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Mosquito transmission of dengue viruses to humans starts with infection of skin resident cells at the biting site. There is great interest in identifying transmission-enhancing factors in mosquito saliva in order to counteract them. Here we report the discovery of high levels of the anti-immune subgenomic flaviviral RNA (sfRNA) in dengue virus 2-infected mosquito saliva. We established that sfRNA is present in saliva using three different methods: northern blot, RT-qPCR and RNA sequencing. We next show that salivary sfRNA is protected in detergent-sensitive compartments, likely extracellular vesicles. In support of this hypothesis, we visualized viral RNAs in vesicles in mosquito saliva and noted a marked enrichment of signal from 3'UTR sequences, which is consistent with the presence of sfRNA. Furthermore, we show that incubation with mosquito saliva containing higher sfRNA levels results in higher virus infectivity in a human hepatoma cell line and human primary dermal fibroblasts. Transfection of 3'UTR RNA prior to DENV2 infection inhibited type I and III interferon induction and signaling, and enhanced viral replication. Therefore, we posit that sfRNA present in salivary extracellular vesicles is delivered to cells at the biting site to inhibit innate immunity and enhance dengue virus transmission. Support for this research came from a fellowship from the McLauglin Family Foundation to TS, scholarships from the Fondation pour la Recherche Médicale (FRM project SPF202110013925) to HM, from the Institut Méditerranéen Hospitalier (IHU, Marseille) to IMSP and from the graduate school French Ministry of Higher Education and Research to FRC and FR, UTMB start-up funds and Cancer Prevention & Research Institute of Texas grant RP200650 to LJK and MKS, the Ministerio de Ciencia e Innovación (PID2020-115696RB-I00) to MAM-R, Cancer Prevention & Research Institute of Texas grant ID RR210018 to GN, Ministry of Education (Singapore) Tier3 grant (MOE2015-T3-1-003) to RMK and JP, a National Medical Research Council (Singapore) ZRRF grant (ZRRF/007/2017) to JP, a French Agence Nationale de la Recherche grant (ANR-20-CE15-0006) to JP, and the Duke-NUS Signature Research Programme funded by the Agency for Science Technology and Research (A*Star Singapore) and NIH/NIAID P01 AI150585 to MAGB. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

International audience; Our study assessed the impact of using ovitraps with pyriproxyfen on mosquito populations and the feasibility of using human saliva samples to test for seroconversion to dengue virus (DENV). We used a quasi-experimental research design by forming the intervention (n = 220) and the control (n = 223) groups in neighboring Taguig City, Philippines, over 4 months. Socio-demographic data, entomological indices, and IgG antibodies against DENV were measured. Associations between the implementation of ovitraps dosed with pyriproxyfen and mosquito densities (percentage positive ovitraps and container indices) and DENV seroconversion were calculated post-intervention in Months 2, 3, and 4. Among the participants recruited at baseline, 17 and 13 were seropositive for dengue (DENV) in the intervention and control groups, respectively. Both entomological indices were lower in the treated area than the control site at post-intervention Months 2, 3, and 4, but not earlier. Dengue seroconversions rates decreased in the treated population, but not significantly so. In conclusion, the use of PPF-treated ovitraps may have impacted the mosquito population, but not seroconversion rates. Compliance in providing saliva samples and the ability to detect IgG antibodies within these samples was encouraging and suggests that further studies on larger populations for longer durations are warranted.

The explosive emergence of Zika virus (ZIKV) across the Pacific and Americas since 2007 was associated with hundreds of thousands of human cases and severe outcomes, including congenital microcephaly caused by ZIKV infection during pregnancy. Although ZIKV was first isolated in Uganda, Africa has so far been exempt from large-scale ZIKV epidemics, despite widespread susceptibility among African human populations. A possible explanation for this pattern is natural variation among populations of the primary vector of ZIKV, the mosquito Aedes aegypti. Globally invasive populations of Ae. aegypti outside of Africa are considered effective ZIKV vectors because they are human specialists with high intrinsic ZIKV susceptibility, whereas African populations of Ae. aegypti across the species’ native range are predominantly generalists with low intrinsic ZIKV susceptibility, making them less likely to spread viruses in the human population. We test this idea by studying a notable exception to the patterns observed across most of Africa: Cape Verde experienced a large ZIKV outbreak in 2015 to 2016. We find that local Ae. aegypti in Cape Verde have substantial human-specialist ancestry, show a robust behavioral preference for human hosts, and exhibit increased susceptibility to ZIKV infection, consistent with a key role for variation among mosquito populations in ZIKV epidemiology. These findings suggest that similar human-specialist populations of Ae. aegypti in the nearby Sahel region of West Africa, which may be expanding in response to rapid urbanization, could serve as effective vectors for ZIKV in the future.

Mosquito bites transmit a number of pathogens via salivary droplets deposited during blood-feeding, resulting in potentially fatal diseases. Little is known about the genomic content of these nanodroplets, including the transmission dynamics of live pathogens. Here we introduce Vectorchip, a low-cost, scalable microfluidic platform enabling high-throughput molecular interrogation of individual mosquito bites. We introduce an ultra-thin PDMS membrane which acts as a biting interface to arrays of micro-wells. Freely-behaving mosquitoes deposit saliva droplets by biting into these micro-wells. By modulating membrane thickness, we observe species-dependent differences in mosquito biting capacity, utilizable for selective sample collection. We demonstrate RT-PCR and focus-forming assays on-chip to detect mosquito DNA, Zika virus RNA, as well as quantify infectious Mayaro virus particles transmitted from single mosquito bites. The Vectorchip presents a promising approach for single-bite-resolution laboratory and field characterization of vector-pathogen communities, and could serve as a powerful early warning sentinel for mosquito-borne diseases., High-throughput molecular surveillance of mosquitoes carrying dangerous pathogens is currently challenging. Here the authors present Vectorchip, a low-cost microfluidic platform enabling multiplexed detection of mosquito DNA, viral RNA and infectious viral particles at single bite resolution.

