Summary: End-to-end next-generation sequencing microbiology data analysis requires a diversity of tools covering bacterial resequencing, de novo assembly, scaffolding, bacterial RNA-Seq, gene annotation and metagenomics. However, the construction of computational pipelines that use different software packages is difficult owing to a lack of interoperability, reproducibility and transparency. To overcome these limitations we present Orione, a Galaxy-based framework consisting of publicly available research software and specifically designed pipelines to build complex, reproducible workflows for next-generation sequencing microbiology data analysis. Enabling microbiology researchers to conduct their own custom analysis and data manipulation without software installation or programming, Orione provides new opportunities for data-intensive computational analyses in microbiology and metagenomics.Availability and implementation: Orione is available online at http://orione.crs4.it.Contact: gianmauro.cuccuru@crs4.itSupplementary information: Supplementary data are available at Bioinformatics online.

Hepatitis C virus (HCV) glycoprotein E2 binds to human cells by interacting with the CD81 molecule, which has been proposed to be the viral receptor. A correlation between binding to CD81 and species permissiveness to HCV infection has also been reported. We have determined the sequence of CD81 from the tamarin, a primate species known to be refractory to HCV infection. Tamarin CD81 (t-CD81) differs from the human molecule at 5 amino acid positions (155, 163, 169, 180, and 196) within the large extracellular loop (LEL), where the binding site for E2 has been located. Soluble recombinant forms of human CD81 (h-CD81), t-CD81, and African green monkey CD81 (agm-CD81) LEL molecules were analyzed by enzyme-linked immunosorbent assay for binding to E2 glycoprotein. Both h-CD81 and t-CD81 molecules were able to bind E2. Competition experiments showed that the two receptors cross-compete and that the t-CD81 binds with stronger affinity than the human molecule. Recently, h-CD81 residue 186 has been characterized as the critical residue involved in the interaction with E2. Recombinant CD81 mutant proteins were expressed to test whether human and tamarin receptors interacted with E2 in a comparable manner. Mutation of residue 186 (F186L) dramatically reduced the binding capability of t-CD81, a result that has already been demonstrated for the human receptor, whereas the opposite mutation (L186F) in agm-CD81 resulted in a neat gain of binding activity. Finally, the in vitro data were confirmed by detection of E2 binding to cotton-top tamarin (Saguinus oedipus) cell line B95-8 expressing endogenous CD81. These results indicate that the binding of E2 to CD81 is not predictive of an infection-producing interaction between HCV and host cells.Hepatitis C virus (HCV) is the major etiologic agent of non-A non-B hepatitis, infecting an estimated 400 million people worldwide (3). A striking feature of HCV infection is the tendency towards a chronic status leading to liver diseases such as chronic hepatitis, cirrhosis, and hepatocellular carcinoma (24). Moreover, HCV infection is also associated with mixed cryoglobulinemia, a B-lymphocyte proliferative disorder (2). HCV is an enveloped virus classified as a Flavivirus (20); its genome is a positive-strand RNA of 9.5 kb, containing a single open reading frame encoding a large polyprotein precursor (5, 6), whose cleavage results in mature structural and nonstructural viral proteins (18). A basic polypeptide (core) and two envelope glycoproteins (E1 and E2) are the putative HCV structural proteins (30). Because of the lack of a cell culture system for virus growth, most of the studies aimed at understanding the biological activity of viral envelope proteins have been performed on recombinant proteins (15,16,27,28). A truncated soluble recombinant form of HCV E2 glycoprotein has been reported to bind the surface of human cells (22) by interacting with the CD81 molecule (9, 19).CD81 is a member of the tetraspanin family, membrane proteins containing four transmembrane domains, a short int...

