of ICC and provide a basis for the serious outcome of this cancer.

Background. Defective hepatitis B virus (HBV) particles, generated from singly spliced HBV RNA, have been detected in chronic carriers of HBV. The present study was designed to quantify the expression of defective HBV (dHBV) and wild-type HBV (wtHBV) genomes in the serum of patients with HBV infection and its relation to the severity of liver disease. Methods. HBV and dHBV loads were determined by quantitative polymerase chain reaction in the serum of 89 untreatedHBV-infectedpatients(31coinfectedwithhumanimmunodeficiencyvirus[HIV]type1)withliverdisease of different stages. The ratio of dHBV DNA to total (wtHBV plus dHBV) HBV DNA (dHBV/HBV ratio) was used to express data independently of the level of viral replication. Results. DespiteaglobalcorrelationbetweendHBVandwtHBVload,thedHBV/HBVratiorangedfrom0.001% to 69%. The variation in dHBV/HBV ratio was independent of HIV coinfection, HBV genotype, and precore mutations. The mean dHBV/HBV ratio was higher in patients with severe liver necrosis and fibrosis. Conclusions. OurdataindicatethatanelevateddHBV/HBVratioisassociatedwithlivernecroinflammationand fibrosis disease, suggesting a regulation of dHBV expression according to the severity of the liver disease. The dHBV/ HBV ratio may help to better define liver disease stage during HBV infection. Persistent human hepatitis B virus (HBV) infection is a major public health problem. The major complications of chronic HBV infection are the development of liver cirrhosis and hepatocellular carcinoma (HCC) [1‐3]. Evidence indicating a direct involvement of HBV in this process is now available [3]; indeed, recent data have demonstrated that serum HBV DNA levels are strong predictorsoftheriskofHCC,independentofhepatitisB e antigen (HBeAg) expression, serum alanine amino

We have developed a new strategy for antiviral peptide discovery by using lyssaviruses (rabies virus and rabies-related viruses) as models. Based on the mimicry of natural bioactive peptides, two genetically encoded combinatorial peptide libraries composed of intrinsically constrained peptides (coactamers) were designed. Proteomic knowledge concerning the functional network of interactions in the lyssavirus transcription-replication complex highlights the phosphoprotein (P) as a prime target for inhibitors of viral replication. We present an integrated, sequential drug discovery process for selection of peptides with antiviral activity directed against the P. Our approach combines (i) an exhaustive two-hybrid selection of peptides binding two phylogenetically divergent lyssavirus P's, (ii) a functional analysis of protein interaction inhibition in a viral reverse genetic assay, coupled with a physical analysis of viral nucleoprotein-P complex by protein chip mass spectrometry, and (iii) an assay for inhibition of lyssavirus infection in mammalian cells. The validity of this strategy was demonstrated by the identification of four peptides exhibiting an efficient antiviral activity. Our work highlights the importance of P as a target in anti-rabies virus drug discovery. Furthermore, the screening strategy and the coactamer libraries presented in this report could be considered, respectively, a general target validation strategy and a potential source of biologically active peptides which could also help to design pharmacologically active peptide-mimicking molecules. The strategy described here is easily applicable to other pathogens.

During a study of the fecal microbiomes from two healthy piglets using high-throughput sequencing (HTS), we identified a viral genome containing an open reading frame encoding a predicted polyprotein of 2,133 amino acids. This novel viral genome displayed the typical organization of picornaviruses, containing three structural proteins (VP0, VP3, and VP1), followed by seven nonstructural proteins (2A, 2B, 2C, 3A, 3B, 3C pro , and 3D pol ). Given its particular relationship with Parechovirus , we propose to name it “Pasivirus” for Pa recho si ster clade virus, with “Swine pasivirus 1” (SPaV1) as the type species. Fecal samples collected at an industrial farm from healthy sows and piglets from the same herd (25 and 75, respectively) with ages ranging from 4 to 28 weeks were analyzed for the presence of SPaV1 by one-step reverse transcription (RT)-PCR targeting a 3D region of 151 bp. SPaV1 was detected in fecal samples from 51/75 healthy piglets (68% of the animals) and in none of the 25 fecal samples from healthy sows, indicating that SPaV1 circulates through enteric infection of healthy piglets. We propose that SPaV1 represents the first member of a novel Picornaviridae genus related to parechoviruses.