Originating from African forests, Zika virus (ZIKV) has now emerged worldwide in urbanized areas, mainly transmitted by Aedes aegypti mosquitoes. Although Aedes albopictus can transmit ZIKV experimentally and was suspected to be a ZIKV vector in Central Africa, the potential of this species to sustain virus transmission was yet to be uncovered until the end of 2019, when several autochthonous transmissions of the virus vectored by Ae. albopictus occurred in France. Aside from these few locally acquired ZIKV infections, most territories colonized by Ae. albopictus have been spared so far. The risk level of ZIKV emergence in these areas remains however an open question. To assess Ae. albopictus’ vector potential for ZIKV and identify key virus outbreak predictors, we built a complete framework using the complementary combination of (i) dose-dependent experimental Ae. albopictus exposure to ZIKV followed by time-dependent assessment of infection and systemic infection rates, (ii) modeling of intra-human ZIKV viremia dynamics, and (iii) in silico epidemiological simulations using an Agent-Based Model. The highest risk of transmission occurred during the pre-symptomatic stage of the disease, at the peak of viremia. At this dose, mosquito infection probability was estimated to be 20%, and 21 days were required to reach the median systemic infection rates. Mosquito population origin, either temperate or tropical, had no impact on infection rates or intra-host virus dynamic. Despite these unfavorable characteristics for transmission, Ae. albopictus was still able to trigger and yield large outbreaks in a simulated environment in the presence of sufficiently high mosquito biting rates. Our results reveal a low but existing epidemic potential of Ae. albopictus for ZIKV, that might explain the absence of large scale ZIKV epidemics so far in territories occupied only by Ae. albopictus. They nevertheless support active surveillance and eradication programs in these territories to maintain the risk of emergence to a low level., Author summary Zika virus (ZIKV) has emerged worldwide and triggered large outbreaks in human populations. While the yellow fever mosquito Aedes aegypti is considered the primary vector of ZIKV, the Asian tiger mosquito Aedes albopictus has been shown experimentally to transmit the virus and has been involved in a few autochthonous transmission in France in 2019. Here, we provide a comprehensive study on the ability of Ae. albopictus mosquitoes to transmit ZIKV by considering the within-host dynamics of ZIKV infection in humans and its impact on both mosquito infection probability and time to mosquito infectiousness. These empirical data were then leveraged by in silico simulations to embed them into their epidemiological context. Our study reveals a low but existing epidemic potential of Ae. albopictus for ZIKV, whatever their tropical or temperate origins. We identified mosquito density as a predictor for ZIKV outbreak occurrence when vectored by Ae. albopictus. Our findings help to explain the absence of large scale ZIKV epidemics in territories occupied by Ae. albopictus but call for active surveillance and eradication programs to maintain the risk of emergence to a low level.

International audience; In a recent review of laboratory tests for diagnosing SARS-CoV-2 infection, Zheng et al. concluded that the detection rate of real-time quantitative PCR was lower than that of computed tomography [1]. But the authors did not discuss the possibility of using saliva sampling. Nasopharyngeal (NP) swabs are the preferred collection vehicles in France and many other countries and several studies have indicated that this method is more sensitive than sampling other sites [2–4]. Saliva sampling is less invasive for patients, less hazardous for health care workers, requires fewer experimented staff and is less expensive for mass testing. However, little is known about any differences in the sensitivities of saliva and NP sampling or how any differences vary with presence or absence of clinical symptoms

International audience; Alzheimer's disease (AD) is an incurable neurodegenerative disease characterized by progressive decline of cognitive abilities. Amyloid beta peptides (Aβ), Tau proteins and the phosphorylated form of the Tau protein, p-Tau, are the core pathological biomarkers of the disease, and their detection for the diagnosis of patients is progressively being implemented. However, to date, their quantification is mostly performed on cerebrospinal fluid (CSF), the collection of which requires an invasive lumbar puncture. Early diagnosis has been shown to be important for disease-modifying treatment, which is currently in development, to limit the progression of the disease. Nevertheless, the diagnosis is often delayed to the point where the disease has already progressed, and the tools currently available do not allow for a systematic follow-up of patients. Thus, the search for a molecular signature of AD in a body fluid such as blood or saliva that can be collected in a minimally invasive way offers hope. A number of methods have been developed for the quantification of core biomarkers, especially in easily accessible fluids such as the blood, that improve their accuracy, specificity and sensitivity. This review summarizes and compares these approaches, focusing in particular on their use for Aβ detection, the earliest biomarker to be modified in the course of AD. The review also discusses biomarker quantification in CSF, blood and saliva and their clinical applications.

The promise of prostate-specific membrane antigen (PSMA)–targeted radionuclide therapy is being demonstrated by a growing number of reports detailing institutional experience with various agents, and prospective clinical trials are in progress to further establish the safety and efficacy of this

International audience; OBJECTIVE:Whereas circulating levels of C-reactive protein (CRP) have been associated with, for example, arterial stiffness, subclinical atherosclerosis and metabolic syndrome, other inflammatory biomarkers with potential interest for these conditions may not be measurable systemically. The predictive value of salivary biomarkers in these contexts has remained largely unexplored. The aim of the present study was to establish the association of different salivary biomarkers of inflammation with subclinical cardiovascular disease.METHODS:Two hundred and fifty-nine individuals were included in the study. Saliva and plasma samples were collected, and each individual underwent carotid ultrasound and measures of pulse wave velocity and blood pressure. Medical history of previous cardiovascular disease, current medications and smoking were collected by questionnaire.RESULTS:Salivary levels of CRP, leukotriene B4 (LTB4), prostaglandin E2 (PGE2), matrix metalloproteinase 9 (MMP-9), creatinine and lysozyme were measured. Salivary levels of CRP were significantly correlated with plasma levels (r = 0.73, P

International audience; During Epstein-Barr virus (EBV) latency, the EBV genome is largely silenced by methylation. This silencing is overturned during the switch to the lytic cycle. A key event is the production of the viral protein Zta which binds to three Zta-response elements (ZRE) from the Rta promoter (Rp), two of which (ZRE2 and ZRE3) include three CpG motifs methylated in the latent genome. The bisulphite pyrosequencing reaction was used to quantify the methylation of ZRE2, ZRE3a, and ZRE3b in EBV-positive cell lines and in ex vivo samples of EBV-related diseases, in order to assess whether the level of methylation in these ZREs could provide additional information to viral DNA load and serology in the characterization of EBV-associated diseases. In PBMC from two patients with infectious mononucleosis, over time Rp became increasingly methylated whereas EBV load decreased. In tonsil from patients with chronic tonsillitis, the methylation was less than in EBV-associated tumors, regardless of the viral load. This was even more striking when only the ZRE3a and ZRE3b were considered since some samples presented unbalanced profiles on ZRE2. EBV reactivation in cell culture showed that the reduction in the overall level of methylation was closely related to the production of unmethylated virions. Thus, an assessment of the level of methylation may help to better characterize EBV replication in PBMC and in biopsies with high EBV load, during infectious mononucleosis and EBV-associated cancers. J. Med. Virol. 88:1814-1820, 2016. © 2016 Wiley Periodicals, Inc.

