A combined rolling-circle amplification (RCA) and sequence-independent single primer amplification (SISPA) approach was applied to four samples of human plasma and one sample of saliva from a cat. This approach permitted the characterization of nine anelloviruses. Most of them were identified as highly divergent strains that were classified into species of the genus Anellovirus. The smallest anellovirus described so far in humans was characterized (2PoSMA, 2002 nt; 'small anellovirus' species). Two highly divergent sequences belonging to the species Torque Teno Mini Virus (LIL-y1, 2887 nt; LIL-y2, 2871 nt), which clustered into a new phylogenetic branch, were also identified in human plasma samples. Finally, two genomes that are separated by a genetic divergence of 46 % were characterized in the cat's saliva, one of these creating a distinct phylogenetic branch (PRA1, 2019 nt). These results highlight the potential of RCA-SISPA for detecting circular (or circularized) genomes.

We compared the performance of SARS-CoV-2 neutralising antibody testing between 12 European laboratories involved in convalescent plasma trials. Raw titres differed almost 100-fold differences between laboratories when blind-testing 15 plasma samples. Calibration of titres in relation to the reference reagent and standard curve obtained by testing a dilution series reduced the inter-laboratory variability ca 10-fold. The harmonisation of neutralising antibody quantification is a vital step towards determining the protective and therapeutic levels of neutralising antibodies.

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Here we describe a practical procedure for sequencing long PCR products. The method relies on ultrasonic shearing of PCR products, resulting in fragments 700-1,000 nt long. Termini are subsequently repaired to obtain blunt ends and 3¢ A-overhangs are added before TA cloning. A predetermined number of clones are sequenced using an insert-independent primer to obtain an overlapping contig covering the full length of the PCR product. This method is cost effective and enables the complete sequencing of any large PCR product in a high-throughput format. Processing of amplified DNA requires 3 h handling time prior to the ligation step, and the clone library is available 2 d later. The complete sequence information is obtained approximately 5 d after the PCR step, depending on the sequencing procedure adopted.

A strictly anaerobic, slightly halophilic and moderately thermophilic, sporulating rod designated strain DVird3 T was isolated from deep-sea hydrothermal vent samples collected at a depth of approximately 800 m on the Atlantic Ocean Ridge. Strain DVird3 T possessed a few laterally inserted flagella, had a DNA G+C content of 33?1 mol% and grew optimally at pH 6?6 and at 45˚C. Growth was observed at temperatures between 20 and 58˚C and at pH values between 5?8 and 8?2. The optimum NaCl concentration for growth was 3 % sea salt (30 g l "1 ); no growth was observed in the presence of 15 or 60 g sea salt l "1 . Strain DVird3 T is heterotrophic and utilizes some sugars and various single amino acids. Acetate was the main fatty acid detected from carbohydrate fermentation, together with H 2 and CO 2 . Gelatin was used as an energy source. It performed the Stickland reaction. Phylogenetically, strain DVird3 T branched with members of cluster XI of the order Clostridiales, with Clostridium halophilum as its closest relative (similarity of 94?6 %). On the basis of its phenotypic, genotypic and phylogenetic characteristics, strain DVird3 T (=DSM 15212 T =CIP 107654 T ) is proposed as the type strain of a novel species of the genus Clostridium, Clostridium caminithermale sp. nov.Deep-sea hydrothermal vents are inhabited by a wide range of microbial communities that comprise free-living microorganisms as well as micro-organisms living in association with invertebrates (Jeanthon, 2000). Among these freeliving micro-organisms are bacteria belonging to the e-subclass of the Proteobacteria, and the significance of their ecological role has been identified, mainly in molecular surveys (Haddad et al., 1995;Jeanthon, 2000;Reysenbach et al., 2000b;Corre et al., 2001). The recent isolation, from deep-sea hydrothermal vents, of moderately thermophilic and thermophilic members of the e-subclass of the Proteobacteria has demonstrated the ability of these microorganisms to grow autotrophically using elemental sulfur as a terminal electron acceptor (Campbell et al., 2001;Alain et al., 2002a). This trait of sulfur reduction has also been found to be a common physiological feature of the wellstudied, anaerobic, chemoautotrophic, thermophilic and hyperthermophilic micro-organisms (domain Archaea) isolated from these peculiar deep marine environments (Jeanthon, 2000). Besides chemoautotrophy and mixotrophy, chemoheterotrophy has also been recognized as a significant type of metabolism within the deep-sea hydrothermal vent anaerobic microbial community (Jeanthon, 2000). Among such trophic microbial groups inhabiting deep environments, little attention has been paid to anaerobic mesophiles and moderate thermophiles in comparison with thermophilic and hyperthermophilic micro-organisms from both domains (Bacteria and Archaea). Therefore, most microbiological studies performed so far have underlined the fact that thermophilic and hyperthermophilic archaea thrive in the hottest part of the ecosystem, with only a few thermophilic anaerobic members o...

Toscana virus (TOSV) is an emerging pathogen in the Mediterranean basin where it causes summertime outbreaks of aseptic meningitis and meningoencephalitis. Many aspects of TOSV biology remain unknown including the possible implication of an amplifying mammalian host besides its vector. The three experiments described here were designed to assess the relationship between TOSV and type-I interferon (IFN) response. The main findings were as follows. First, TOSV growth in Vero cells is sensitive to an antiviral state induced by low-dose addition of exogenous IFN beta (IFN-β) (10IU/ml). Second, no IFN-β mRNA or IFN-β was detectable after infection of HeLa and 293T cells by TOSV. Finally, TOSV inhibits IFN-β production induced by Sendaï virus, a well known inducer of IFN-β production. In addition to showing that TOSV can inhibit the IFN-β response, these findings suggest that anti-IFN capability is maintained by regular contact with that of a mammalian host.

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