Identification of novel reassortant mammalian orthoreoviruses from bats in Slovenia.

BACKGROUND: Recently, mammalian orthoreoviruses (MRVs) were detected for the first time in European bats, and the closely related strain SI-MRV01 was isolated from a child with severe diarrhoea in Slovenia. Genetically similar strains have also been reported from other mammals, which reveals their wide host distribution. The aim of this study was to retrospectively investigate the occurrence and genetic diversity of MRVs in bats in Slovenia, from samples obtained throughout the country in 2008 to 2010, and in 2012 and to investigate the occurrence of the novel SI-MRV01 MRV variant in Slovenian bats. RESULTS: The detection of MRVs in bat guano was based on broad-range RT-PCR and specific bat MRV real-time RT-PCR. Subsequently, MRV isolates were obtained from cell culture propagation, with detailed molecular characterisation through whole-genome sequencing. Overall, bat MRVs were detected in 1.9% to 3.8% of bats in 2008, 2009 and 2012. However, in 2010 the prevalence was 33.0%, which defined an outbreak of the single SI-MRV01 strain. Here, we report on the identification of five MRV isolates of different serotypes that are designated as SI-MRV02, SI-MRV03, SI-MRV04, SI-MRV05 and SI-MRV06. There is high genetic variability between these characterised isolates, with evident genome reassortment seen across their genome segments. CONCLUSIONS: In conclusion, we have confirmed the presence of the SI-MRV01 strain in a Slovenian bat population. Moreover, according to genetic characterisation of S1 genome segment, all three MRV serotypes were present in the bat population. In this study, five independent MRV isolates were obtained and detailed whole genome analysis revealed high diversity between them. This study generates new information about the epidemiology and molecular characteristics of emerging bat MRV variants, and provides important molecular data for further studies of their pathogenesis and evolution.

The colonization of an irradiated environment: the case of microbial biofilm in a nuclear reactor.

The investigation of the microbial community change in the biofilm, growing on the walls of a containment tank of TRIGA nuclear reactor revealed a thriving community in an oligotrophic and heavy-metal-laden environment, periodically exposed to high pulses of ionizing radiation (IR). We observed a vertical IR resistance/tolerance stratification of microbial genera, with higher resistance and less diversity closer to the reactor core. One of the isolated Bacillus strains survived 15 kGy of combined gamma and proton radiation, which was surprising. It appears that there is a succession of genera that colonizes or re-colonizes new or IR-sterilized surfaces, led by Bacilli and/or Actinobacteria, upon which a photoautotrophic and diazotrophic community is established within a fortnight. The temporal progression of the biofilm community was evaluated also as a proxy for microbial response to radiological contamination events. This indicated there is a need for better dose-response models that could describe microbial response to contamination events. Overall, TRIGA nuclear reactor offers a unique insight into IR microbiology and provides useful means to study relevant microbial dose-thresholds during and after radiological contamination.

Metrological evaluation of DNA extraction method effects on the bacterial microbiome and resistome in sputum.

Targeted high-throughput sequencing (HTS) has revolutionized the way we look at bacterial communities. It can be used for the species-specific detection of bacteria as well as for the determination of the microbiome and resistome and can be applied to samples from almost any environment. However, the results of targeted HTS can be influenced by many factors, which poses a major challenge for its use in clinical diagnostics. In this study, we investigated the impact of the DNA extraction method on the determination of the bacterial microbiome and resistome by targeted HTS using principles from metrology and diagnostics such as repeatability and analytical sensitivity. Sputum samples spiked with Acinetobacter baumannii, Klebsiella pneumoniae, and Pseudomonas aeruginosa at three different concentrations (103-106 cells/mL) were used. DNA was extracted from each sample on 2 separate days in three replicates each using three different extraction methods based on cetrimonium bromide, magnetic beads, and silica membranes. All three spiked bacteria were detected in sputum, and the DNA extraction method had no significant effect on detection. However, the DNA extraction method had significant effects on the composition of the microbiome and the resistome. The sequencing results were repeatable in the majority of cases. The silica membrane-based DNA extraction kit provided the most repeatable results and the highest diversity of the microbiome and resistome. Targeted HTS has been shown to be a reliable tool for determining the microbiome and resistome; however, the method of DNA extraction should be carefully selected to minimize its impact on the results. IMPORTANCE: High-throughput sequencing (HTS) is one of the crucial new technologies that gives us insights into previously hidden parts of microbial communities. The DNA extraction method is an important step that can have a major impact on the results, and understanding this impact is of paramount importance for their reliable interpretation. Our results are of great value for the interpretation of sputum microbiome and resistome results obtained by targeted HTS. Our findings allow for a more rational design of future microbiome studies, which would lead to higher repeatability of results and easier comparison between different laboratories. This could also facilitate the introduction of targeted HTS in clinical microbiology for reliable identification of pathogenic bacteria and testing for antimicrobial resistance (AMR). As AMR is a major threat to public health, the improved methods for determining AMR would bring great benefits to both the healthcare system and society as a whole.