International audience; In the ANRS IPERGAY pre-exposure prophylaxis (PrEP) trial, a single dose of tenofovir disoproxil fumarate and emtricitabine was taken orally 2-24 h before sexual intercourse. A sub-study was conducted to assess the pharmacokinetics of tenofovir and emtricitabine in blood, saliva and rectal tissue following this initial oral intake. Methods: Plasma, PBMC, saliva and rectal tissue sampling was performed over 24 h in 12 seronegative men before enrolment in the ANRS IPERGAY trial, following a single dose of 600 mg tenofovir disoproxil fumarate/400 mg emtricitabine. Ex vivo HIV infectibility of rectal biopsies was also assessed. Results: The median plasma T max of tenofovir (median C max : 401 mg/L) and emtricitabine (median C max : 2868 mg/L) was obtained 1 h (range: 0.5-4) and 2 h (range: 1-4) after dosing, respectively. The median C 24 of tenofovir and emtricitabine was 40 and 63 mg/L, respectively. The median PBMC tenofovir diphosphate and emtricitabine triphosphate levels were 12.2 and 16.7 fmol/10 6 cells and 2800 and 2000 fmol/10 6 cells at 2 and 24 h after dosing, respectively. Saliva/plasma AUC 0-24 ratios were 2% and 17% for tenofovir and emtricitabine, respectively. Emtricitabine was detected in rectal tissue 30 min after dosing, whereas tenofovir was only detectable at 24 h. Ex vivo HIV infectibility assays of rectal biopsies showed partial protection after dosing (P,0.07). Discussion: A single high dose of oral tenofovir disoproxil fumarate/emtricitabine provides rapid and high blood levels of tenofovir and emtricitabine, with rapid diffusion of emtricitabine in saliva and rectal tissue.

Background Rapid diagnostic tests (RDTs) have been commercialized in order to help physicians in dengue diagnosis. Until recently, only blood samples were used for those tests but it has been shown in several studies that urine and saliva can also be employed for dengue diagnosis. RDTs for the detection of NS1 antigen and anti-dengue IgG, IgM and IgA in urine and saliva specimens have thus been developed by Standard Diagnostics. The aim of this study was to evaluate the performances these new commercial assays. Methods Two panels of clinical specimens were used: one for the evaluation of the NS1-detection devices and the second for the evaluation of the antibody-detection kits. Each panel consisted of urine and saliva specimens collected sequentially from 86 patients with a confirmed dengue infection. A total of 291 saliva and 440 urine samples were included in the NS1-evaluation panel and 530 saliva and 528 urine specimens constituted the antibody-evaluation panel. All samples were tested in parallel by in-house ELISAs and by the commercial RDTs. Results The RDTs demonstrated an overall sensitivity of 15.5 %/27.9 %/10.7 % for NS1/IgG/IgA detection in urine samples and 20.4 %/ 34.8 %/11 %/6.2 % for NS1/IgG/IgM/IgA detection in saliva samples. Compared to the in-house NS1 ELISA, the results obtained with the NS1 RDT demonstrated a good correlation with urine samples (kappa coefficient: 0.88) but not with saliva specimens (kappa coefficient: 0.28). RDTs designed for antibody detection in saliva and urine were extremely specific (100 %), but less sensitive than the in-house ELISAs (i.e., reduction of the overall sensitivity by 12.2 % for the RDT designed for IgG detection in urine and by 23.7 % for the RDT detecting anti-DENV IgM in saliva). IgM were not detected in urine, either by RDT or ELISA. Conclusions Although the RDTs evaluated here offer an apparently attractive approach for dengue diagnosis, this study suggests that these new commercial kits would require further improvement to increase the sensitivity. Electronic supplementary material The online version of this article (doi:10.1186/s12879-016-1551-x) contains supplementary material, which is available to authorized users.

International audience; BACKGROUND:The number of human deaths due to rabies is currently underestimated to be 55,000 deaths per year. Biological diagnostic methods for confirmation of rabies remain limited, because testing on postmortem cerebral samples is the reference method, and in many countries, sampling brain tissue is rarely practiced. There is a need for a reliable method based on a simple collection of nonneural specimens.METHODS:A new reverse-transcription, heminested polymerase chain reaction (RT-hnPCR) protocol was standardized at 3 participating centers in Cambodia, Madagascar, and France. Fifty-one patients from Cambodia, Madagascar, Senegal, and France were prospectively enrolled in the study; 43 (84%) were ultimately confirmed as having rabies. A total of 425 samples were collected from these patients during hospitalization. We studied the accuracy of the diagnosis by comparing the results obtained with use of biological fluid specimens (saliva and urine) and skin biopsy specimens with the results obtained with use of the standard rabies diagnostic procedure performed with a postmortem brain biopsy specimen.RESULTS:The data obtained indicate a high specificity (100%) of RT-hnPCR and a higher sensitivity (>/=98%) when the RT-hnPCR was performed with skin biopsy specimens than when the test was performed with fluid specimens, irrespective of the time of collection (i.e., 1 day after the onset of symptoms or just after death). Also, a sensitivity of 100% was obtained with the saliva sample when we analyzed at least 3 successive samples per patient.CONCLUSIONS:Skin biopsy specimens should be systematically collected in cases of encephalitis of unknown origin. These samples should be tested by RT-hnPCR immediately to confirm rabies; if the technique is not readily available locally, the samples should be tested retrospectively for epidemiological purposes.

Background West Nile virus (WNV) is a highly pathogenic flavivirus transmitted by Culex spp. mosquitoes. In North America (NA), lineage 1 WNV caused the largest outbreak of neuroinvasive disease to date, while a novel pathogenic lineage 2 strain circulates in southern Europe. To estimate WNV lineage 2 epidemic potential it is paramount to know if mosquitoes from currently WNV-free areas can support further spread of this epidemic. Methodology/Principal Findings We assessed WNV vector competence of Culex pipiens mosquitoes originating from north-western Europe (NWE) in direct comparison with those from NA. We exposed mosquitoes to infectious blood meals of lineage 1 or 2 WNV and determined the infection and transmission rates. We explored reasons for vector competence differences by comparing intrathoracic injection versus blood meal infection, and we investigated the influence of temperature. We found that NWE mosquitoes are highly competent for both WNV lineages, with transmission rates up to 25%. Compared to NA mosquitoes, transmission rates for lineage 2 WNV were significantly elevated in NWE mosquitoes due to better virus dissemination from the midgut and a shorter extrinsic incubation time. WNV infection rates further increased with temperature increase. Conclusions/Significance Our study provides experimental evidence to indicate markedly different risk levels between both continents for lineage 2 WNV transmission and suggests a degree of genotype-genotype specificity in the interaction between virus and vector. Our experiments with varying temperatures explain the current localized WNV activity in southern Europe, yet imply further epidemic spread throughout NWE during periods with favourable climatic conditions. This emphasizes the need for intensified surveillance of virus activity in current WNV disease-free regions and warrants increased awareness in clinics throughout Europe., Author Summary West Nile virus (WNV) is on the rise in Europe, with increasing numbers of human cases of neurological disease and death since 2010. However, it is currently unknown whether or not WNV will continue to spread to north-western Europe (NWE), in a fashion similar to the WNV epidemic sweep in the United States (1999–2004). The presence of competent mosquitoes is a strict requirement for WNV transmission, but no laboratory studies have been conducted with the new European lineage 2 WNV outbreak strain. Our study is the first to investigate transmissibility in NWE Culex pipiens for lineage 2 WNV in a systematic, direct comparison with North American Culex pipiens and with the lineage 1 WNV strain. We demonstrate that European mosquitoes are highly competent for both WNV lineages, which underscores the epidemic potential of WNV in Europe. However, the transmission rate for lineage 2 WNV was significantly lower in North American mosquitoes, which indicates different risk levels between both continents for lineage 2 but not lineage 1 WNV. Based on our result, we propose that WNV surveillance in mosquitoes and birds must be intensified in Europe to allow early detection, timely intervention strategies and prevent outbreaks of WNV neurological disease.

