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Background: Leishmania (L.) spp are intracellular eukaryotic parasites responsible for cutaneous or visceral leishmaniasis, replicating predominantly in macrophages (MF). In C57BL/6 mice virulence with L. amazonensis has been associated with inhibition of Th1 immune responses and an uncontrolled lesion development, whereas DBA/2 mice control any lesion. Parasitic clearance by the MFs requires the activation of proper immune responses. One of the immune related genes expressed in immune cells including MF, codes for osteopontin (OPN). OPN is a secreted glycoprotein, acting as an immune regulator. Its implication in promoting Th1 immunity in response to infectious microorganisms and its known protective effect against viral and bacterial infections via activation of the immune response, render OPN a molecule of interest in the study of the host response to L. amazonensis., Results: We examined the host response to L. amazonensis of opn mutant and wild type C57BL/6 mice. Bone marrow derived MFs were infected with the parasites in vitro, and opn mutant and wild type mice were inoculated in vivo by intradermal injection in the ears. The DBA/2 strain known to control L. amazonensis infection was also used for comparison. Our data indicate that the parasites increased opn gene expression and OPN protein while parasitic proliferation was contained in the presence of OPN. In the presence of parasites the expression of inflammation-related transcripts was inhibited. Interleukin-1-beta (IL-1β), and transcripts of the NLR-family (NLRC4, NLRP3) were down regulated after L. amazonensis infection. In the absence of OPN, the inhibition by the parasites of IL-1β transcripts was less efficient and a pyroptosis-like cell phenotype was detected in vitro, suggesting a central role of OPN in the host-response to L. amazonensis. Similarly, in vivo, in the absence of OPN, while the clinical inflammatory phenotype is more severe, an increase of these transcripts was observed., Conclusions: L. amazonensis infection induces opn gene expression and protein, which in turn participates in shaping the host response to the parasites, seemingly by decreasing the activation of inflammation. OPN, further evaluated as a target for Leishmaniasis control represents an additional interest in improving vaccination strategies against the parasites.

Visceral leishmaniasis is an insidious neglected disease with worldwide distribution. It is caused by parasites from the Leishmania donovani complex, which are able to be transmitted by different species of phlebotomine sand flies and to infect numerous mammal hosts. Despite the high number of people infected or at risk, and the remarkable quantity of studies focusing on this disease, a proper experimental model to efficiently decipher the infectious process of visceral leishmaniasis taking into account the nuances of parasite’s virulence and the duration of the infection is still lacking. Therefore, using golden Syrian hamsters and BALB/c mice, state-of-the-art genetic manipulation applied on a fully virulent L. donovani strain and in vivo imaging approaches, we describe herein three benefits for experimental visceral leishmaniasis: (i) the development of a double transfected bioluminescent (firefly luciferase) and fluorescent (E2-crimson) virulent strain of L. donovani (Ld1S_luci_E2-crimson), favoring a wide range of both in vivo and in vitro investigations, (ii) the establishment of a non-invasive mouse model to evaluate the infectious process during visceral leishmaniasis and the parasite’s virulence in real time, allowing longitudinal studies with the same animals, and (iii) the elaboration of a suitable method to reinstate (and verify anew) the virulence in a population of attenuated parasites, by recovering persistent parasites from chronic infected mice. Consequently, these results open up new perspectives on the study of visceral leishmaniasis, especially in the fields of therapeutics and vaccinology, since the model described herein renders now possible long-lasting follow up studies, with easy and accurate day-by-day verifications of the infection status along with a reduced number of laboratory animals., Trial Registration: ClinicalTrials.gov 2013-0047.

Zika virus (ZIKV) viremia is reported as low and transient; however, these estimates rely on limited data. We report RNA loads in sera collected from symptomatic patients during the 2013-2014 French Polynesian ZIKV outbreak. We performed molecular detection of ZIKV RNA in sera from 747 patients presenting with suspected acute phase ZIKV infection. Among patients with confirmed infection, we analyzed the duration of viremia, assessed viral RNA loads and recorded the main clinical symptoms. A total of 210/747 (28.1%) sera tested positive using a ZIKV-specific RT-PCR. Viral RNA loads in symptomatic patients that ranged from 5 to 3.7 × 10 6  copies/mL (mean 9.9 × 10 4  copies/mL) were not related to a particular clinical presentation, and were significantly lower than those previously obtained from asymptomatic ZIKV infected blood donors. The rate of detection of ZIKV RNA in sera from suspected cases of acute phase ZIKV infection was low. ZIKV RNA loads were lower in symptomatic patients compared to asymptomatic blood donors and were lower than RNA loads usually reported in dengue infections. As there is no abrupt onset of symptoms in ZIKV infections, we suggest that infected patients sought for medical attention when viremia was already decreasing or had resolved., (© 2016 The Authors. Journal of Medical Virology Published by Wiley Periodicals, Inc.)