High similarity of novel orthoreovirus detected in a child hospitalized with acute gastroenteritis to mammalian orthoreoviruses found in bats in Europe.

Mammalian orthoreoviruses (MRVs) are known to cause mild enteric and respiratory infections in humans. They are widespread and infect a broad spectrum of mammals. We report here the first case of an MRV detected in a child with acute gastroenteritis, which showed the highest similarity to an MRV reported recently in European bats. An examination of a stool sample from the child was negative for most common viral and bacterial pathogens. Reovirus particles were identified by electron microscopic examination of both the stool suspension and cell culture supernatant. The whole-genome sequence was obtained with the Ion Torrent next-generation sequencing platform. Prior to sequencing, the stool sample suspension and cell culture supernatant were pretreated with nucleases and/or the convective interaction medium (CIM) monolithic chromatographic method to purify and concentrate the target viral nucleic acid. Whole-genome sequence analysis revealed that the Slovenian SI-MRV01 isolate was most similar to an MRV found in a bat in Germany. High similarity was shared in all genome segments, with nucleotide and amino acid identities between 93.8 to 99.0% and 98.4 to 99.7%, respectively. It was shown that CIM monolithic chromatography alone is an efficient method for enriching the sample in viral particles before nucleic acid isolation and next-generation sequencing application.

Detection of the GPI-anchorless prion protein fragment PrP226* in human brain.

BACKGROUND: The accumulation of the misfolded forms of cellular prion protein, i.e. prions (PrPSc), in the brain is one of the crucial characteristics of fatal neurodegenerative disorders, called transmissible spongiform encephalopathies (TSEs). Cellular prion protein is normally linked to the cell surface by the glycosylphosphatidylinositol (GPI) anchor. There is accumulating evidence that the GPI-anchorless prion protein may act as an accelerator of formation and propagation of prions. In the TSE affected human brain we have previously discovered a novel GPI-anchorless prion protein fragment, named PrP226*, which ends with the tyrosine 226. This fragment can be labeled specifically by the monoclonal antibody V5B2. METHODS: We developed a DELFIA based assay for quick and sensitive detection of the PrP226* fragment in human brain tissue homogenates. By calculating the ratio between the signals of native (N) and denatured (D) samples applied to the assay we were able to observe significant difference between 24 TSE affected brains and 10 control brains. The presence of PrP226* in brain tissue was confirmed by western blot. RESULTS: Our results demonstrate that PrP226* is present in small quantities in healthy human brain, whereas in degenerated brain it accumulates in prion aggregates, proportionally to PrPSc. Samples with high D/N ratio generally comprised more proteinase K resistant PrP, while no correlation was found between the quantity of PrP226* and standard classification of Creutzfeldt-Jakob disease (CJD). CONCLUSIONS: In the present study we show that the PrP226* fragment accumulates in prion aggregates and after being released from them by a denaturation procedure, could serve as a proteinase K digestion independent biomarker for human TSEs. The PrP226* assay described in this paper offers a tool to follow and study this unique anchorless PrP fragment in various parts of human brain and possibly also in other tissues and body fluids.

Dissecting giant hailstones: A glimpse into the troposphere with its diverse bacterial communities and fibrous microplastics.

The formation of giant hailstones is a rare weather event that has devastating consequences in inhabited areas. This hazard has been occurring more frequently and with greater size of hailstones in recent years, and thus needs to be better understood. While the generally accepted mechanism is thought to be a process similar to the formation of smaller hailstones but with exceptional duration and stronger updrafts, recent evidence suggests that biotic and abiotic factors also influence the growth of these unusually large ice chunks. In this study, we improved these findings by determining the distribution of a wide variety of these factors throughout the hail volume and expanding the search to include new particles that are common in the environment and are of anthropogenic origin. We melted the concentric layers of several giant hailstones that fell to the ground over a small region in Slovenia in 2019. The samples, up to 13 cm in diameter, were analyzed for biotic and abiotic constituents that could have influenced their formation. Using 16S rRNA-based metagenomics approaches, we identified a highly diverse bacterial community, and by using scanning electron microscopy and Raman spectroscopy, we found natural and synthetic fibers concentrated in the cores of the giant hailstones. For the first time, we were able to detect the existence of microplastic fibers in giant hailstones and determine the changes in the distribution of sand within the volume of the samples. Our results suggest that changes in the composition of hail layers and their great diversity are important factors that should be considered in research. It also appears that anthropogenic microfiber pollutants were a significant factor in the formation of the giant hailstones analyzed in this study.