International audience; Rabies virus, a neurotropic lyssavirus responsible for unavoidable fatal encephalitis, is transmitted by saliva of infected animals through bite, scratch or licking of broken skin or a mucous membrane. Infection can be prevented by timely prevention (wash for several minutes, antisepsis and vaccination completed by antirabies immunoglobulins [Ig] according to the severity of exposure). The 55,000 human deaths estimated annually worldwide result mainly from uncontrolled canine rabies in enzootic countries (particularly in Africa and in Asia), attributable to a lack of resources or interest for this disease. Bat rabies, henceforth first cause of human's rabies in many countries in America, affects a very small number of individuals but seems more difficult to control. Shortened vaccine protocols, rationalized use of Ig and development of products of substitution should enhance access of exposed patients to prevention. Finally, research on the biological cycle, the pathogeny and on escape of virus-induced mechanisms from the immune system should continue to pave the way for presently unknown treatments of clinical rabies.

International audience; Rabies virus, a neurotropic lyssavirus responsible for unavoidable fatal encephalitis, is transmitted by saliva of infected animals through bite, scratch or licking of broken skin or a mucous membrane. Infection can be prevented by timely prevention (wash for several minutes, antisepsis and vaccination completed by antirabies immunoglobulins [Ig] according to the severity of exposure). The 55,000 human deaths estimated annually worldwide result mainly from uncontrolled canine rabies in enzootic countries (particularly in Africa and in Asia), attributable to a lack of resources or interest for this disease. Bat rabies, henceforth first cause of human's rabies in many countries in America, affects a very small number of individuals but seems more difficult to control. Shortened vaccine protocols, rationalized use of Ig and development of products of substitution should enhance access of exposed patients to prevention. Finally, research on the biological cycle, the pathogeny and on escape of virus-induced mechanisms from the immune system should continue to pave the way for presently unknown treatments of clinical rabies.

Background The chikungunya (CHIK) outbreak that struck La Reunion Island in 2005 was preceded by few human cases of Dengue (DEN), but which surprisingly did not lead to an epidemic as might have been expected in a non-immune population. Both arboviral diseases are transmitted to humans by two main mosquito species, Aedes aegypti and Aedes albopictus. In the absence of the former, Ae. albopictus was the only species responsible for viral transmission on La Reunion Island. This mosquito is naturally super-infected with two Wolbachia strains, wAlbA and wAlbB. While Wolbachia does not affect replication of CHIK virus (CHIKV) in Ae. albopictus, a similar effect was not observed with DEN virus (DENV). Methods/Principal Findings To understand the weak vectorial status of Ae. albopictus towards DENV, we used experimental oral infections of mosquitoes from La Reunion Island to characterize the impact of Wolbachia on DENV infection. Viral loads and Wolbachia densities were measured by quantitative PCR in different organs of Ae. albopictus where DENV replication takes place after ingestion. We found that: (i) Wolbachia does not affect viral replication, (ii) Wolbachia restricts viral density in salivary glands, and (iii) Wolbachia limits transmission of DENV, as infectious viral particles were only detected in the saliva of Wolbachia-uninfected Ae. albopictus, 14 days after the infectious blood-meal. Conclusions We show that Wolbachia does not affect the replication of DENV in Ae. albopictus. However, Wolbachia is able to reduce viral infection of salivary glands and limit transmission, suggesting a role of Wolbachia in naturally restricting the transmission of DENV in Ae. albopictus from La Reunion Island. The extension of this conclusion to other Ae. albopictus populations should be investigated., Author Summary Aedes albopictus is an invasive species that is expanding its natural range of geographic distribution. While it was previously considered a secondary vector of different arboviruses, this mosquito species is involved in the most recent outbreaks of chikungunya but contributes weakly to dengue outbreaks. Ae. albopictus naturally carries two strains of the bacterium Wolbachia, wAlbA and wAlbB. Present in 20% of insect species, Wolbachia is an obligate intracellular symbiont mainly transmitted through females. When inoculated into some mosquito hosts, Wolbachia is able to shorten the adult life span and to block arbovirus transmission. We have previously shown that Wolbachia is not capable of limiting chikungunya replication in the mosquito vector. In this study, we show that the native Wolbachia is able to limit dengue transmission by restricting the delivery of infectious viral particles from the mosquito saliva when biting. Therefore, our results might explain the low vector competence of Ae. albopictus for dengue, and thus its weak contribution as an epidemic dengue vector.

Background Since the major outbreak in 2007 in the Yap Island, Zika virus (ZIKV) causing dengue-like syndromes has affected multiple islands of the South Pacific region. In May 2015, the virus was detected in Brazil and then spread through South and Central America. In December 2015, ZIKV was detected in French Guiana and Martinique. The aim of the study was to evaluate the vector competence of the mosquito spp. Aedes aegypti and Aedes albopictus from the Caribbean (Martinique, Guadeloupe), North America (southern United States), South America (Brazil, French Guiana) for the currently circulating Asian genotype of ZIKV isolated from a patient in April 2014 in New Caledonia. Methodology/Principal Findings Mosquitoes were orally exposed to an Asian genotype of ZIKV (NC-2014-5132). Upon exposure, engorged mosquitoes were maintained at 28°±1°C, a 16h:8h light:dark cycle and 80% humidity. 25–30 mosquitoes were processed at 4, 7 and 14 days post-infection (dpi). Mosquito bodies (thorax and abdomen), heads and saliva were analyzed to measure infection, dissemination and transmission, respectively. High infection but lower disseminated infection and transmission rates were observed for both Ae. aegypti and Ae. albopictus. Ae. aegypti populations from Guadeloupe and French Guiana exhibited a higher dissemination of ZIKV than the other Ae. aegypti populations examined. Transmission of ZIKV was observed in both mosquito species at 14 dpi but at a low level. Conclusions/Significance This study suggests that although susceptible to infection, Ae. aegypti and Ae. albopictus were unexpectedly low competent vectors for ZIKV. This may suggest that other factors such as the large naïve population for ZIKV and the high densities of human-biting mosquitoes contribute to the rapid spread of ZIKV during the current outbreak., Author Summary Zika virus (ZIKV) is an emerging mosquito-borne arbovirus causing dengue-like symptoms. This virus was commonly detected in Africa and Asia. Since its emergence in Yap Island in Micronesia in 2007, ZIKV reemerged in the South Pacific region in 2013 and ultimately reached the American continent in 2015. The human biting mosquito Aedes aegypti and the less anthropophilic Aedes albopictus have been incriminated as vectors of ZIKV. Our study showed that American populations of Ae. aegypti and Ae. albopictus were able to become infected and disseminate ZIKV within the mosquito general cavity at early days (4, 7) post-infection (dpi). Nevertheless, transmission was unexpectedly low and only detected at 14 dpi. Our findings will help in designing more adapted vector control strategies and limiting the impact of a new emerging threat on human health in the Americas as did the chikungunya in 2014.