Background: Leptospirosis is a highly endemic bacterial zoonosis in French Polynesia (FP). Nevertheless, data on the epidemiology of leptospirosis in FP are scarce. We conducted molecular studies on Leptospira isolated from humans and the potential main animal reservoirs in order to identify the most likely sources for human infection., Methodology/principal Findings: Wild rats (n = 113), farm pigs (n = 181) and domestic dogs (n = 4) were screened for Leptospira infection in Tahiti, the most populated island in FP. Positive samples were genotyped and compared to Leptospira isolated from human cases throughout FP (n = 51), using secY, 16S and LipL32 sequencing, and MLST analysis. Leptospira DNA was detected in 20.4% of rats and 26.5% of pigs. We identified two Leptospira species and three sequence types (STs) in animals and humans: Leptospira interrogans ST140 in pigs only and L. interrogans ST17 and Leptospira borgpetersenii ST149 in humans and rats. Overall, L. interrogans was the dominant species and grouped into four clades: one clade including a human case only, two clades including human cases and dogs, and one clade including human cases and rats. All except one pig sample showed a unique L. interrogans (secY) genotype distinct from those isolated from humans, rats and dogs. Moreover, LipL32 sequencing allowed the detection of an additional Leptospira genotype in pigs, clearly distinct from the previous ones., Conclusions/significance: Our data confirm rats as a major potential source for human leptospirosis in FP. By contrast to what was expected, farm pigs did not seem to be a major reservoir for the Leptospira genotypes identified in human patients. Thus, further investigations will be required to determine their significance in leptospirosis transmission in FP.

Characterizing the clinical, immunological and parasitological features associated with visceral leishmaniasis is complex. It involves recording in real time and integrating quantitative multi-parametric data sets from parasite infected host tissues. Although several models have been used, hamsters are considered the bona fide experimental model for Leishmania donovani studies. To study visceral leishmaniasis in hamsters we generated virulent transgenic L. donovani that stably express a reporter luciferase protein. Two complementary methodologies were combined to follow the infectious process: in vivo imaging using luciferase-expressing Leishmania and real time RT-PCR to quantify both Leishmania and host transcripts. This approach allows us: i) to assess the clinical outcome of visceral leishmaniasis by individual monitoring of hamster weight, ii) to follow the parasite load in several organs by real time analysis of the bioluminescence in vivo and through real time quantitative PCR analysis of amastigote parasite transcript abundance ex vivo, iii) to evaluate the immunological responses triggered by the infection by quantifying hamster transcripts on the same samples and iv) to limit the number of hamsters selected for further analysis. The overall data highlight a correlation between the transcriptional cytokine signatures of hamster affected tissues and the amastigote burden fluctuations, thus providing new insights into the immunopathological process driven by L. donovani in the tissues of mammalian hosts. Finally, they suggest organ-specific immune responses., (Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.)

Background: Saliva and urine have been used for arthropod-borne viruses molecular detection but not yet for chikungunya virus (CHIKV). We investigated the use of saliva and urine for molecular detection of CHIKV during the French Polynesian outbreak., Methods: During the French Polynesian chikungunya outbreak (2014-2015), we collected the same day blood and saliva samples from 60 patients with probable chikungunya (47 during the 1st week post symptoms onset and 13 after), urine was available for 39 of them. All samples were tested using a CHIKV reverse-transcription PCR., Results: Forty eight patients had confirmed chikungunya. For confirmed chikungunya presenting during the 1st week post symptoms onset, CHIKV RNA was detected from 86.1 % (31/36) of blood, 58.3 % (21/36) of saliva and 8.3 % (2/24) of urine. Detection rate of CHIKV RNA was significantly higher in blood compared to saliva. For confirmed chikungunya presenting after the 1st week post symptoms onset, CHIKV RNA was detected from 8.3 % (1/12) of blood, 8.3 % (1/12) of saliva and 0 % (0/8) of urine., Conclusions: In contrast to Zika virus (ZIKV), saliva did not increased the detection rate of CHIKV RNA during the 1st week post symptoms onset. In contrast to ZIKV, dengue virus and West Nile virus, urine did not enlarged the window of detection of CHIKV RNA after the 1st week post symptoms onset. Saliva can be used for molecular detection of CHIKV during the 1st week post symptoms onset only if blood is impossible to collect but with a lower sensitivity compared to blood.