International audience; The human skin is a complex ecosystem that hosts a heterogeneous flora. Until recently, the diversity of the cutaneous microbiota was mainly investigated for bacteria through culture based assays subsequently confirmed by molecular techniques. There are now many evidences that viruses represent a significant part of the cutaneous flora as demonstrated by the asymptomatic carriage of beta and gamma-human papillomaviruses on the healthy skin. Furthermore, it has been recently suggested that some representatives of the Polyomavirus genus might share a similar feature. In the present study, the cutaneous virome of the surface of the normal-appearing skin from five healthy individuals and one patient with Merkel cell carcinoma was investigated through a high throughput metagenomic sequencing approach in an attempt to provide a thorough description of the cutaneous flora, with a particular focus on its viral component. The results emphasize the high diversity of the viral cutaneous flora with multiple polyomaviruses, papillomaviruses and circoviruses being detected on normal-appearing skin. Moreover, this approach resulted in the identification of new Papillomavirus and Circovirus genomes and confirmed a very low level of genetic diversity within human polyomavirus species. Although viruses are generally considered as pathogen agents, our findings support the existence of a complex viral flora present at the surface of healthy-appearing human skin in various individuals. The dynamics and anatomical variations of this skin virome and its variations according to pathological conditions remain to be further studied. The potential involvement of these viruses, alone or in combination, in skin proliferative disorders and oncogenesis is another crucial issue to be elucidated.

Dengue viruses (DENV) are characterized by extensive genetic diversity and can be organized in multiple, genetically distinct lineages that arise and die out on a regular basis in regions where dengue is endemic. A fundamental question for understanding DENV evolution is the relative extent to which stochastic processes (genetic drift) and natural selection acting on fitness differences among lineages contribute to lineage diversity and turnover. Here, we used a set of recently collected and archived low-passage DENV-1 isolates from Thailand to examine the role of mosquito vector-virus interactions in DENV evolution. By comparing the ability of 23 viruses isolated on different dates between 1985 and 2009 to be transmitted by a present-day Aedes aegypti population from Thailand, we found that a major clade replacement event in the mid-1990s was associated with virus isolates exhibiting increased titers in the vector's hemocoel, which is predicted to result in a higher probability of transmission. This finding is consistent with the hypothesis that selection for enhanced transmission by mosquitoes is a possible mechanism underlying major DENV clade replacement events. There was significant variation in transmission potential among isolates within each clade, indicating that in addition to vector-driven selection, other evolutionary forces act to maintain viral genetic diversity. We conclude that occasional adaptive processes involving the mosquito vector can drive major DENV lineage replacement events.

International audience

International audience; Dengue virus (DENV) causes the major arboviral disease of the tropics, characterized in its severe forms by signs of hemorrhage and plasma leakage. DENV encodes a nonstructural glycoprotein, NS1, that associates with intracellular membranes and the cell surface. NS1 is eventually secreted as a soluble hexamer from DENV-infected cells and circulates in the bloodstream of infected patients. Extracellular NS1 has been shown to modulate the complement system and to enhance DENV infection, yet its structure and function remain essentially unknown. By combining cryoelectron microscopy analysis with a characterization of NS1 amphipathic properties, we show that the secreted NS1 hexamer forms a lipoprotein particle with an open-barrel protein shell and a prominent central channel rich in lipids. Biochemical and NMR analyses of the NS1 lipid cargo reveal the presence of triglycerides, bound at an equimolar ratio to the NS1 protomer, as well as cholesteryl esters and phospholipids, a composition evocative of the plasma lipoproteins involved in vascular homeostasis. This study suggests that DENV NS1, by mimicking or hijacking lipid metabolic pathways, contributes to endothelium dysfunction, a key feature of severe dengue disease.