We report on a case of imported human rabies in Portugal, in July 2011 in a woman who presented initially complaining of back pain, without relating exposure to animal bites. She had travelled from Portugal to Bissau, Guinea-Bissau, in April where she had been bitten by a dog on 1 May. She was diagnosed with rabies on 26 July and died two weeks later in spite of being treated following the Milwaukee protocol.

Background Until 2008, human rabies had never been reported in French Guiana. On 28 May 2008, the French National Reference Center for Rabies (Institut Pasteur, Paris) confirmed the rabies diagnosis, based on hemi-nested polymerase chain reaction on skin biopsy and saliva specimens from a Guianan, who had never travelled overseas and died in Cayenne after presenting clinically typical meningoencephalitis. Methodology/Principal Findings Molecular typing of the virus identified a Lyssavirus (Rabies virus species), closely related to those circulating in hematophagous bats (mainly Desmodus rotundus) in Latin America. A multidisciplinary Crisis Unit was activated. Its objectives were to implement an epidemiological investigation and a veterinary survey, to provide control measures and establish a communications program. The origin of the contamination was not formally established, but was probably linked to a bat bite based on the virus type isolated. After confirming exposure of 90 persons, they were vaccinated against rabies: 42 from the case's entourage and 48 healthcare workers. To handle that emergence and the local population's increased demand to be vaccinated, a specific communications program was established using several media: television, newspaper, radio. Conclusion/Significance This episode, occurring in the context of a Department far from continental France, strongly affected the local population, healthcare workers and authorities, and the management team faced intense pressure. This observation confirms that the risk of contracting rabies in French Guiana is real, with consequences for population educational program, control measures, medical diagnosis and post-exposure prophylaxis., Author Summary Until 2008, rabies had never been described within the French Guianan human population. Emergence of the first case in May 2008 in this French Overseas Department represented a public health event that markedly affected the local population, healthcare workers and public health authorities. The antirabies clinic of French Guiana, located at Institut Pasteur de la Guyane, had to reorganize its functioning to handle the dramatically increased demand for vaccination. A rigorous epidemiological investigation and a veterinary study were conducted to identify the contamination source, probably linked to a bat bite, and the exposed population. Communication was a key factor to controlling this episode and changing the local perception of this formerly neglected disease. Because similar clinical cases had previously been described, without having been diagnosed, medical practices must be adapted and the rabies virus should be sought more systematically in similarly presenting cases. Sharing this experience could be useful for other countries that might someday have to manage such an emergence.

International audience; BACKGROUND: Chronic stress during pregnancy has been associated with worsened maternal and fetal outcomes. Acute stress immediately before spinal anaesthesia for caesarean section may contribute to hypotension. Therefore objective measures of acute stress may help identify women at risk of adverse outcomes. Salivary alpha-amylase is a stress biomarker that has so far been poorly investigated during pregnancy. The reference change value is the difference between two sequential results that must be exceeded for a change to be considered clinically relevant. Our first aim was to determine if salivary alpha-amylase increased in pregnant patients when subjected to the stress of transfer to the operating room. Our second aim was to determine if changes in salivary alpha-amylase were likely to be clinically significant by measuring reference change value in healthy volunteers. METHODS: In 15 pregnant patients undergoing planned caesarean section under spinal anaesthesia, salivary alpha-amylase, systolic blood pressure, heart rate, and immediate anxiety were measured on the morning of surgery on the ward and again in the operating room. The reference change value was calculated from 18 healthy volunteers. RESULTS: A median 220% increase in salivary alpha-amylase activity (P=0.0015) and a 17% increase in systolic blood pressure (P=0.0006) were observed between the ward and operating room. No changes of immediate anxiety or heart rate were observed. Reference change value was ±76% in volunteers and 13 of the 15 pregnant patients had a salivary alpha-amylase increase greater than the reference change value. CONCLUSION: When pregnant women are taken to the operating room, a clinically and statistically significant increase in salivary alpha-amylase was observed. Further studies are required to define its clinical usefulness.

International audience; Candida yeasts colonize the human oral cavity as commensals or opportunistic pathogens. They may be isolated from water circulating in dental unit waterlines mixed with traces of saliva mainly because of the dysfunction of antiretraction valves. This study deals with the growth ability of Candida albicans, Candida glabrata and Candida parapsilosis in tap water with saliva (0-20% v/v). Results show that C. glabrata is the most susceptible species in tap water. Furthermore, saliva promotes both survival and proliferation of the three studied Candida species in tap water.

International audience; Important data obtained in mice raise the possibility that immunization against the saliva of sand flies could protect from leishmaniasis. Sand fly saliva stimulates the production of specific antibodies in individuals living in endemic areas of parasite transmission. To characterize the humoral immune response against the saliva of Phlebotomus papatasi in humans, we carried out a prospective study on 200 children living in areas of Leishmania major transmission. We showed that 83% of donors carried anti-saliva IgG antibodies, primarily of IgG4 isotype. Positive sera reacted differentially with seven salivary proteins. The protein PpSP30 was prominently recognized by all the sera. The salivary proteins triggered the production of various antibody isotypes. Interestingly, the immunodominant PpSP30 was recognized by all IgG subclasses, whereas PpSP12 was not by IgG4. Immunoproteomic analyses may help to identify the impact of each salivary protein on the L. major infection and to select potential vaccine candidates.

International audience; Cushing's syndrome is a rare condition in the general population (1/1000000) and is even less common during pregnancy. We report the case of a patient cared at 27 weeks of gestation (wg) for hypertension and electolyte disturbances. Cushing's syndrome was confirmed by salivary cortisol and ACTH assessment. RMN revealed a 9 cm left adrenal tumor. Severe hypertension and electolyte disturbances on the one hand, and diagnostic uncertainty on the other hand, imposed adrenalectomy at 29 wg. Twelve days later, fetal distress led to a caesarian section and birth of a well being male baby.

