Employing Constrained Nonnegative Matrix Factorization for Microstructure Segmentation.

Materials characterization using electron backscatter diffraction (EBSD) requires indexing the orientation of the measured region from Kikuchi patterns. The quality of Kikuchi patterns can degrade due to pattern overlaps arising from two or more orientations, in the presence of defects or grain boundaries. In this work, we employ constrained nonnegative matrix factorization to segment a microstructure with small grain misorientations, (<1∘), and predict the amount of pattern overlap. First, we implement the method on mixed simulated patterns-that replicates a pattern overlap scenario, and demonstrate the resolution limit of pattern mixing or factorization resolution using a weight metric. Subsequently, we segment a single-crystal dendritic microstructure and compare the results with high-resolution EBSD. By utilizing weight metrics across a low-angle grain boundary, we demonstrate how very small misorientations/low-angle grain boundaries can be resolved at a pixel level. Our approach constitutes a versatile and robust tool, complementing other fast indexing methods for microstructure characterization.

A simple, fast, and cost-effective smartphone-based digital imaging method for quantification of lidocaine hydrochloride in pharmaceutical formulations.

OBJECTIVE: A simple, highly specific, accurate and fast method by smartphone-based digital imaging was developed for estimating lidocaine hydrochloride in pharmaceutical formulations. MATERIAL AND METHODS: To obtain the images, a Galaxy A03 Core smartphone and an image acquisition device developed in the laboratory were used to control the incident factors in reproducibility of the measurements. The processing of the images was carried out with the Color Grab application. Finally, the absorbance values were calculated using the RGB intensity values of blank, standard, and sample solutions. The proposed method was compared with spectroscopic and chromatographic methods. RESULTS: The reaction between copper and lidocaine hydrochloride was characterized, showing better results in an equimolar ratio and maintaining the pH of the solution above 11.5. The use of the device for the capture of digital images allowed to control those sensitive parameters for reproducibility so that the analytical measurements showed adequate precision and accuracy. Validation of the main parameters of the method showed compliance with acceptance criteria. The application of the method for the analysis of injectable samples achieved reliable results, which were statistically similar to other reference instrumental methods. CONCLUSION: The proposed method presented figures of merit in relation to linearity, precision, selectivity, accuracy, and robustness; it was carried out by designing and manufacturing a device for capturing digital images on a smartphone, which were analyzed to obtain RGB intensity values. These data are finally used to calculate absorbance values of solutions. All these elements provide this work with innovative characteristics in the field of analysis for control of pharmaceutical formulations.

Solid-state quantum chemistry with ΘΦ (ThetaPhi): Spin-liquids, superconductors, and magnetic superstructures made computationally available.

We present a standalone ΘΦ (ThetaPhi) package capable to read the results of ab initio DFT/PAW quantum-chemical solid-state calculations processed through various tools projecting them to the atomic basis states as an input and to perform on top of this an analysis of so derived electronic structure which includes (among other options) the possibility to obtain a superconducting (Bardeen-Cooper-Schrieffer, BCS), spin-liquid (resonating valence bond, RVB) states/phases as solutions of the electronic structure problem along with the magnetically ordered phases with an arbitrary pitch (magnetic superstructure) vector. Remarkably, different solutions of electronic-structure problems come out as temperature-dependent (exemplified by various superconducting and spin-liquid phases) which feature is as well implemented. All that is exemplified by model calculations on 1D chain, 2D square lattice as well as on more realistic superconducting doped graphene, magnetic phases of iron, and spin-liquid and magnetically ordered states of a simplest nitrogen-based copper pseudo-oxide, CuNCN, resembling socalled metal-oxide framework (MOF) phases by the atomic interlinkage.

Leishmania major RUVBL1 has a hexameric conformation in solution and, in the presence of RUVBL2, forms a heterodimer with ATPase activity.

