The molecular targets of ivermectin and lotilaner in the human louse Pediculus humanus humanus: New prospects for the treatment of pediculosis.

Control of infestation by cosmopolitan lice (Pediculus humanus) is increasingly difficult due to the transmission of parasites resistant to pediculicides. However, since the targets for pediculicides have no been identified in human lice so far, their mechanisms of action remain largely unknown. The macrocyclic lactone ivermectin is active against a broad range of insects including human lice. Isoxazolines are a new chemical class exhibiting a strong insecticidal potential. They preferentially act on the γ-aminobutyric acid (GABA) receptor made of the resistant to dieldrin (RDL) subunit and, to a lesser extent on glutamate-gated chloride channels (GluCls) in some species. Here, we addressed the pediculicidal potential of isoxazolines and deciphered the molecular targets of ivermectin and the ectoparasiticide lotilaner in the human body louse species Pediculus humanus humanus. Using toxicity bioassays, we showed that fipronil, ivermectin and lotilaner are efficient pediculicides on adult lice. The RDL (Phh-RDL) and GluCl (Phh-GluCl) subunits were cloned and characterized by two-electrode voltage clamp electrophysiology in Xenopus laevis oocytes. Phh-RDL and Phh-GluCl formed functional homomeric receptors respectively gated by GABA and L-glutamate with EC50 values of 16.0 µM and 9.3 µM. Importantly, ivermectin displayed a super agonist action on Phh-GluCl, whereas Phh-RDL receptors were weakly affected. Reversally, lotilaner strongly inhibited the GABA-evoked currents in Phh-RDL with an IC50 value of 40.7 nM, whereas it had no effect on Phh-GluCl. We report here for the first time the insecticidal activity of isoxazolines on human ectoparasites and reveal the mode of action of ivermectin and lotilaner on GluCl and RDL channels from human lice. These results emphasize an expected extension of the use of the isoxazoline drug class as new pediculicidal agents to tackle resistant-louse infestations in humans.

Molecular and Functional Characterization of GABA Receptor Subunits GRD and LCCH3 from Human Louse Pediculus Humanus Humanus.

Human louse Pediculus humanus is a cosmopolitan obligatory blood-feeding ectoparasite causing pediculosis and transmitting many bacterial pathogens. Control of infestation is difficult due to the developed resistance to insecticides that mainly target GABA (γ-aminobutyric acid) receptors. Previous work showed that Pediculus humanus humanus (Phh) GABA receptor subunit resistance to dieldrin (RDL) is the target of lotilaner, a synthetic molecule of the isoxazoline chemical class. To enhance our understanding of how insecticides act on GABA receptors, two other GABA receptor subunits were cloned and characterized: three variants of Phh-grd (glycine-like receptor of Drosophila) and one variant of Phh-lcch3 (ligand-gated chloride channel homolog 3). Relative mRNA expression levels of Phh-rdl, Phh-grd, and Phh-lcch3 revealed that they were expressed throughout the developmental stages (eggs, larvae, adults) and in the different parts of adult lice (head, thorax, and abdomen). When expressed individually in the Xenopus oocyte heterologous expression system, Phh-GRD1, Phh-GRD2, Phh-GRD3, and Phh-LCCH3 were unable to reconstitute functional channels, whereas the subunit combinations Phh-GRD1/Phh-LCCH3, Phh-GRD1/Phh-RDL, and Phh-LCCH3/Phh-RDL responded to GABA in a concentration-dependent manner. The three heteromeric receptors were similarly sensitive to the antagonistic effect of picrotoxin and fipronil, whereas Phh-GRD1/Phh-RDL and Phh-LCCH3/Phh-RDL were respectively about 2.5-fold and 5-fold more sensitive to ivermectin than Phh-GRD1/Phh-LCCH3. Moreover, the heteropentameric receptor constituted by Phh-GRD1/Phh-LCCH3 was found to be permeable and highly sensitive to the extracellular sodium concentration. These findings provided valuable additions to our knowledge of the complex nature of GABA receptors in human louse that could help in understanding the resistance pattern to commonly used pediculicides. SIGNIFICANCE STATEMENT: Human louse is an ectoparasite that causes pediculosis and transmits several bacterial pathogens. Emerging strains developed resistance to the commonly used insecticides, especially those targeting GABA receptors. To understand the molecular mechanisms underlying this resistance, two subunits of GABA receptors were cloned and described: Phh-grd and Phh-lcch3. The heteromeric receptor reconstituted with the two subunits was functional in Xenopus oocytes and sensitive to commercially available insecticides. Moreover, both subunits were transcribed throughout the parasite lifecycle.