Background Assessment exposure and immunity to malaria is an important step in the fight against the disease. Increased malaria infection in non-immune travellers under anti-malarial chemoprophylaxis, as well as the implementation of malaria elimination programmes in endemic countries, raises new issues that pertain to these processes. Notably, monitoring malaria immunity has become more difficult in individuals showing low antibody (Ab) responses or taking medications against the Plasmodium falciparum blood stages. Commonly available techniques in malaria seroepidemiology have limited sensitivity, both against pre-erythrocytic, as against blood stages of the parasite. Thus, the aim of this study was to develop a sensitive tool to assess the exposure to malaria or to bites from the vector Anopheles gambiae, despite anti-malarial prophylactic treatment. Methods Ab responses to 13 pre-erythrocytic P. falciparum-specific peptides derived from the proteins Lsa1, Lsa3, Glurp, Salsa, Trap, Starp, CSP and Pf11.1, and to 2 peptides specific for the Anopheles gambiae saliva protein gSG6 were tested. In this study, 253 individuals from three Senegalese areas with different transmission intensities and 124 European travellers exposed to malaria during a short period of time were included. Results The multiplex assay was optimized for most but not all of the antigens. It was rapid, reproducible and required a small volume of serum. Proportions of Ab-positive individuals, Ab levels and the mean number of antigens (Ags) recognized by each individual increased significantly with increases in the level of malaria exposure. Conclusion The multiplex assay developed here provides a useful tool to evaluate immune responses to multiple Ags in large populations, even when only small amounts of serum are available, or Ab titres are low, as in case of travellers. Finally, the relationship of Ab responses with malaria endemicity levels provides a way to monitor exposure in differentially exposed autochthonous individuals from various endemicity areas, as well as in travellers who are not immune, thus indirectly assessing the parasite transmission and malaria risk in the new eradication era.

Noroviruses have been recognized to be the predominant agents of nonbacterial gastroenteritis outbreaks in humans, and their transmission via contaminated shellfish consumption has been demonstrated. Norovirus laboratory experiments, volunteer challenge studies, and community gastroenteritis outbreak investigations have identified human genetic susceptibility factors related to histo-blood group antigen expression. Following a banquet in Brittany, France, in February 2008, gastroenteritis cases were linked to oyster consumption. This study identified an association of the norovirus illnesses with histo-blood group expression, and oyster contamination with norovirus was confirmed by qualitative and quantitative analyses. The secretor phenotype was associated with illness, especially for the non-A subgroup. The study showed that, in addition to accidental climatic events that may lead to oyster contamination, illegal shellfish collection and trading are also risk factors associated with outbreaks.

International audience; One of the major therapeutic strategy in cystic fibrosis aims at developing modulators of cystic fibrosis transmembrane conductance regulator (CFTR) channels. We recently discovered methylglyoxal alpha-aminoazaheterocycle adducts, as a new family of CFTR inhibitors. In a structure-activity relationship study, we have now identified GPact-11a, a compound able not to inhibit but to activate CFTR. Here, we present the effect of GPact-11a on CFTR activity using in vitro (iodide efflux, fluorescence imaging and patch-clamp recordings), ex vivo (short-circuit current measurements) and in vivo (salivary secretion) experiments. We report that GPact-11a: 1) is an activator of CFTR in several airway epithelial cell lines; 2) activates rescued F508del-CFTR in nasal, tracheal, bronchial, pancreatic cell lines and in human CF ciliated epithelial cells, freshly dissociated from lung samples; 3) stimulates ex vivo the colonic chloride secretion and increases in vivo the salivary secretion in cftr(+/+) but not cftr(-/-) mice; and 4) is selective for CFTR because its effect is inhibited by CFTR(inh)-172, GlyH-101, glibenclamide and GPinh-5a. To conclude, this work identifies a selective activator of wild-type and rescued F508del-CFTR. This nontoxic and water-soluble agent represents a good candidate, alone or in combination with a F508del-CFTR corrector, for the development of a CFTR modulator in cystic fibrosis.

International audience; To optimize malaria control, WHO has prioritised the need for new indicators to evaluate the efficacy of malaria vector control strategies. The gSG6-P1 peptide from gSG6 protein of Anopheles gambiae salivary glands was previously designed as a specific salivary sequence of malaria vector species. It was shown that the quantification of human antibody (Ab) responses to Anopheles salivary proteins in general and especially to the gSG6-P1 peptide was a pertinent biomarker of human exposure to Anopheles. The present objective was to validate this indicator in the evaluation of the efficacy of Insecticide Treated Nets (ITNs). A longitudinal evaluation, including parasitological, entomological and immunological assessments, was conducted on children and adults from a malaria-endemic area before and after the introduction of ITNs. Significant decrease of anti-gSG6-P1 IgG response was observed just after the efficient ITNs use. Interestingly, specific IgG Ab level was especially pertinent to evaluate a short-time period of ITNs efficacy and at individual level. However, specific IgG rose back up within four months as correct ITN use waned. IgG responses to one salivary peptide could constitute a reliable biomarker for the evaluation of ITN efficacy, at short- and long-term use, and provide a valuable tool in malaria vector control based on a real measurement of human-vector contact.

Background Buruli ulcer, the third mycobacterial disease after tuberculosis and leprosy, is caused by the environmental mycobacterium M. ulcerans. Various modes of transmission have been suspected for this disease, with no general consensus acceptance for any of them up to now. Since laboratory models demonstrated the ability of water bugs to transmit M. ulcerans, a particular attention is focused on the transmission of the bacilli by water bugs as hosts and vectors. However, it is only through detailed knowledge of the biodiversity and ecology of water bugs that the importance of this mode of transmission can be fully assessed. It is the objective of the work here to decipher the role of water bugs in M. ulcerans ecology and transmission, based on large-scale field studies. Methodology/Principal Findings The distribution of M. ulcerans-hosting water bugs was monitored on previously unprecedented time and space scales: a total of 7,407 water bugs, belonging to large number of different families, were collected over one year, in Buruli ulcer endemic and non endemic areas in central Cameroon. This study demonstrated the presence of M. ulcerans in insect saliva. In addition, the field results provided a full picture of the ecology of transmission in terms of biodiversity and detailed specification of seasonal and regional dynamics, with large temporal heterogeneity in the insect tissue colonization rate and detection of M. ulcerans only in water bug tissues collected in Buruli ulcer endemic areas. Conclusion/Significance The large-scale detection of bacilli in saliva of biting water bugs gives enhanced weight to their role in M. ulcerans transmission. On practical grounds, beyond the ecological interest, the results concerning seasonal and regional dynamics can provide an efficient tool in the hands of sanitary authorities to monitor environmental risks associated with Buruli ulcer., Author Summary Buruli ulcer, caused by Mycobacterium ulcerans, is a devastating skin disease. Most cases of Buruli ulcer occur in poor rural communities. As a result, treatment is frequently sought too late and about 25% of those infected—particularly children—become permanently disabled. Outbreaks of Buruli ulcer have always been associated with swampy areas. However, the route(s) of bacillus transmission is (are) still unclear. This Mycobacterium species resides in water where it colonizes many ecological niches such as aquatic plants, herbivorous animals and predatory/carnivorous insects. For several years the role of water bugs as hosts and vectors of M. ulcerans was suspected and was demonstrated under laboratory conditions. The aim of this work was to further assess the role of water bugs as hosts and vectors of M. ulcerans in environmental context. This work identifies several water bug families as hosts of M. ulcerans in Buruli ulcer endemic area. The detection of bacilli in saliva of human biting insects provides further evidence for their role in M. ulcerans transmission. Interestingly, three of these insects are good flyers, and as such could participate in M. ulcerans dissemination.