ATPases belonging to the AAA+ superfamily are associated with diverse cellular activities and are mainly characterized by a nucleotide-binding domain (NBD) containing the Walker A and Walker B motifs. AAA+ proteins have a range of functions, from DNA replication to protein degradation. Rvbs, also known as RUVBLs, are AAA+ ATPases with one NBD domain and were described from human to yeast as participants of the R2TP (Rvb1-Rvb2-Tah1-Pih1) complex. Although essential for the assembly of multiprotein complexes-containing DNA and RNA, the protozoa Rvb orthologs are less studied. For the first time, this work describes the Rvbs from Leishmania major, one of the causative agents of Tegumentar leishmaniasis in human. Recombinant LmRUVBL1 and LmRUVBL2 his-tagged proteins were successfully purified and investigated using biophysical tools. LmRUVBL1 was able to form a well-folded elongated hexamer in solution, while LmRUVBL2 formed a large aggregate. However, the co-expression of LmRUVBL1 and LmRUVBL2 assembled the proteins into an elongated heterodimer in solution. Thermo-stability and fluorescence experiments indicated that the LmRUVBL1/2 heterodimer had ATPase activity in vitro. This is an interesting result because hexameric LmRUVBL1 alone had low ATPase activity. Additionally, using independent SL-RNAseq libraries, it was possible to show that both proteins are expressed in all L. major life stages. Specific antibodies obtained against LmRUVBLs identified the proteins in promastigotes and metacyclics cell extracts. Together, the results here presented are the first step towards the characterization of Leishmania Rvbs, and may contribute to the development of possible strategies to intervene against leishmaniasis, a neglected tropical disease of great medical importance.

Rotavirus Species B Encodes a Functional Fusion-Associated Small Transmembrane Protein.

Rotavirus is an important cause of diarrheal disease in young mammals. Rotavirus species A (RVA) causes most human rotavirus diarrheal disease and primarily affects infants and young children. Rotavirus species B (RVB) has been associated with sporadic outbreaks of human adult diarrheal disease. RVA and RVB are predicted to encode mostly homologous proteins but differ significantly in the proteins encoded by the NSP1 gene. In the case of RVB, the NSP1 gene encodes two putative protein products of unknown function, NSP1-1 and NSP1-2. We demonstrate that human RVB NSP1-1 mediates syncytium formation in cultured human cells. Based on sequence alignment, NSP1-1 proteins from species B, G, and I contain features consistent with fusion-associated small transmembrane (FAST) proteins, which have previously been identified in other genera of the Reoviridae family. Like some other FAST proteins, RVB NSP1-1 is predicted to have an N-terminal myristoyl modification. Addition of an N-terminal FLAG peptide disrupts NSP1-1-mediated fusion. NSP1-1 from a human RVB mediates fusion of human cells but not hamster cells and, thus, may serve as a species tropism determinant. NSP1-1 also can enhance RVA replication in human cells, both in single-cycle infection studies and during a multicycle time course in the presence of fetal bovine serum, which inhibits rotavirus spread. These findings suggest potential yet untested roles for NSP1-1 in RVB species tropism, immune evasion, and pathogenesis.IMPORTANCE While species A rotavirus is commonly associated with diarrheal disease in young children, species B rotavirus has caused sporadic outbreaks of adult diarrheal disease. A major genetic difference between species A and B rotaviruses is the NSP1 gene, which encodes two proteins for species B rotavirus. We demonstrate that the smaller of these proteins, NSP1-1, can mediate fusion of cultured human cells. Comparison with viral proteins of similar function provides insight into NSP1-1 domain organization and fusion mechanism. These comparisons suggest that there is a fatty acid modification at the amino terminus of the protein, and our results show that an intact amino terminus is required for NSP1-1-mediated fusion. NSP1-1 from a human virus mediates fusion of human cells, but not hamster cells, and enhances species A rotavirus replication in culture. These findings suggest potential, but currently untested, roles for NSP1-1 in RVB host species tropism, immune evasion, and pathogenesis.

Advances on the Structure of the R2TP/Prefoldin-like Complex.

Cellular stability, assembly and activation of a growing list of macromolecular complexes require the action of HSP90 working in concert with the R2TP/Prefoldin-like (R2TP/PFDL) co-chaperone. RNA polymerase II, snoRNPs and complexes of PI3-kinase-like kinases, a family that includes the ATM, ATR, DNA-PKcs, TRAPP, SMG1 and mTOR proteins, are among the clients of the HSP90-R2TP system. Evidence links the R2TP/PFDL pathway with cancer, most likely because of the essential role in pathways commonly deregulated in cancer. R2TP forms the core of the co-cochaperone and orchestrates the recruitment of HSP90 and clients, whereas prefoldin and additional prefoldin-like proteins, including URI, associate with R2TP, but their function is still unclear. The mechanism by which R2TP/PFLD facilitates assembly and activation of such a variety of macromolecular complexes is poorly understood. Recent efforts in the structural characterization of R2TP have started to provide some mechanistic insights. We summarize recent structural findings, particularly how cryo-electron microscopy (cryo-EM) is contributing to our understanding of the architecture of the R2TP core complex. Structural differences discovered between yeast and human R2TP reveal unanticipated complexities of the metazoan R2TP complex, and opens new and interesting questions about how R2TP/PFLD works.