International audience; The enzyme tissue non-specific alkaline phosphatase (TNAP) belongs to the ectophosphatase family. It is present in large amounts in bone in which it plays a role in mineralization but little is known about its function in other tissues. Arguments are accumulating for its involvement in the brain, in particular in view of the neurological symptoms accompanying human TNAP deficiencies. We have previously shown, by histochemistry, alkaline phosphatase (AP) activity in monkey brain vessels and parenchyma in which AP exhibits specific patterns. Here, we clearly attribute this activity to TNAP expression rather than to other APs in primates (human and marmoset) and in rodents (rat and mouse). We have not found any brain-specific transcripts but our data demonstrate that neuronal and endothelial cells exclusively express the bone TNAP transcript in all species tested, except in mouse neurons in which liver TNAP transcripts have also been detected. Moreover, we highlight the developmental regulation of TNAP expression; this also acts during neuronal differentiation. Our study should help to characterize the regulation of the expression of this ectophosphatase in various cell types of the central nervous system.

International audience; Similar to many other host-pathogen interactions, the vector competence of Aedes aegypti for dengue viruses appears to be determined by genotype-by-genotype interactions, whereby the outcome of infection depends on the specific combination of mosquito and virus genotypes. This can complicate efforts to dissect the genetic basis of vector competence in nature because it obscures mapping between genotype and phenotype and brings into question the notion of universal mosquito resistance or susceptibility. Conversely, it offers novel opportunities to better define compatible vector-pathogen associations based on integration of both vector and pathogen genomics, which should eventually improve understanding of pathogen transmission dynamics and the risk of vector-borne disease emergence.

International audience; Background: Dengue virus is transmitted by mosquitoes and has four serotypes. Cross-protection to other serotypes lasting for a few months is observed following infection with one serotype. There is evidence that low-affinity T and/or B cells from primary infections contribute to the severe syndromes often associated with secondary dengue infections. such pronounced immune-mediated enhancement suggests a dengue-specific pattern of immune cell activation. This study investigates the acute and early convalescent B cell response leading to the generation of cross-reactive and neutralizing antibodies following dengue infection.Methodology/Principal Findings : We assayed blood samples taken from dengue patients with primary or secondary infection during acute disease and convalescence and compared them to samples from patients presenting with non-dengue related fever. Dengue induced massive early plasmablast formation, which correlated with the appearance of polyclonal, cross-reactive IgG for both primary and secondary infection. Surprisingly, the contribution of IgG to the neutralizing titer 4-7 days after fever onset was more than 50% even after primary infection.Conclusions/Significance : Poly-reactive and virus serotype cross-reactive IgG are an important component of the innate response in humans during both primary and secondary dengue infection, and "innate specificities" seem to constitute part of the adaptive response in dengue. While of potential importance for protection during secondary infection, cross-reactive B cells will also compete with highly neutralizing B cells and possibly interfere with their development.

© 2011 Wolters Kluwer Health | Lippincott Williams & Wilkins, OBJECTIVE: The contribution of naive CD4+ T cells to the pool of HIV-infected cells remains poorly described. This study aimed at evaluating HIV infection in naive T-cell subsets in viremic and HAART-treated patients, together with various parameters implicated in naive T-cell homeostasis, in order to better understand infection in these subsets. DESIGN AND METHODS: HIV provirus was quantified in various FACS-sorted CD4/CD8 T-cell subsets [recent thymic emigrants (RTEs), non-RTE naives and memory T cells] purified from peripheral blood cells of untreated viremic and HAART-treated aviremic HIV-infected patients. HIV proviral DNA was quantified using a highly sensitive real-time PCR assay allowing detection of one HIV copy in 10⁵ cells. Intrathymic precursor T-cell proliferation and circulating T-cell cycling were, respectively, evaluated through measurement of the sj/βTREC ratio (signal joint T-Cell Receptor Excision Circle frequency divided by DβJβTREC frequency) and Ki-67 expression. Plasma interleukin (IL)-7 concentrations were measured by ELISA. RESULTS: RTEs and non-RTEs were equally HIV infected. Altogether, naive CD4+ T cells represented 0.24%-60% of the infected cells. In contrast, HIV DNA was undetectable in naive CD8+ T cells. RTE infection rate directly correlated with IL-7 plasma levels (r = 0.607, P = 0.0035) but was independent from plasma viral load, peripheral T-cell cycling and intrathymic precursor T-cell proliferation. CONCLUSION: We demonstrated that RTEs are effectively HIV infected. The similar infection rate observed in RTEs and other naive T cells, its relationship with plasma IL-7 levels, together with the lack of correlation between RTE infection and either thymic or peripheral proliferation, strongly suggests that RTE infection occurs either late during thymopoiesis or early on during their extrathymic maturation.

