Complete genome sequence, taxonomic assignment, and comparative analysis of the untranslated regions of the Modoc virus, a flavivirus with no known vector

We recently developed a model for flavivirus infection in mice and hamsters using the Modoc virus (MODV), a flavivirus with no known vector (P. Leyssen, A. Van Lommel, C. Drosten, H. Schmitz, E. De Clercq, and J. Neyts, 2001, Virology 279, 27-37). We now present the coding and noncoding sequence of MODV. The Modoc virus genome was determined to be 10,600 nucleotides in length with a single open reading frame extending from nucleotides 110 to 10,234, encoding 3374 amino acids. The deduced gene order of the single open reading frame is C-prM-E-NS1-NS2A-NS2B-NS3-NS4A-NS4B-NS5, which is exactly the same as that of the mosquito- and tick-borne flaviviruses. It is flanked by a 5'- and 3'-untranslated region (UTR) of 109 and 366 nucleotides, respectively. Alignment of the MODV amino acid sequence with that of 20 other flaviviruses revealed several regions with high sequence similarity corresponding to functionally important domains (e.g., the serine protease/helicase/NTPase of NS3 and the methyltransferase/RNA-dependent RNA polymerase of NS5) and conserved sites for proteolytic cleavage by viral and cellular proteases. Phylogenetic analysis of the entire coding region confirmed the classification of MODV within the flaviviruses with no known vector, which is in agreement with previous findings based on partial NS5 sequences. A detailed comparative analysis of the putative folding patterns of the 5'- and 3'-UTR of MODV and of the tick- and mosquito-borne viruses was carried out. Structural elements in the 5'- and 3' UTR of MODV that are preserved among vector-borne flaviviruses were noted and so were structural elements distinguishing the MODV UTRs from mosquito-borne and tick-borne flaviviruses. Also the putative secondary structure of circularized MODV RNA is presented.     

Molecular diagnosis of group B coltiviruses infections.

The group-B of genus Coltivirus encompasses isolates from humans, ticks or mosquitoes collected in Indonesia and China. Subgroup-B1 includes the strain JKT/dsR-7075 and subgroup-B2 strains JKT/dsR-6423, JKT/dsR-6969, JKT/dsR-7043 and the Banna virus. Data are described for the PCR-based diagnosis of infection by group B coltiviruses. Sets of primers were designed from the sequences of the 7th, 9th and 12th viral segments and RT PCR assays were developed. Consensus primers permitted the detection of all known isolates of subgroup 1 or 2. Viral strains could be characterised further using primers specific for type B2a or B2b, or based on the length of the amplification products. All primers gave negative results when using RNAs from Orbiviruses or Group-A coltiviruses. These primers permitted the detection of Group-B coltiviruses-RNA in infected mouse blood at the acute stage of the disease. Accordingly, they could be used for the diagnosis of infection in humans.     

Is citrate theophylline adenosine dipyridamole (CTAD) better than citrate to survey unfractionated heparin treatment? Has delayed centrifugation a real impact on this survey?

Journal of Thrombosis and Thrombolysis - Unfractionated heparin (UFH) is the main anticoagulant used in intensive care unit. The anticoagulant effect is monitored by activated partial thrombin time...     

Bayesian modelling of daphnid responses to time-varying cadmium exposure in laboratory aquatic microcosms

Experiments were carried out to test the effects of cadmium on five aquatic species in 2-L indoor freshwater/sediment microcosms. Experimental data were collected over 21 days in static conditions, i.e. the microcosms evolved without water renewal. Because of speciation, the total cadmium concentration in water decreased with time. Here we present a focus on Daphnia magna responses. For the three life history traits we considered (survival, growth and reproduction), mathematical effect models were built based on threshold stress functions involving no effect concentrations (NECs). These models took the time-varying conditions of exposure into account through a time-recurrent formalism. Within a Bayesian framework, four kinds of data were fitted simultaneously (exposure, survival, growth and reproduction), using an appropriate error model for each endpoint. Hence, NECs were determined as well as their associated estimation uncertainty. Through this modelling approach, we demonstrate that thresholds for stress functions can be successfully inferred even in experimental setup more complex than standard bioassays.     