Differential roles of putative arginine fingers of AAA+ ATPases Rvb1 and Rvb2.

The evolutionarily conserved AAA+ ATPases Rvb1 and Rvb2 proteins form a heteromeric complex (Rvb1/2) required for assembly or remodeling of macromolecular complexes in essential cellular processes ranging from chromatin remodeling to ribosome biogenesis. Rvb1 and Rvb2 have a high degree of sequence and structural similarity, and both contain the classical features of ATPases of their clade, including an N-terminal AAA+ subdomain with the Walker A motif, an insertion domain that typically interacts with various binding partners, and a C-terminal AAA+ subdomain containing a Walker B motif, the Sensor I and II motifs, and an arginine finger. In this study, we find that despite the high degree of structural similarity, Rvb1 and Rvb2 have distinct active sites that impact their activities and regulation within the Rvb1/2 complex. Using a combination of biochemical and genetic approaches, we show that replacing the homologous arginine fingers of Rvb1 and Rvb2 with different amino acids not only has distinct effects on the catalytic activity of the complex, but also impacts cell growth, and the Rvb1/2 interactions with binding partners. Using molecular dynamics simulations, we find that changes near the active site of Rvb1 and Rvb2 cause long-range effects on the protein dynamics in the insertion domain, suggesting a molecular basis for how enzymatic activity within the catalytic site of ATP hydrolysis can be relayed to other domains of the Rvb1/2 complex to modulate its function. Further, we show the impact that the arginine finger variants have on snoRNP biogenesis and validate the findings from molecular dynamics simulations using a targeted genetic screen. Together, our results reveal new aspects of the regulation of the Rvb1/2 complex by identifying a relay of long-range molecular communication from the ATPase active site of the complex to the binding site of cofactors. Most importantly, our findings suggest that despite high similarity and cooperation within the same protein complex, the two proteins have evolved with unique properties critical for the regulation and function of the Rvb1/2 complex.

Detection and genetic diversity of porcine rotavirus A, B and C in eastern Australian piggeries.

Rotaviruses (RV) have a high prevalence in piggeries worldwide and are one of the major pathogens causing severe diarrhoea in young pigs. RV species A, B, and C have been linked to piglet diarrhoea in Australian pig herds, but their genetic diversity has not been studied in detail. Based on sequencing of the structural viral protein 7 (VP7) RVA G genotypes G3, G4 and G5, and RVC types G1, G3, G5, and G6 have been identified in Australian piggeries in previous studies. Although occurrence of RVB was reported in Australia in 1988, no further genetic analysis has been conducted. To improve health management decisions in Australian pig herds, more information on RV prevalence and genetic diversity is needed. Here, 243 enteric samples collected from 20 pig farms within Eastern Australia were analysed for the presence of RV in different age groups using a novel PCR-based multiplex assay (Pork MultiPath™ enteric panel). RVA, RVB, and RVC were detected in 10, 14, and 14 farms, respectively. Further sequencing of VP7 in selected RV-positive samples revealed G genotypes G2, G5, G9 (RVA), G6, G8, G14, G16, G20 (RVB), and G1, G3, G5, G6 (RVC) present. RVA was only detected in young (<10 weeks old) pigs whereas RVB and RVC were also detected in older animals (>11 weeks old). Interestingly, RVB and RVC G-type occurrence differed between age groups. In conclusion, this study provides new insights on the prevalence and diversity of different RV species in pig herds of Eastern Australia whilst demonstrating the ability of the Pork MultiPath™ technology to accurately differentiate between these RV species.

Development of a Quadruplex RT-qPCR for the Detection of Porcine Rotaviruses and the Phylogenetic Analysis of Porcine RVH in China.