Interferon-α (IFN-α)–based therapy is presently the standard treatment for hepatitis C virus (HCV)–infected patients. Despite good effectiveness, this cytokine is associated with major side effects, including significant lymphopenia, that limits its use for HIV/HCV-coinfected patients. Interleukin-7 (IL-7) has recently shown therapeutic potential and safety in several clinical trials designed to demonstrate T-cell restoration in immunodeficient patients. The purpose of this study was to evaluate, in simian immunodeficiency virus-infected rhesus macaques, the relevance of IL-7 therapy as a means to overcoming IFN-α–induced lymphopenia. We showed that low-dose IFN-α treatment induced strong lymphopenia in chronically infected monkeys. In contrast, high-dose IFN-α treatment stimulated IL-7 production, leading to increased circulating T-cell counts. Moreover, IL-7 therapy more than abrogated the lymphopenic effect of low-dose IFN-α. Indeed, the association of both cytokines resulted in increased circulating T-cell counts, in particular in the naive compartments, as a consequence of central and peripheral homeostatic functions of the IL-7. Finally, reduced PD-1 expression by memory CD8+ T cells and transient T-cell repertoire diversification were observed under IL-7 therapy. Our data strongly suggest that IL-7 immunotherapy will be of substantial benefit in the treatment of HIV/HCV coinfection and should enhance the likelihood of HCV eradication in poorly responding patients.