GaHV-2 ICP22 protein is expressed from a bicistronic transcript regulated by three GaHV-2 microRNAs.

Herpesviruses have a lifecycle consisting of successive lytic, latent and reactivation phases. Only three infected cell proteins (ICPs) have been described for the oncogenic Marek's disease virus (or Gallid herpes virus 2, GaHV-2): ICP4, ICP22 and ICP27. We focus here on ICP22, confirming its cytoplasmic location and showing that ICP22 is expressed during productive phases of the lifecycle, via a bicistronic transcript encompassing the US10 gene. We also identified the unique promoter controlling ICP22 expression, and its core promoter, containing functional responsive elements including E-box, ETS-1 and GATA elements involved in ICP22 transactivation. ICP22 gene expression was weakly regulated by DNA methylation and activated by ICP4 or ICP27 proteins. We also investigated the function of GaHV-2 ICP22. We found that this protein repressed transcription from its own promoter and from those of IE ICP4 and ICP27, and the late gK promoter. Finally, we investigated posttranscriptional ICP22 regulation by GaHV-2 microRNAs. We found that mdv1-miR-M5-3p and -M1-5p downregulated ICP22 mRNA expression during latency, whereas, unexpectedly, mdv1-miR-M4-5p upregulated the expression of the protein ICP22, indicating a tight regulation of ICP22 expression by microRNAs.

Marek's disease herpesvirus: a model of vaccine-dependent adaptation.

Marek's disease (MD) virus (MDV) is an alphaherpesvirus that causes a rapid-onset T-cell lymphoma in chickens. In order to preserve the viability of poultry industry, non sterilizing vaccines have been used since fifty years, preventing lymphoma development but leading to an imperfect control of MD. Vaccination has been accompanied with the increase in virulence of MDV forcing the development of new vaccine formulations. Several loci of MDV genome are variable and have evolved in link with virulence of MDV strains. It has been shown that MDV is in fact constituted by a dynamic population of genetic variants with a distribution linked to viral strain phenotype. In this context, we have shown that CVI988/Rispens vaccine, still the most efficient one against hypervirulent MDV strains, is composed of twenty variants, variable from one batch to another, evolving likely as RNA virus quasispecies.

Functional endogenous viral elements in the genome of the parasitoid wasp Cotesia congregata: insights into the evolutionary dynamics of bracoviruses.

Bracoviruses represent the most complex endogenous viral elements (EVEs) described to date. Nudiviral genes have been hosted within parasitoid wasp genomes since approximately 100 Ma. They play a crucial role in the wasp life cycle as they produce bracovirus particles, which are injected into parasitized lepidopteran hosts during wasp oviposition. Bracovirus particles encapsidate multiple dsDNA circles encoding virulence genes. Their expression in parasitized caterpillars is essential for wasp parasitism success. Here, we report on the genomic organization of the proviral segments (i.e. master sequences used to produce the encapsidated dsDNA circles) present in the Cotesia congregata parasitoid wasp genome. The provirus is composed of a macrolocus, comprising two-thirds of the proviral segments and of seven dispersed loci, each containing one to three segments. Comparative genomic analyses with closely related species gave insights into the evolutionary dynamics of bracovirus genomes. Conserved synteny in the different wasp genomes showed the orthology of the proviral macrolocus across different species. The nudiviral gene odv-e66-like1 is conserved within the macrolocus, suggesting an ancient co-localization of the nudiviral genome and bracovirus proviral segments. By contrast, the evolution of proviral segments within the macrolocus has involved a series of lineage-specific duplications.

The bracovirus genome of the parasitoid wasp Cotesia congregata is amplified within 13 replication units, including sequences not packaged in the particles.