Rotavirus A species (RVA), RVB, RVC, and RVH are four species of rotaviruses (RVs) that are prevalent in pig herds, and co-infections occur frequently. In this study, a quadruplex real-time quantitative RT-PCR (RT-qPCR) for the simultaneous detection of four porcine RVs was developed by designing specific primers and probes based on the VP6 gene of RVA, RVB, RVC, and RVH, respectively. The method showed high specificity and could only detect RVA, RVB, RVC, and RVH, without cross-reaction with other porcine viruses; showed excellent sensitivity, with a limit of detection (LOD) of 1.5 copies/µL for each virus; showed good repeatability, with intra-assay coefficients of variation (CVs) of 0.15-1.14% and inter-assay CVs of 0.07-0.96%. A total of 1447 clinical fecal samples from Guangxi province in China were tested using the developed quadruplex RT-qPCR. The results showed that RVA (42.71%, 618/1447), RVB (26.95%, 390/1447), RVC (42.92%, 621/1447), and RVH (13.68%, 198/1447) were simultaneously circulating in the pig herds, and the co-infection rate of different species of rotaviruses was found to be up to 44.01% (579/1447). The clinical samples were also detected using one previously reported method, and the coincidence rate of the detection results using two methods was more than 99.65%. The phylogenetic tree based on the VP6 gene sequences of RVH revealed that the porcine RVH strains from Guangxi province belonged to the genotype I5, which was closely related to Japanese and Vietnamese strains. In summary, an efficient, sensitive, and accurate method for the detection and differentiation of RVA, RVB, RVC, and RVH was developed and applied to investigate the prevalence of porcine RVs in Guangxi province, China. This study is the first to report the prevalence of porcine RVH in China.

Biochemical and biophysical characterization of the RVB-1/RVB-2 protein complex, the RuvBL/RVB homologues in Neurospora crassa.

The RVB proteins, composed of the conservative paralogs, RVB1 and RVB2, belong to the AAA+ (ATPases Associated with various cellular Activities) protein superfamily and are present in archaea and eukaryotes. The most distinct structural features are their ability to interact with each other forming the RVB1/2 complex and their participation in several macromolecular protein complexes leading them to be involved in many biological processes. We report here the biochemical and biophysical characterization of the Neurospora crassa RVB-1/RVB-2 complex. Chromatographic analyses revealed that the complex (APO) predominantly exists as a dimer in solution although hexamers were also observed. Nucleotides influence the oligomerization state, while ATP favors hexamers formation, ADP favors the formation of multimeric states, likely dodecamers, and the Molecular Dynamics (MD) simulations revealed the contribution of certain amino acid residues in the nucleotide stabilization. The complex binds to dsDNA fragments and exhibits ATPase activity, which is strongly enhanced in the presence of DNA. In addition, both GFP-fused proteins are predominantly nuclear, and their nuclear localization signals (NLS) interact with importin-α (NcIMPα). Our findings show that some properties are specific of the fungus proteins despite of their high identity to orthologous proteins. They are essential proteins in N. crassa, and the phenotypic defects exhibited by the heterokaryotic strains, mainly related to growth and development, indicate N. crassa as a promising organism to investigate additional biological and structural aspects of these proteins.

Isolation and whole protein characterization of species A and B bovine rotaviruses from Chinese calves.

Rotaviruses (RVs) account for severe diarrhea in children and young animals globally. In the current study, the fecal samples of diarrheic calves from a beef farm in Inner Mongolia were screened for RVA by ELISA and RT-PCR, followed by culture of three positive RVA samples in the MA-104 cell line. After 10 blind passages, cytopathic effects (CPE) appeared as detachment, granulation, and clustering of the inoculated cells. The virus isolates were identified by RT-PCR (VP6 gene RVA) and ESI-LC-MS/MS for whole protein sequencing. The protein sequences demonstrated the presence of two strains from species A rotavirus and one RVB strain; RVA/Cow-tc/CHN/35333/2019/G6P[5] was mixed with one RVB strain (RVB/Cow-tc/CHN/35334/2019/G5P[3]) in two samples, and RVA/Cow-tc/CHN/10927/2019/G8P[7] was found in one sample. They are of genotype constellations (G6-P[5]-I2-R2-C2-M2-A3-N2-T6-E2-H3), (G8-P[7]-I5-R1-C1- M2-A1-N1-T1-E1-H1), and (G5-P[3]-I3-R5-C5-A5-N4-H5), respectively. Besides, phylogenetic analysis of the obtained sequences demonstrated viral evolution.

NMR assignment and solution structure of the external DII domain of the yeast Rvb2 protein.

We report the nearly complete 1H, 15N and 13C resonance assignment and the solution structure of the external DII domain of the yeast Rvb2 protein, a member of the AAA+ATPase superfamily.

Genome Characterization of a Pathogenic Porcine Rotavirus B Strain Identified in Buryat Republic, Russia in 2015.