In this essay, I argue that unraveling thefull genetic architecture (i.e., the number,position, effect, and interactions amonggenes underlying phenotypic variation)and molecular landscape of host–pathogeninteractions can only be achieved byaccounting for their genetic specificity.Indeed, the outcome of host–pathogeninteractions often depends on the specificpairing of host and pathogen genotypes[1]. In such cases, the infection phenotypedoes not merely result from additive effectsof host and pathogen genotypes, but alsofrom a specific interaction between thetwo genomes (Box 1). This specific com-ponent, which can be measured by theinteraction term in a two-way statisticalanalysis of phenotypic variation as afunction of host and pathogen genotypes,is referred to as a genotype-by-genotype(G6G) interaction [1]. By analogy togenotype-by-environment (G6E) interac-tions that occur when different genotypesrespond differently to environmentalchange, G6G interactions occur whenthe response of host genotypes differsacross pathogen genotypes. Although theconcept of G6G interactions has mostlybeen used by evolutionary ecologists todescribe the specificity of host immunedefenses against pathogens [2], it can beapplied to any phenotype resulting fromthe specific interaction between two ge-nomes. The general definition of G6Ginteractions allows its use to characterizephenotypes ranging from macroscopictraits such as lifespan [3] to the level ofgene expression [4]. Here, the geneticspecificity of host–pathogen associations isdefined in the sense of G6G interactions.This definition differs from that of immu-nological specificity, which is the ability ofa host to recognize and mount an immuneresponse against a particular pathogengenotype or antigen. Whereas immuno-logical specificity often depends on infec-tion history (i.e., past exposure to apathogen), genetic specificity describesthe intrinsic compatibility between hostand pathogen genotypes and occurs inde-pendently of infection history.In some instances, the specificity ofhost–pathogen associations can be ex-plained to a large extent by major genesof hosts and pathogens, as in the gene-for-gene model of plant–pathogen compati-bility [5,6]. In general, however, multiplegenes and epistatic interactions amongthese genes determine the infection out-come [7–9]. A recent meta-analysis of 500published studies reporting quantitativetrait loci (QTL) for host resistance topathogens in plants and animals revealedthat the genetic architecture of this traitvaries dramatically across different combi-nations of host and pathogen genotypes[9]. Thus, different host–pathogen associ-ations involve different QTL and epistaticinteractions, indicating that a substantialportion of phenotypic variation derivesfrom the specific interaction between thetwo genomes. This is made even morecomplex when multiple pathogen speciesor strains infect the same host [10] and/orwhen G6G interactions are environment-dependent [11,12].It is striking that, to date, quantitativegenetic studies of host–pathogen systemshave neglected the specific component ofthe interaction. Dissecting the geneticarchitecture of complex infection traitshas traditionally relied on QTL mappingstrategies [7,9] and more recently onassociation analyses of candidate genepolymorphisms [8]. A major caveat ofthese QTL mapping and associationstudies is that they focus on either thehost or the pathogen genome. Becausethey consider variation in only one of thetwo interacting organisms, these studiesignore specific host genome by pathogengenome interactions. In order to fullydissect the genetic architecture and ex-plore the molecular landscape of host–pathogen interactions, it will be necessaryto account for the specific component ofthe relationship. This should be madepossible by recent developments in molec-ular strategies combining host and patho-gen genetics [13–15] and in quantitativegenetic models of host–pathogen interac-tions allowing detection of host QTL bypathogen QTL interactions [16,17]. Ad-vantage could also be taken from existingmethods for analysis of gene–gene andgene–environment interactions [18–21]. Acritical (and limiting) aspect for investigat-ing genetic specificity is the need toinclude different combinations of hostand pathogen genotypes in the experi-mental design.From a fundamental standpoint, im-proved knowledge of the genetic architec-ture of host–pathogen specificity hasimportant implications for our under-standing of the ecology and evolution ofhost–pathogen associations. The geneticspecificity of host–pathogen interactions isthought to promote the maintenance ofhost and pathogen genetic diversity viafrequency-dependent coevolutionary cy-cles [22–25], which in turn favor higherrates of mutation, recombination, andsexual reproduction [26]. Unraveling thegenetic architecture and molecular land-scape of host–pathogen specificity, com-bined with molecular evolution analyses,will shed light on the mechanistic basis ofthe infection process and the biochemistryof host–pathogen recognition [27–30].The genetic model and precise epistaticinteractions underlying host–pathogenspecificity are critical determinants of

International audience; The dramatic global expansion of Aedes albopictus in the last three decades has increased public health concern because it is a potential vector of numerous arthropod-borne viruses (arboviruses), including the most prevalent arboviral pathogen of humans, dengue virus (DENV). Ae. aegypti is considered the primary DENV vector and has repeatedly been incriminated as a driving force in dengue's worldwide emergence. What remains unresolved is the extent to which Ae. albopictus contributes to DENV transmission and whether an improved understanding of its vector status would enhance dengue surveillance and prevention. To assess the relative public health importance of Ae. albopictus for dengue, we carried out two complementary analyses. We reviewed its role in past dengue epidemics and compared its DENV vector competence with that of Ae. aegypti. Observations from "natural experiments" indicate that, despite seemingly favorable conditions, places where Ae. albopictus predominates over Ae. aegypti have never experienced a typical explosive dengue epidemic with severe cases of the disease. Results from a meta-analysis of experimental laboratory studies reveal that although Ae. albopictus is overall more susceptible to DENV midgut infection, rates of virus dissemination from the midgut to other tissues are significantly lower in Ae. albopictus than in Ae. aegypti. For both indices of vector competence, a few generations of mosquito colonization appear to result in a relative increase of Ae. albopictus susceptibility, which may have been a confounding factor in the literature. Our results lead to the conclusion that Ae. albopictus plays a relatively minor role compared to Ae. aegypti in DENV transmission, at least in part due to differences in host preferences and reduced vector competence. Recent examples of rapid arboviral adaptation to alternative mosquito vectors, however, call for cautious extrapolation of our conclusion. Vector status is a dynamic process that in the future could change in epidemiologically important ways.