International audience; For the fight against malaria, the World Health Organization (WHO) has emphasized the need for indicators to evaluate the efficacy of vector-control strategies. This study investigates a potential immunological marker, based on human antibody responses to Anopheles saliva, as a new indicator to evaluate the efficacy of insecticide-treated nets (ITNs). Parasitological, entomological, and immunological assessments were carried out in children and adults from a malaria-endemic region of Angola before and after the introduction of ITNs. Immunoglobulin G (IgG) levels to An. gambiae saliva were positively associated with the intensity of An. gambiae exposure and malaria infection. A significant decrease in the anti-saliva IgG response was observed after the introduction of ITNs, and this was associated with a drop in parasite load. This study represents the first stage in the development of a new indicator to evaluate the efficacy of malaria vector-control strategies, which could apply in other arthropod vector-borne diseases.

Centre Hospitalier Universitaire (CHU) Limoges, Centre National de Reference du CMV, Laboratoire de Bactériologie-Virologie-Hygiène, Limoges, France; International audience; BACKGROUND: The polymorphism of genes encoding CMV envelope protein is used for strain classification and may influence pathogenesis and/or infectivity. CMV genotyping is usually based on sequencing or acrylamide gel-RFLP, but these methods are not suited to rapid screening of large populations. OBJECTIVES: We developed a high-throughput method to analyze CMV strains diversity and to detect multiple-strain infection in a large population of toddlers (six daycare centers (DCC) and an emergency unit (EU)). METHODS: We developed a new PCR-RFLP method coupled with capillary electrophoresis fragment detection for UL55-gB, UL75-gH and UL73-gN genotyping. To detect gB recombinants, gpUL55 typing was applied to two variable zones (NTerminal and central). We applied this method to 212 CMV-positive saliva samples and controlled the results by direct sequencing of PCR products. RESULTS: We identified 112 strains, that fell into eight groups in UL55-gB, two groups in UL75-gH, and seven groups in UL73-gN. The 79 samples from the emergency unit contained 30 strains, 28 children harboring 2 strains. The samples (n=133) from the six daycare centers contained respectively 4, 1, 6, 1 and 11 strains. Fifteen percent of strains were UL55-gB recombinants. CONCLUSION: Our new method can simultaneously determine gB, gH and gN genotypes and offers more precise classification of CMV strains than previous RFLP-based methods. This could constitute the basis for a new classification, particularly in UL55-gB. Easy direct identification of multiple strains and recombinants in pathological samples could facilitate large epidemiologic studies.

International audience

International audience; BACKGROUND: Cortisol hypersecretion is characteristic of acute clinical depression, but little is known in fully recovered, non-treated elderly persons with a lifetime history of depression. This study was designed to examine patterns of diurnal cycle of cortisol in an elderly cohort without current depression or treatment for depression according to whether the person has or has not experienced a previous episode of depression or co-morbid depression with anxiety. METHODS: Cortisol secretion was evaluated in 162 community-dwelling elderly on a stressful and a non-stressful day (basal level). Past depression and anxiety disorders were assessed using a standardized psychiatric examination based on DSM-IV criteria (the Mini International Neuropsychiatric Interview). RESULTS: Antidepressant-free persons with a history of non-co-morbid major depression (6.8% of the sample) showed basal cortisol hypersecretion compared to those with depression and anxiety (8.6%) or controls. Several hours after exposure to a stressful situation, controls showed a sustained increase in cortisol secretion, which was not observed in persons with a history of depression. Persons with a history of depression with anxiety showed a similar cortisol secretion at baseline to controls but a heightened response to stressful situation; a pattern comparable to that observed in subjects with pure anxiety disorders (16.7%). CONCLUSION: An abnormal HPA response persists even after effective treatment for depression. A history of co-morbid depression and anxiety gives rise to changes characteristic of anxiety alone. Our findings suggest that cortisol abnormalities may be trait markers for vulnerability to depression and for the differentiation of depression and depression with co-morbid anxiety.

International audience; The rhythm of plasma melatonin concentration is currently the most accurate marker of the endogenous human circadian pacemaker. A number of methods exist to estimate circadian phase and amplitude from the observed melatonin rhythm. However, almost all these methods are limited because they depend on the shape and amplitude of the melatonin pulse, which vary among individuals and can be affected by environmental influences, especially light. Furthermore, these methods are not based on the underlying known physiology of melatonin secretion and clearance, and therefore cannot accurately quantify changes in secretion and clearance observed under different experimental conditions. A published physiologically-based mathematical model of plasma melatonin can estimate synthesis onset and offset of melatonin under dim light conditions. We amended this model to include the known effect of melatonin suppression by ocular light exposure and to include a new compartment to model salivary melatonin concentration, which is widely used in clinical settings to determine circadian phase. This updated model has been incorporated into an existing mathematical model of the human circadian pacemaker and can be used to simulate experimental protocols under a number of conditions.

International audience; BACKGROUND: Buruli ulcer is a severe human skin disease caused by Mycobacterium ulcerans. This disease is primarily diagnosed in West Africa with increasing incidence. Antimycobacterial drug therapy is relatively effective during the preulcerative stage of the disease, but surgical excision of lesions with skin grafting is often the ultimate treatment. The mode of transmission of this Mycobacterium species remains a matter of debate, and relevant interventions to prevent this disease lack (i) the proper understanding of the M. ulcerans life history traits in its natural aquatic ecosystem and (ii) immune signatures that could be correlates of protection. We previously set up a laboratory ecosystem with predatory aquatic insects of the family Naucoridae and laboratory mice and showed that (i) M. ulcerans-carrying aquatic insects can transmit the mycobacterium through bites and (ii) that their salivary glands are the only tissues hosting replicative M. ulcerans. Further investigation in natural settings revealed that 5%-10% of these aquatic insects captured in endemic areas have M. ulcerans-loaded salivary glands. In search of novel epidemiological features we noticed that individuals working close to aquatic environments inhabited by insect predators were less prone to developing Buruli ulcers than their relatives. Thus we set out to investigate whether those individuals might display any immune signatures of exposure to M. ulcerans-free insect predator bites, and whether those could correlate with protection. METHODS AND FINDINGS: We took a two-pronged approach in this study, first investigating whether the insect bites are protective in a mouse model, and subsequently looking for possibly protective immune signatures in humans. We found that, in contrast to control BALB/c mice, BALB/c mice exposed to Naucoris aquatic insect bites or sensitized to Naucoris salivary gland homogenates (SGHs) displayed no lesion at the site of inoculation of M. ulcerans coated with Naucoris SGH components. Then using human serum samples collected in a Buruli ulcer-endemic area (in the Republic of Benin, West Africa), we assayed sera collected from either ulcer-free individuals or patients with Buruli ulcers for the titre of IgGs that bind to insect predator SGH, focusing on those molecules otherwise shown to be retained by M. ulcerans colonies. IgG titres were lower in the Buruli ulcer patient group than in the ulcer-free group. CONCLUSIONS: These data will help structure future investigations in Buruli ulcer-endemic areas, providing a rationale for research into human immune signatures of exposure to predatory aquatic insects, with special attention to those insect saliva molecules that bind to M. ulcerans.