Leishmania/L. major was identified as the etiological agent of human localized cutaneous leishmaniasis. L. major metacyclic promastigotes/MP - the infectious form transmitted by sand flies - were enriched from axenically-derived cultures and inoculated into the dermis of mice (10(3) or 10(4) luciferase-expressing L. major MP inoculated into the C57BL/6 mouse ear pinna). Quantitative readout assays were then combined with imaging of this L. major-hosting skin site and established i) that a specific period of time - depending upon the L. major load used for the inoculation - is required for the L. major-hosting ear pinna to be continuously populated by a balanced population of functional regulatory and effector T lymphocytes, and that ii) this balance coincides with persisting low numbers of amastigotes in more or less rapidly healing skin. This approach also established that, whatever the MP inoculum load delivered to the primary site, the immune processes that reduce the L. major amastigote population also account for concomitant immunity, namely remodelling of the secondary site - where 10(4) MP were delivered - as a clinically silent niche hosting a small L. major population., (© 2013. Published by Elsevier Ireland Ltd. All rights reserved.)

Although artificial C2-H2 zinc fingers can be designed to recognize specific DNA sequences, it remains unclear to which extent nuclear receptor C4 zinc fingers can be tailored to bind novel DNA elements. Steroid receptors bind as dimers to palindromic response elements differing in the two central base pairs of repeated motifs. Predictions based on one amino acid-one base-pair relationships may not apply to estrogen receptors (ERs), which recognize the two central base pairs of estrogen response elements (EREs) via two charged amino acids, each contacting two bases on opposite DNA strands. Mutagenesis of these residues, E203 and K210 in ERalpha, indicated that both contribute to ERE binding. Removal of the electric charge and steric constraints associated with K210 was required for full loss of parental DNA-binding specificity and recognition of novel sequences by E203 mutants. Although some of the new binding profiles did not match predictions, the double mutation E203R-K210A generated as predicted a mutant ER that was transcriptionally active on palindromes of PuGCTCA motifs, but not on consensus EREs. This study demonstrates the feasibility of designing C4 zinc finger mutants with novel DNA-binding specificity, but also uncovers limitations of this approach.

Tagging of viral capsid proteins is a powerful tool to study viral assembly; it also raises the possibility of using viral particles to present exogenous epitopes in vaccination or gene therapy strategies. The ability of reoviruses to induce strong mucosal immune response and their large host range and low pathogenicity in humans are some of the advantages of using reoviruses in such applications. In the present study, the feasibility of introducing foreign epitopes, "tags", to the sigma3 protein, a major component of the reovirus outer capsid, was investigated. Among eight different positions, the amino-terminal end of the protein appeared as the best location to insert exogenous sequences. Additional amino acids at this position do not preclude interaction with the micro1 protein, the other major constituent of the viral outer capsid, but strongly interfere with micro1 to micro1C cleavage. Nevertheless, the tagged sigma3 protein was still incorporated to virions upon recoating of infectious subviral particles to which authentic sigma3 protein was removed by proteolysis, indicating that micro1 cleavage is not a prerequisite for outer capsid assembly. The recently published structure of the sigma3- micro1 complex suggests that the amino-terminally inserted epitope could be exposed at the outer surface of viral particles.

We have characterized a novel mutation of the human AR, G577R, associated with partial androgen insensitivity syndrome. G577 is the first amino acid of the P box, a region crucial for the selectivity of receptor/DNA interaction. Although the equivalent amino acid in the GR (also Gly) is not involved in DNA interaction, the residue at the same position in the ER (Glu) interacts with the two central base pairs in the PuGGTCA motif. Using a panel of 16 palindromic probes that differ in these base pairs (PuGNNCA) in gel shift experiments with either the AR DNA-binding domain or the full length receptor, we observed that the G577R mutation does not induce binding to probes that are not recognized by the wild-type AR. However, binding to the four PuGNACA elements recognized by the wild-type AR was affected to different degrees, resulting in an altered selectivity of DNA response element recognition. In particular, AR-G577R did not interact with PuGGACA palindromes. Modeling of the complex between mutant AR and PuGNACA motifs indicates that the destabilizing effect of the mutation is attributable to a steric clash between the C beta of Arg at position 1 of the P box and the methyl group of the second thymine residue in the TGTTCPy arm of the palindrome. In addition, the Arg side chain can interact with G or T at the next position (PuGCACA and PuGAACA elements, respectively). The presence of C is not favorable, however, because of incompatible charges, abrogating binding to the PuGGACA element. Transactivation of several natural or synthetic promoters containing PuGGACA motifs was drastically reduced by the G577R mutation. These data suggest that androgen target genes may be differentially affected by the G577R mutation, the first natural mutation characterized that alters the selectivity of the AR/DNA interaction. This type of mutation may thus contribute to the diversity of phenotypes associated with partial androgen insensitivity syndrome.