Background HIV infection leads to a progressive depletion of uninfected CD4 cells. We showed that NKp44L, a cellular ligand for an activating NK receptor is over-expressed during HIV infection and is correlated with both CD4 depletion and increase in viral load. NKp44L+CD4+ cells are highly sensitive to the NK lysis activity. However, HIV-infected cells are resistant to NK killing, suggesting that HIV-1 develop escape mechanisms to counteract NK cytotoxicity.

We characterized the localization, phenotype, and some functions of plasmacytoid dendritic cells (pDCs) in the human spleen. pDCs were localized in the marginal zone and the periarteriolar region. Some were also found in the red pulp. pDCs were immature by phenotypic labeling, consistently with their capacity to internalize Dextran in a functional assay. In spleens from HIV-infected patients with thrombocytopenic purpura, these characteristics were unaffected. However, an accumulation of pDCs, but not myeloid dendritic cells (mDCs), was observed in some HIV+ patients, correlating with high proviral loads. Moreover, although undetectable in most HIV− patients, interferon-α (IFN-α) production was evidenced in situ and by flow cytometry in most HIV+ patients. IFN-α was located in the marginal zone. Surprisingly, IFN-α colocalized only with few pDCs, but rather with other cells, including T and B lymphocytes, mDCs, and macrophages. Therefore, pDCs accumulated in spleens from HIV+ patients with high proviral loads, but they did not seem to be the main IFN-α producers.

International audience; The TAR RNA binding Protein, TRBP, inhibits the activity of the interferon-induced protein kinase R (PKR), whereas the PKR activator, PACT, activates its function. TRBP and PACT also bind to each other through their double-stranded RNA binding domains (dsRBDs) and their Medipal domains, which may influence their activity on PKR. In a human immunodeficiency virus (HIV) long terminal repeat-luciferase assay, PACT unexpectedly reversed PKR-mediated inhibition of gene expression. In a translation inhibition assay in HeLa cells, PACT lacking the 13 C-terminal amino acids (PACTDelta13), but not full-length PACT, activated PKR and enhanced interferon-mediated repression. In contrast, in the astrocytic U251MG cells that express low TRBP levels, both proteins activate PKR, but PACTDelta13 is stronger. Immunoprecipitation assays and yeast two-hybrid assays show that TRBP and PACTDelta13 interact very weakly due to a loss of binding in the Medipal domain. PACT-induced PKR phosphorylation was restored in Tarbp2(-/-) murine tail fibroblasts and in HEK293T or HeLa cells when TRBP expression was reduced by RNA interference. In HEK293T and HeLa cells, arsenite, peroxide, and serum starvation-mediated stresses dissociated the TRBP-PACT interaction and increased PACT-induced PKR activation, demonstrating the relevance of this control in a physiological context. Our results demonstrate that in cells, TRBP controls PACT activation of PKR, an activity that is reversed by stress.

International audience; The quantitation of human hepatitis B virus (HBV) in the serum of infected patients is recommended to characterize the course of chronic HBV infection. The aim of this prospective study was to evaluate the performance of the Abbott RealTime PCR assay for HBV DNA quantitation by comparison with the standard Versant HBV DNA 3.0 assay. The better sensitivity and broader dynamic range of HBV DNA quantitation using the Abbott RealTime PCR assay was confirmed by the study of 362 serum samples from 311 patients. In addition, data analysis revealed the concordance of HBV DNA quantitations between the two assays. When this evaluation was assessed as a function of HBV genotype, there was discordance for HBV genotype C samples. Thus, we performed an in-house PCR to confirm the discrepancy observed regarding the HBV genotypes. The in-house PCR results agreed better with the Abbott RealTime PCR method when compared with the standard hybridization assay. In conclusion, the wide dynamic range of HBV DNA quantitation achieved with the Abbott RealTime PCR assay makes it appropriate for the clinical monitoring of HBV infected patients. However, a change of HBV DNA quantitation method could influence results on the follow-up of HBV genotype C infected patients.