An outbreak of enteric disease of unknown etiology with 60% morbidity and 8% mortality in weaning piglets occurred in November 2015 on a farm in Buryat Republic, Russia. Metagenomic sequencing revealed the presence of rotavirus B in feces from diseased piglets while no other pathogens were identified. Clinical disease was reproduced in experimentally infected piglets, yielding the 11 RVB gene segments for strain Buryat15, with an RVB genotype constellation of G12-P[4]-I13-R4-C4-M4-A8-N10-T4-E4-H7. This genotype constellation has also been identified in the United States. While the Buryat15 VP7 protein lacked unique amino acid differences in the predicted neutralizing epitopes compared to the previously published swine RVB G12 strains, this report of RVB in Russian swine increases our epidemiological knowledge on the global prevalence and genetic diversity of RVB.

Regulation of Rvb1/Rvb2 by a Domain within the INO80 Chromatin Remodeling Complex Implicates the Yeast Rvbs as Protein Assembly Chaperones.

The hexameric AAA+ ATPases Rvb1 and Rvb2 (Rvbs) are essential for diverse processes ranging from metabolic signaling to chromatin remodeling, but their functions are unknown. While originally thought to act as helicases, recent proposals suggest that Rvbs act as protein assembly chaperones. However, experimental evidence for chaperone-like behavior is lacking. Here, we identify a potent protein activator of the Rvbs, a domain in the Ino80 ATPase subunit of the INO80 chromatin-remodeling complex, termed Ino80INS. Ino80INS stimulates Rvbs' ATPase activity by 16-fold while concomitantly promoting their dodecamerization. Using mass spectrometry, cryo-EM, and integrative modeling, we find that Ino80INS binds asymmetrically along the dodecamerization interface, resulting in a conformationally flexible dodecamer that collapses into hexamers upon ATP addition. Our results demonstrate the chaperone-like potential of Rvb1/Rvb2 and suggest a model where binding of multiple clients such as Ino80 stimulates ATP-driven cycling between hexamers and dodecamers, providing iterative opportunities for correct subunit assembly.

Heterocyclic pharmacochemistry of new rhinovirus antiviral agents: A combined computational and experimental study.

Rhinovirus (RV), member of the Enterovirus genus, is known to be involved in more than half of the common colds. Through advances in molecular biology, rhinoviruses have also been associated with exacerbations of chronic pulmonary diseases (e.g. asthma, chronic obstructive pulmonary disease (COPD) and cystic fibrosis). In the current investigation, we develop a novel series of 4,5-dimethoxybenzyl derivatives that potently inhibits rhinovirus replication. Compound (S)-7f blocks RV-B14 replication with an EC50 value of 0.25 µM and shows a low toxicity in HeLa cells (CC50 > 271 µM). Enantioseparation followed by an absolute configuration determination by a Mosher's method revealed the interest of enantiopure compounds. Molecular docking studies permitted the identification of key biological interactions within the drug-binding pocket and an in silico drug-like study revealed a good potential for the development of these derivatives.

Pih1p-Tah1p Puts a Lid on Hexameric AAA+ ATPases Rvb1/2p.

The Saccharomyces cerevisiae (Sc) R2TP complex affords an Hsp90-mediated and nucleotide-driven chaperone activity to proteins of small ribonucleoprotein particles (snoRNPs). The current lack of structural information on the ScR2TP complex, however, prevents a mechanistic understanding of this biological process. We characterized the structure of the ScR2TP complex made up of two AAA+ ATPases, Rvb1/2p, and two Hsp90 binding proteins, Tah1p and Pih1p, and its interaction with the snoRNP protein Nop58p by a combination of analytical ultracentrifugation, isothermal titration calorimetry, chemical crosslinking, hydrogen-deuterium exchange, and cryoelectron microscopy methods. We find that Pih1p-Tah1p interacts with Rvb1/2p cooperatively through the nucleotide-sensitive domain of Rvb1/2p. Nop58p further binds Pih1p-Tahp1 on top of the dome-shaped R2TP. Consequently, nucleotide binding releases Pih1p-Tah1p from Rvb1/2p, which offers a mechanism for nucleotide-driven binding and release of snoRNP intermediates.

Features of the Chaperone Cellular Network Revealed through Systematic Interaction Mapping.