The relationship between parasitoid wasps and polydnaviruses constitutes one of the few known mutualisms between viruses and eukaryotes. Viral particles are injected with the wasp eggs into parasitized larvae, and the viral genes thus introduced are used to manipulate lepidopteran host physiology. The genome packaged in the particles is composed of 35 double-stranded DNA (dsDNA) circles produced in wasp ovaries by amplification of viral sequences from proviral segments integrated in tandem arrays in the wasp genome. These segments and their flanking regions within the genome of the wasp Cotesia congregata were recently isolated, allowing extensive mapping of amplified sequences. The bracovirus DNAs packaged in the particles were found to be amplified within more than 12 replication units. Strikingly, the nudiviral cluster, the genes of which encode particle structural components, was also amplified, although not encapsidated. Amplification of bracoviral sequences was shown to involve successive head-to-head and tail-to-tail concatemers, which was not expected given the nudiviral origin of bracoviruses.

Evolutionary mechanisms driving the evolution of a large polydnavirus gene family coding for protein tyrosine phosphatases.

BACKGROUND: Gene duplications have been proposed to be the main mechanism involved in genome evolution and in acquisition of new functions. Polydnaviruses (PDVs), symbiotic viruses associated with parasitoid wasps, are ideal model systems to study mechanisms of gene duplications given that PDV genomes consist of virulence genes organized into multigene families. In these systems the viral genome is integrated in a wasp chromosome as a provirus and virus particles containing circular double-stranded DNA are injected into the parasitoids' hosts and are essential for parasitism success. The viral virulence factors, organized in gene families, are required collectively to induce host immune suppression and developmental arrest. The gene family which encodes protein tyrosine phosphatases (PTPs) has undergone spectacular expansion in several PDV genomes with up to 42 genes. RESULTS: Here, we present strong indications that PTP gene family expansion occurred via classical mechanisms: by duplication of large segments of the chromosomally integrated form of the virus sequences (segmental duplication), by tandem duplications within this form and by dispersed duplications. We also propose a novel duplication mechanism specific to PDVs that involves viral circle reintegration into the wasp genome. The PTP copies produced were shown to undergo conservative evolution along with episodes of adaptive evolution. In particular recently produced copies have undergone positive selection in sites most likely involved in defining substrate selectivity. CONCLUSION: The results provide evidence about the dynamic nature of polydnavirus proviral genomes. Classical and PDV-specific duplication mechanisms have been involved in the production of new gene copies. Selection pressures associated with antagonistic interactions with parasitized hosts have shaped these genes used to manipulate lepidopteran physiology with evidence for positive selection involved in adaptation to host targets.

Polydnaviruses of braconid wasps derive from an ancestral nudivirus.

Many species of parasitoid wasps inject polydnavirus particles in order to manipulate host defenses and development. Because the DNA packaged in these particles encodes almost no viral structural proteins, their relation to viruses has been debated. Characterization of complementary DNAs derived from braconid wasp ovaries identified genes encoding subunits of a viral RNA polymerase and structural components of polydnavirus particles related most closely to those of nudiviruses--a sister group of baculoviruses. The conservation of this viral machinery in different braconid wasp lineages sharing polydnaviruses suggests that parasitoid wasps incorporated a nudivirus-related genome into their own genetic material. We found that the nudiviral genes themselves are no longer packaged but are actively transcribed and produce particles used to deliver genes essential for successful parasitism in lepidopteran hosts.

The epidemiology of the highly pathogenic H5N1 avian influenza in Mute Swan (Cygnus olor) and other Anatidae in the Dombes region (France), 2006.

In February 2006, a highly pathogenic avian influenza (HPAI) H5N1 virus was isolated from Common Pochards (Aythia ferina) in the Dombes region of France, an important migrating and wintering waterfowl area. Thereafter, HPAI H5N1 virus was isolated from 39 swab pools collected from dead waterfowl found in the Dombes, but only from three pooled samples collected outside of this area but located on the same migration flyway. A single turkey farm was infected in the Dombes. The epizootic lasted 2 mo and was restricted to the Dombes area. Virus-positive pools were detected in 20 of 1,200 ponds and infected Mute Swans (Cygnus olor) represented 82% of the virus-positive pools. Other infected species included Common Pochard (n=4), Grey Heron (Ardea cinerea, n=1), Eurasian Buzzard (Buteo buteo, n=1), and Greylag Goose (Anser anser, n=1). Despite intensive monitoring during and after the outbreak, HPAI H5N1 virus was not isolated from healthy wild birds. Our results are consistent with an HPAI H5N1-virus introduction into the Dombes via migrating ducks. These birds could have been pushed west by a severe cold spell in central Europe where the virus had already been detected. The Mute Swan served as an excellent epidemiologic sentinel during this outbreak; swans appear to be highly sensitive to infection with these viruses and swan mortality was easy to detect. During the outbreak, the mortality rates for wild birds remained moderate and the virus affected a limited number of species.