International audience; Human immunodeficiency virus (HIV-1) and hepatitis B virus (HBV) spread via similar transmission pathways, and infection by HBV occurs in up to 32% of HIV-1 cases. Here, we describe the successful expression of novel recombinant HIV-1/HBV virus-like particles (VLPs) in Nicotiana tabacum and Arabidopsis thaliana. The production levels and quality of the recombinant VLPs were comparable in the two plants, showing that parameters intrinsic to the recombinant proteins determined their assembly into VLPs. These heterologous VLPs can be used in a bivalent anti-HIV-1/-HBV vaccine, administrated via ingestion of transgenic plants.

International audience; Hepatocellular carcinoma (HCC) is a major cause of cancer worldwide. Proteomic studies provide opportunities to uncover targets for the diagnosis and treatment of this disease. However, in HCC developing in a setting of cirrhosis, the detection of proteome alterations may be hampered by the increased cellular heterogeneity of tissue when analysing global liver homogenates. The aim of this study was to evaluate whether the identification of proteome alterations in these HCC cases was improved when the differential protein profile between tumour and non-tumour areas of liver was determined using hepatocytes isolated by laser microdissection (LM). Differential profiles established with LM-hepatocytes and liver section homogenates using 2-DE and MS exhibited noticeable differences: 30% of the protein spots with deregulated expression in tumorous LM-samples did not display any modification in homogenates; conversely 15% of proteins altered in tumorous homogenates were not impaired in LM-hepatocytes. These alterations resulted from the presence in cirrhotic liver of fibrotic stroma which displayed a protein pattern different from that determined in LM-hepatocytes. In conclusion, our data demonstrate the interest of LM in distinguishing between fibrotic and hepatocyte proteome alterations and thus the benefit of LM to proteome studies of HCC developing in a context of cirrhosis.

We identified sequences from two distantly related papillomaviruses in genital warts from two Burmeister's porpoises, including a PV antigen-positive specimen, and characterized Phocoena spinipinnis papillomavirus type 1 (PsPV-1). The PsPV-1 genome comprises 7879 nt and presents unusual features. It lacks an E7, an E8 and a bona fide E5 open reading frame (ORF) and has a large E6 ORF. PsPV-1 L1 ORF showed the highest percentage of nucleotide identity (54–55 %) with human papillomavirus type 5, bovine papillomavirus type 3 (BPV-3) and Tursiops truncatus papillomavirus type 2 (TtPV-2). This warrants the classification of PsPV-1 as the prototype of the genus Omikronpapillomavirus. PsPV-1 clustered with TtPV-2 in the E6 and E1E2 phylogenetic trees and with TtPV-2 and BPV-3 in the L2L1 tree. This supports the hypothesis that PV evolution may not be monophyletic across all genes.

BackgroundA Chikungunya (CHIK) outbreak hit La Réunion Island in 2005-2006. The implicated vector was Aedes albopictus. Here, we present the first study on the susceptibility of Ae. albopictus populations to sympatric CHIKV isolates from La Réunion Island and compare it to other virus/vector combinations.Methodology and findingsWe orally infected 8 Ae. albopictus collections from La Réunion and 3 from Mayotte collected in March 2006 with two Chikungunya virus (CHIKV) from La Réunion: (i) strain 05.115 collected in June 2005 with an Alanine at the position 226 of the glycoprotein E1 and (ii) strain 06.21 collected in November 2005 with a substitution A226V. Two other CHIKV isolates and four additional mosquito strains/species were also tested. The viral titer of the infectious blood-meal was 10(7) plaque forming units (pfu)/mL. Dissemination rates were assessed by immunofluorescent staining on head squashes of surviving females 14 days after infection. Rates were at least two times higher with CHIKV 06.21 compared to CHIKV 05.115. In addition, 10 individuals were analyzed every day by quantitative RT-PCR. Viral RNA was quantified on (i) whole females and (ii) midguts and salivary glands of infected females. When comparing profiles, CHIKV 06.21 produced nearly 2 log more viral RNA copies than CHIKV 05.115. Furthermore, females infected with CHIKV 05.115 could be divided in two categories: weakly susceptible or strongly susceptible, comparable to those infected by CHIKV 06.21. Histological analysis detected the presence of CHIKV in salivary glands two days after infection. In addition, Ae. albopictus from La Réunion was as efficient vector as Ae. aegypti and Ae. albopictus from Vietnam when infected with the CHIKV 06.21.ConclusionsOur findings support the hypothesis that the CHIK outbreak in La Réunion Island was due to a highly competent vector Ae. albopictus which allowed an efficient replication and dissemination of CHIKV 06.21.