A comprehensive view of molecular chaperone function in the cell was obtained through a systematic global integrative network approach based on physical (protein-protein) and genetic (gene-gene or epistatic) interaction mapping. This allowed us to decipher interactions involving all core chaperones (67) and cochaperones (15) of Saccharomyces cerevisiae. Our analysis revealed the presence of a large chaperone functional supercomplex, which we named the naturally joined (NAJ) chaperone complex, encompassing Hsp40, Hsp70, Hsp90, AAA+, CCT, and small Hsps. We further found that many chaperones interact with proteins that form foci or condensates under stress conditions. Using an in vitro reconstitution approach, we demonstrate condensate formation for the highly conserved AAA+ ATPases Rvb1 and Rvb2, which are part of the R2TP complex that interacts with Hsp90. This expanded view of the chaperone network in the cell clearly demonstrates the distinction between chaperones having broad versus narrow substrate specificities in protein homeostasis.

The Structure of the R2TP Complex Defines a Platform for Recruiting Diverse Client Proteins to the HSP90 Molecular Chaperone System.

The R2TP complex, comprising the Rvb1p-Rvb2p AAA-ATPases, Tah1p, and Pih1p in yeast, is a specialized Hsp90 co-chaperone required for the assembly and maturation of multi-subunit complexes. These include the small nucleolar ribonucleoproteins, RNA polymerase II, and complexes containing phosphatidylinositol-3-kinase-like kinases. The structure and stoichiometry of yeast R2TP and how it couples to Hsp90 are currently unknown. Here, we determine the 3D organization of yeast R2TP using sedimentation velocity analysis and cryo-electron microscopy. The 359-kDa complex comprises one Rvb1p/Rvb2p hetero-hexamer with domains II (DIIs) forming an open basket that accommodates a single copy of Tah1p-Pih1p. Tah1p-Pih1p binding to multiple DII domains regulates Rvb1p/Rvb2p ATPase activity. Using domain dissection and cross-linking mass spectrometry, we identified a unique region of Pih1p that is essential for interaction with Rvb1p/Rvb2p. These data provide a structural basis for understanding how R2TP couples an Hsp90 dimer to a diverse set of client proteins and complexes.

The AAA+ proteins Pontin and Reptin enter adult age: from understanding their basic biology to the identification of selective inhibitors.

Pontin and Reptin are related partner proteins belonging to the AAA+ (ATPases Associated with various cellular Activities) family. They are implicated in multiple and seemingly unrelated processes encompassing the regulation of gene transcription, the remodeling of chromatin, DNA damage sensing and repair, and the assembly of protein and ribonucleoprotein complexes, among others. The 2nd International Workshop on Pontin and Reptin took place at the Instituto de Tecnologia Química e Biológica António Xavier in Oeiras, Portugal on October 10-12, 2014, and reported significant new advances on the mechanisms of action of these two AAA+ ATPases. The major points under discussion were related to the mechanisms through which these proteins regulate gene transcription, their roles as co-chaperones, and their involvement in pathophysiology, especially in cancer and ciliary biology and disease. Finally, they may become anticancer drug targets since small chemical inhibitors were shown to produce anti-tumor effects in animal models.

Detection of rotavirus species A, B and C in domestic mammalian animals with diarrhoea and genotyping of bovine species A rotavirus strains.

Rotaviruses (RVs) are a major cause of neonatal diarrhoea in humans and animals worldwide. In this study, 425 faecal samples were collected between 1999 and 2013 from diarrhoeic livestock and companion animals at different locations in Germany and tested for RVs. A previously published real-time RT-PCR assay was optimized for detection of a larger variety of RV species A (RVA) strains, and real-time RT-PCR assays for detection of RV species B (RVB) and C (RVC) were newly developed. The detection limits of the assays were 1.54×10(2), 3.95×10(2) and 3.60×10(3) genome copies for RVA, RVB and RVC, respectively. RVA was identified in 85.2% of bovine samples, 51.2% of porcine samples, 50.0% of feline samples, 43.2% of equine samples and 39.7% of canine samples. RVB was found in 3.0% of bovine samples, 2.7% of equine samples and 1.6% of porcine samples. RVC was detected in 31.0% of porcine samples, 21.7% of feline samples, 9.0% of canine samples and 6.0% of bovine samples. For genotyping, 101 RVA-positive bovine samples were further analysed by semi-nested RT-PCR. Genotype combination G6P[5] was most frequently detected (67.3% of samples), followed by G6P[11] (13.9%), G10P[5] (4.0%), G8P[11] (3.0%), G6P[1] (1.0%), and G10P[11] (1.0%). Mixed RVA infections were detected in 5.9% of samples; no or incomplete typing was possible in 4.0% of the samples. This first overview on RV species and RVA genotypes in diarrhoeic livestock and companion animals from Germany indicates a broad circulation of a large variety of RVs.