Human Autosomal Recessive Osteopetrosis (ARO) is a genetically heterogeneous disorder caused by reduced bone resorption by osteoclasts. In 2000, we found that mutations in the TCIRG1 gene encoding for a subunit of the proton pump (V-ATPase) are responsible for more than one-half of ARO cases. Since then, five additional genes have been demonstrated to be involved in the pathogenesis of the disease, leaving approximately 25% of cases that could not be associated with a genotype. Very recently, a mutation in the sorting nexin 10 (SNX10) gene, whose product is suggested to interact with the proton pump, has been found in 3 consanguineous families of Palestinian origin, thus adding a new candidate gene in patients not previously classified. Here we report the identification of 9 novel mutations in this gene in 14 ARO patients from 12 unrelated families of different geographic origin. Interestingly, we define the molecular defect in three cases of ''Västerbottenian osteopetrosis,'' named for the Swedish Province where a higher incidence of the disease has been reported. In our cohort of more than 310 patients from all over the world, SNX10-dependent ARO constitutes 4% of the cases, with a frequency comparable to the receptor activator of NF-kB ligand (RANKL), receptor activator of NF-kB (RANK) and osteopetrosisassociated transmembrane protein 1 (OSTM1)-dependent subsets. Although the clinical presentation is relatively variable in severity, bone seems to be the only affected tissue and the defect can be almost completely rescued by hematopoietic stem cell transplantation (HSCT). These results confirm the involvement of the SNX10 gene in human ARO and identify a new subset with a relatively favorable prognosis as compared to TCIRG1-dependent cases. Further analyses will help to better understand the role of SNX10 in osteoclast physiology and verify whether this protein might be considered a new target for selective antiresorptive therapies. ß

Tamarins (Saguinus species) infected by GB virus B (GBV-B) have recently been proposed as an acceptable surrogate model for hepatitis C virus (HCV) infection.The availability of infectious genomic molecular clones of both viruses will permit chimeric constructs to be tested for viability in animals. Studies in cells with parental and chimeric constructs would also be very useful for both basic research and drug discovery. For this purpose, a convenient host cell type supporting replication of in vitro-transcribed GBV-B RNA should be identified. We constructed a GBV-B subgenomic selectable replicon based on the sequence of a genomic molecular clone proved to sustain infection in tamarins. The corresponding in vitro-transcribed RNA was used to transfect the Huh7 human hepatoma cell line, and intracellular replication of transfected RNA was shown to occur, even though in a small percentage of transfected cells, giving rise to antibiotic-resistant clones. Sequence analysis of GBV-B RNA from some of those clones showed no adaptive mutations with respect to the input sequence, whereas the host cells sustained higher GBV-B RNA replication than the original Huh7 cells. The enhancement of replication depending on host cell was shown to be a feature common to the majority of clones selected. The replication of GBV-B subgenomic RNA was susceptible to inhibition by known inhibitors of HCV to a level similar to that of HCV subgenomic RNA.A problem that remains unsolved in the search for new therapeutic agents against hepatitis C virus (HCV) infection (12,20,25) is the availability of a small-animal model suitable for pharmacological studies, since the known host range of this virus includes only humans and chimpanzees. An interesting murine model based on the repopulation of mouse liver with human HCV hepatocytes has recently been proposed as sustaining HCV infection and yielding significantly high serum titers (17). The current version of this model, however, presents several drawbacks, including the difficulty of identifying an unlimited source of human hepatocytes and the low success rate, due in part to the peculiar genetic background of the mice used. Albeit very promising, this model needs improvement to be turned into a method accessible to most of the laboratories concerned.In the absence of a direct and convenient small-animal model, over the last few years a model for HCV infection alternative to the chimp has been proposed by various research groups. That model is based on the use of a surrogate virushost system, tamarins (Saguinus species) infected by GB virus B (GBV-B). A growing body of data about the enzymatic activity of GBV-B proteins has corroborated the hypothesis that useful information for research on anti-HCV drugs can be derived from experiments with GBV-B in tamarins. The identification of in vivo infectious cDNA has provided an indispensable tool to engineer the virus genome by insertion of HCV regions of interest. The availability of a small nonhuman primate GBV-B host and the perspective of using c...

The strong similarity between GB virus B (GBV-B) and hepatitis C virus (HCV) makes tamarins infected by GBV-B an acceptable surrogate animal model for HCV infection. Even more attractive, for drug discovery purposes, is the idea of constructing chimeric viruses by inserting HCV genes of interest into a GBV-B genome frame. To accomplish this, infectious cDNA clones of both viruses must be available. The characterization of several HCV molecular clones capable of infecting chimpanzees has been published, whereas only one infectious GBV-B clone inducing hepatitis in tamarins has been reported so far. Here we describe the infection of tamarins by intrahepatic injection of RNA transcribed from a genomic GBV-B clone (FL-3) and transmission of the disease from infected to naive tamarins via serum inoculation. The disease resulting from both direct and secondary infection was characterized for viral RNA titre and hepatitis parameters as well as for viral RNA distribution in the hepatic tissue. Host humoral immune response to GBV-B antigens was also monitored. The progression of the disease was compared to that induced by intravenous injection of different amounts of the non-recombinant virus.