Strategies for the sequence determination of viral dsRNA genomes

The genetic study of viruses having dsRNA genomes is hampered by the technical difficulty of complete sequence determination of dsRNA. Optimised methods are described here for sequencing dsRNAs, which meet three different situations: (1) genomes that can be obtained in fairly high amounts (>20 ng per separated segment); (2) genomes with limited amounts of RNA that can be detected by electrophoretic gel separation and staining; (3) genomes that cannot be detected by electrophoretic gel separation and staining. These methods include improved Single Primer Amplification Technique protocols, an adaptation of the SMART methodology, and a new method permitting the selective enzymatic removal of dsRNA segments. Strategies permitting adaptation of these protocols to the full-length determination of dsRNA viral genomes are described. Each of the protocols is described for sequence determination of a chosen dsRNA virus.     

Quelle place pour la recherche des anticorps antiphosphatidyléthanolamine dans la suspicion du syndrome des antiphospholipides séronégatif ?

PurposeAntiphospholipid syndrome (APS) is a clinico-biological syndrome, which associates vascular injury and persisting antiphospholipid antibodies (aPL). Patients with clinical symptoms of APS but without aPL are defined as “seronegative APS” (SNAPS). The aim of this study was to evaluate antiphosphatidylethanolamine antibody (aPE) investigation in patients with SNAPS suspicion.MethodsThis retrospective study was conducted in patients with SNAPS suspicion. A homemade enzyme-linked immunosorbent assay (ELISA) was used to search for aPE. The results of this homemade method were compared with those from a global screening ELISA.ResultsTwo hundred twenty-eight patients with SNAPS suspicion were included. Among them, 58.3% had a thrombotic event. The homemade ELISA found positive persisting aPE in 23 patients (10%): 15 with a thrombotic event, 6 with obstetrical morbidity and 2 with a combined event. The global screening ELISA was positive in only 11 of these 23 patients (47.8%).ConclusionThese results suggest the implication of aPE in SNAPS.     

Thrombin generation profile in non-thrombotic factor V Leiden carriers

Journal of Thrombosis and Thrombolysis - Factor V Leiden (FVL) mutation is the most common genetic risk factor for venous thromboembolism. In families with a history of thrombosis, FVL can be...     

Survival data analyses in ecotoxicology: critical effect concentrations,methods and models. What should we use?

In ecotoxicology, critical effect concentrations are the most common indicators to quantitatively assess risks for species exposed to contaminants. Three types of critical effect concentrations are classically used: lowest/ no observed effect concentration (LOEC/NOEC), LC _x (x% lethal concentration) and NEC (no effect concentration). In this article, for each of these three types of critical effect concentration, we compared methods or models used for their estimation and proposed one as the most appropriate. We then compared these critical effect concentrations to each other. For that, we used nine survival data sets corresponding to D. magna exposition to nine different contaminants, for which the time-course of the response was monitored. Our results showed that: (i) LOEC/NOEC values at day 21 were method-dependent, and that the Cochran–Armitage test with a step-down procedure appeared to be the most protective for the environment; (ii) all tested concentration–response models we compared gave close values of LC₅₀ at day 21, nevertheless the Weibull model had the lowest global mean deviance; (iii) a simple threshold NEC-model both concentration and time dependent more completely described whole data (i.e. all timepoints) and enabled a precise estimation of the NEC. We then compared the three critical effect concentrations and argued that the use of the NEC might be a good option for environmental risk assessment.     

Comparison of bioassays with different exposure time patterns: the added value of dynamic modelling in predictive ecotoxicology

The purpose of this study was to compare Daphnia magna responses to cadmium between two toxicity experiments performed in static and flow-through conditions. As a consequence of how water was renewed, the two experiments were characterised by two different exposure time patterns for daphnids, time-varying and constant, respectively. Basing on survival, growth and reproduction, we addressed the questions of organism development and sensitivity to cadmium. Classical analysis methods are not designed to deal with the time dimension and therefore not suitable to compare effects of different exposure time patterns. We used instead a dynamic modelling framework taking all timepoints and the time course of exposure into account, making comparable the results obtained from our two experiments. This modelling framework enabled us to detect an improvement of organism development in flow-through conditions compared to static ones and infer similar sensitivity to cadmium for both exposure time patterns.