International audience; Immature HIV particles bud from infected cells after assembly at the cytoplasmic side of cellular membranes. This assembly is driven by interactions between Gag polyproteins. Mature particles, each containing a characteristic conical core, are later generated by proteolytic maturation of Gag in the virion. The C-terminal domain of the HIV-1 capsid protein (C-CA) has been shown to contain oligomerization determinants essential for particle assembly. Here we report the 1.7-angstrom-resolution crystal structure of C-CA in complex with a peptide capable of inhibiting immature-and mature-like particle assembly in vitro. The peptide inserts as an amphipathic alpha-helix into a conserved hydrophobic groove of C-CA, resulting in formation of a compact five-helix bundle with altered dimeric interactions. This structure thus reveals the details of an allosteric site in the HIV capsid protein that can be targeted for antiviral therapy.

Many genes involved in the immune response of Anopheles gambiae, the main malaria vector in Africa, have been identified, but whether naturally occurring polymorphisms in these genes underlie variation in resistance to the human malaria parasite, Plasmodium falciparum, is currently unknown. Here we carried out a candidate gene association study to identify single nucleotide polymorphisms (SNPs) associated with natural resistance to P. falciparum. A. gambiae M form mosquitoes from Cameroon were experimentally challenged with three local wild P. falciparum isolates. Statistical associations were assessed between 157 SNPs selected from a set of 67 A. gambiae immune-related genes and the level of infection. Isolate-specific associations were accounted for by including the effect of the isolate in the analysis. Five SNPs were significantly associated to the infection phenotype, located within or upstream of AgMDL1, CEC1, Sp PPO activate, Sp SNAKElike, and TOLL6. Low overall and local linkage disequilibrium indicated high specificity in the loci found. Association between infection phenotype and two SNPs was isolate-specific, providing the first evidence of vector genotype by parasite isolate interactions at the molecular level. Four SNPs were associated to either oocyst presence or load, indicating that the genetic basis of infection prevalence and intensity may differ. The validity of the approach was verified by confirming the functional role of Sp SNAKElike in gene silencing assays. These results strongly support the role of genetic variation within or near these five A. gambiae immune genes, in concert with other genes, in natural resistance to P. falciparum. They emphasize the need to distinguish between infection prevalence and intensity and to account for the genetic specificity of vector-parasite interactions in dissecting the genetic basis of Anopheles resistance to human malaria., Author Summary Anopheles gambiae is the main malaria vector in Africa, transmitting the parasite when it blood feeds on human hosts. The parasite undergoes several developmental stages in the mosquito to complete its life cycle, during which time it is confronted by the mosquito's immune system. The resistance of mosquitoes to malaria infection is highly variable in wild populations and is known to be under strong genetic control, but to date the specific genes responsible for this variation remain to be identified. The present study uncovers variations in A. gambiae immune genes that are associated with natural resistance to Plasmodium falciparum, the deadliest human malaria parasite. The association of some mosquito genetic loci with the level of infection depended on the P. falciparum isolate, suggesting that resistance is determined by interactions between the genome of the mosquito and that of the parasite. This finding highlights the need to account for the natural genetic diversity of malaria parasites in future research on vector-parasite interactions. The loci uncovered in this study are potential targets for developing novel malaria control strategies based on natural mosquito resistance mechanisms.