Unfolding the evolutionary story of polydnaviruses.

Polydnaviruses (PDVs) are fascinating viruses. Described in thousands of parasitoid wasp species they are unique viruses having both a segmented DNA genome in viral particles and an integrated form that persists as a provirus in the wasp genome. Parasitoid wasps inject their eggs in another insect host typically a lepidopteran. In these host-parasitoid interactions, the virus particles are co-injected along with the eggs and are essential to ensure wasp parasitism success. PDVs do not replicate in the lepidopteran host, but expression of viral gene products confers protection from the host immune defence response. Two genera of PDVs phylogenetically unrelated exist, the bracoviruses (BVs) and the ichnoviruses (IVs), associated with braconid and ichneumonid wasps, respectively. New data on the genomes of two bracoviruses (Microplitis demolitor BV and Cotesia congregata BV) and an ichnovirus associated with Campoletis sonorensis (CsIV) offers us new elements to discuss the central questions concerning the origin of these viral entities and how they have evolved. The results of sequencing approaches indicate that the tens of millions of years of mutualistic associations between PDVs and wasps have had a strong impact on PDV genomes that now ressemble eukaryotic regions both in organization and gene content.

Genome sequence of a polydnavirus: insights into symbiotic virus evolution.

Little is known of the fate of viruses involved in long-term obligatory associations with eukaryotes. For example, many species of parasitoid wasps have symbiotic viruses to manipulate host defenses and to allow development of parasitoid larvae. The complete nucleotide sequence of the DNA enclosed in the virus particles injected by a parasitoid wasp revealed a complex organization, resembling a eukaryote genomic region more than a viral genome. Although endocellular symbiont genomes have undergone a dramatic loss of genes, the evolution of symbiotic viruses appears to be characterized by extensive duplication of virulence genes coding for truncated versions of cellular proteins.

Stromal cell-derived factor 1 regulates primitive hematopoiesis by suppressing apoptosis and by promoting G(0)/G(1) transition in CD34(+) cells: evidence for an autocrine/paracrine mechanism.

The stromal cell-derived factor 1 (SDF-1) chemokine has various effects on hematopoietic cell functions. Its role in migration and homing of hematopoietic progenitors is currently well established. Previously it was shown that SDF-1 stimulates myeloid progenitor proliferation in synergy with cytokines. Results of this study indicate that SDF-1 alone promotes survival of purified CD34(+) cells from human unmobilized peripheral blood (PB) by counteracting apoptosis as demonstrated by its capacity to reduce DNA fragmentation, annexin-V(+) cell number, and APO2.7 detection and to modulate bcl-2 homolog protein expression. The study demonstrates that SDF-1, produced by sorted CD34(+)CD38(+) cells and over-released in response to cell damage, exerts an antiapoptotic effect on CD34(+) cells through an autocrine/paracrine regulatory loop. SDF-1 participates in the autonomous survival of circulating CD34(+) cells and its effect required activation of the phosphotidyl inositol 3 kinase (PI3-K)/Akt axis. Cell sorting based on Hoechst/pyroninY fluorescences shows that SDF-1 production is restricted to cycling CD34(+) cells. SDF-1 triggers G(0) quiescent cells in G(1) phase and, in synergy with thrombopoietin or Steel factor, makes CD34(+) cells progress through S+G(2)/M phases of cell cycle. By assessing sorted CD34(+)CD38(-) and CD34(+)CD38(+) in semisolid culture, the study demonstrates that SDF-1 promotes survival of clonogenic progenitors. In conclusion, the results are the first to indicate a role for endogenous SDF-1 in primitive hematopoiesis regulation as a survival and cell cycle priming factor for circulating CD34(+) cells. The proposal is made that SDF-1 may contribute to hematopoiesis homeostasis by participating in the autonomous survival and cycling of progenitors under physiologic conditions and by protecting them from cell aggression in stress situations.