Purpose: Our goal was to define tumor and saliva gene methylation profile of head and neck squamous cell carcinoma and to evaluate its prognostic significance and its biomarker potential for early detection of relapse. Experimental Design: We prospectively analyzed 11 genes by methylation-specific PCR on primary tumors, histologically normal adjacent mucosa, and saliva from 90 French patients at diagnosis and during follow-up as well as on 30 saliva specimens from control-matched patients with nonmalignant head and neck pathology. Five additional genes were analyzed on 50 tumors of the series. Results: Methylation of TIMP3, ECAD, p16, MGMT, DAPK, and RASSF1 was the most frequently observed in tumors and paired saliva samples were analyzed at diagnosis, with an excellent agreement between both samples. At least one of these six genes was methylated in >75% of the samples without additional positive samples when other genes were analyzed. Methylation profile was similar in newly diagnosed and second primary cancers. Aberrant methylation was not associated with a worse prognosis. Ninety percent of normal adjacent mucosa and all control saliva samples were negative. Twenty-two patients were followed after treatment; abnormal methylation was detectable in the saliva of five patients few months before clinical and 2-deoxy-2[18F]fluoro-d-glucose-positron emission tomography signs of relapse, allowing curable surgery. Saliva samples were negative for the 17 other patients: 16 were in remission and only 1 relapsed. Conclusions: Gene methylation in saliva is a promising biomarker for the follow-up and early detection of still curable relapses of head and neck squamous cell carcinoma patients.

International audience; The rhythm of plasma melatonin concentration is currently the most accurate marker of the endogenous human circadian pacemaker. A number of methods exist to estimate circadian phase and amplitude from the observed melatonin rhythm. However, almost all these methods are limited because they depend on the shape and amplitude of the melatonin pulse, which vary among individuals and can be affected by environmental influences, especially light. Furthermore, these methods are not based on the underlying known physiology of melatonin secretion and clearance, and therefore cannot accurately quantify changes in secretion and clearance observed under different experimental conditions. A published physiologically-based mathematical model of plasma melatonin can estimate synthesis onset and offset of melatonin under dim light conditions. We amended this model to include the known effect of melatonin suppression by ocular light exposure and to include a new compartment to model salivary melatonin concentration, which is widely used in clinical settings to determine circadian phase. This updated model has been incorporated into an existing mathematical model of the human circadian pacemaker and can be used to simulate experimental protocols under a number of conditions.

International audience; Apyrase activity is present in the saliva of haematophagous arthropods. It is related to blood-feeding because of the apyrase ability to hydrolyse ADP, a key component of platelet aggregation. Five apyrases with apparent molecular masses of 88, 82, 79, 68 and 67 kDa were identified in the saliva of the vector of Chagas disease, Triatoma infestans. The large size observed during purification of these enzymes suggested oligomerization. In the present study, we confirmed, using gel-filtration and analytical ultracentrifugation, the presence of apyrase oligomers with molecular masses of 200 kDa in the saliva. Electrophoretic analyses showed that disulphide bonds were involved in homo-oligomerization. In addition, heterogeneity in disulphide bonds and in pI was detected, with the pI ranging from 4.9 to 5.4. The present study gives the first insights into the quaternary structure of soluble apyrases.

A real-time quantitative PCR assay has been developed to measure human herpesvirus 6 (HHV-6) DNA in biological specimens. The assay sensitivity was 10 copies of DNA per well, with a linear dynamic range of 10 to 10 7 copies of HHV-6 DNA. Intra- and interassay variations were, respectively, 0.88 and 0.8% for samples containing 10 2 DNA copies, 0.99 and 0.96% for samples containing 10 4 copies, and 0.76 and 0.9% for samples containing 10 6 copies. Among 34 saliva samples from healthy subjects, 26 were found to contain HHV-6 DNA (76.5%; median, 23,870 copies/ml), and following a single freeze-thaw cycle, 25 of the same samples were found to be positive for HHV-6 DNA, although at a statistically significantly lower concentration (median, 3,497 copies/ml). The assay enabled detection of HHV-6 DNA in lymph node biopsies from patients with Hodgkin's disease (HD) (13 of 37 patients [35.1%]), B-cell neoplasms (8 of 36 patients [22.2%]), and T- or NK-cell neoplasms (3 of 13 patients [23.1%]), with concentrations ranging from 100 to 864,640 HHV-6 copies per μg of DNA (HHV-6B being found in every case except two). All HD patients infected with HHV-6 presented clinically with the nodular sclerosis subtype of HD. The real-time quantitative PCR assay developed here was simple to perform and was sensitive over a wide range of HHV-6 concentrations. It therefore appears to be of potential value in clinical investigation or diagnosis of HHV-6 infection.

Comment in: "Toward an activist agenda for monitoring virus emergence."https://doi.org/10.1016/j.chom.2014.05.014; International audience; The high replication and mutation rates of RNA viruses can result in the emergence of new epidemic variants. Thus, the ability to follow host-specific evolutionary trajectories of viruses is essential to predict and prevent epidemics. By studying the spatial and temporal evolution of chikungunya virus during natural transmission between mosquitoes and mammals, we have identified viral evolutionary intermediates prior to emergence. Analysis of virus populations at anatomical barriers revealed that the mosquito midgut and salivary gland pose population bottlenecks. By focusing on virus subpopulations in the saliva of multiple mosquito strains, we recapit-ulated the emergence of a recent epidemic strain of chikungunya and identified E1 glycoprotein mutations with potential to emerge in the future. These mutations confer fitness advantages in mosquito and mammalian hosts by altering virion stability and fusogenic activity. Thus, virus evolutionary tra-jectories can be predicted and studied in the short term before new variants displace currently circulating strains.