Next-generation sequencing to characterise pyrazinamide resistance in Mycobacterium tuberculosis isolates from two Balkan countries.

OBJECTIVES: Next-generation sequencing (NGS) can provide a comprehensive analysis of the genetic alterations most commonly linked with pyrazinamide (PZA) resistance. However, there are no studies reporting the molecular background of PZA resistance in Mycobacterium tuberculosis (TB) isolates from the Balkan Peninsula. We aimed to examine the feasibility of full-length analysis of a gene linked with PZA resistance (pncA) using Ion Torrent technology compared with phenotypic BACTEC MGIT 960 drug susceptibility testing (DST) in clinical TB isolates from two countries of the Balkan Peninsula. METHODS: Between 1996 and 2017, we retrospectively selected 61 TB isolates. To identify gene variants related to drug resistance in genomic DNA extracted from TB isolates, AmpliSeq libraries were generated automatically using an AmpliSeq™ Kit for Chef DL8 and Ion AmpliSeq TB Research Panel. RESULTS: Of the 61 TB isolates, 56 were phenotypically resistant to any antibiotic. Among them, 38/56 isolates (67.9%) were phenotypically resistant to pyrazinamide, and pncA mutations were detected in 34/38 cases (89.5%). A mutation in the pncA promoter region was the most prevalent genetic alteration, detected in eight TB isolates. Comparison of NGS data with conventional BACTEC MGIT 960 DST revealed very strong agreement (91.8%) between the two methods in identifying PZA resistance, with high sensitivity (89.5%) and specificity (95.7%) for NGS. CONCLUSION: Detection of PZA resistance using NGS appears to be a valuable tool for surveillance of TB drug resistance in the Balkan Peninsula, with great potential to provide useful information at least 1 weak earlier than is possible with phenotypic DST.

Transporters and Efflux Pumps Are the Main Mechanisms Involved in Staphylococcus epidermidis Adaptation and Tolerance to Didecyldimethylammonium Chloride.

Staphylococcus epidermidis cleanroom strains are often exposed to sub-inhibitory concentrations of disinfectants, including didecyldimethylammonium chloride (DDAC). Consequently, they can adapt or even become tolerant to them. RNA-sequencing was used to investigate adaptation and tolerance mechanisms of S. epidermidis cleanroom strains (SE11, SE18), with S. epidermidis SE11Ad adapted and S. epidermidis SE18To tolerant to DDAC. Adaptation to DDAC was identified with up-regulation of genes mainly involved in transport (thioredoxin reductase [pstS], the arsenic efflux pump [gene ID, SE0334], sugar phosphate antiporter [uhpT]), while down-regulation was seen for the Agr system (agrA, arC, agrD, psm, SE1543), for enhanced biofilm formation. Tolerance to DDAC revealed the up-regulation of genes associated with transporters (L-cysteine transport [tcyB]; uracil permease [SE0875]; multidrug transporter [lmrP]; arsenic efflux pump [arsB]); the down-regulation of genes involved in amino-acid biosynthesis (lysine [dapE]; histidine [hisA]; methionine [metC]), and an enzyme involved in peptidoglycan, and therefore cell wall modifications (alanine racemase [SE1079]). We show for the first time the differentially expressed genes in DDAC-adapted and DDAC-tolerant S. epidermidis strains, which highlight the complexity of the responses through the involvement of different mechanisms.

Anti-idiotypic antibodies: a new approach in prion research.

BACKGROUND: In certain cases, anti-idiotypic antibodies that recognize an antigen-combining site of an antibody can mimic the structure and/or function of certain nominal antigens. This feature makes them particularly useful if conventional experimental approaches fail to fulfil expectations, especially when the molecule of interest is infectious, toxic or difficult to isolate and purify. We suggest the application of an anti-idiotype concept to the field of prion biology, with the aim of evoking a humoral immune response against the pathological isoform of the prion protein (PrPSc). Different ways to induce anti-idiotypic responses were studied in mice and chickens using various forms of V5B2, a PrPSc-specific monoclonal antibody we have described previously. RESULTS: The preparation of anti-idiotypic monoclonal antibodies was achieved with well-defined strategies of immunization, selection and subsequent characterization. Our results demonstrate that it is possible to induce a strong anti-idiotypic immune response against the V5B2 monoclonal antibody in both xenogeneic and syngeneic experimental systems. From the competition seen between polyclonal and monoclonal anti-idiotypic antibodies and the original immunogen, the P1 peptide, and even more importantly, the ultimate target antigen, PrPSc, we conclude that selected antibodies bind to the antigen-combining site of the V5B2 monoclonal antibody and might even resemble the PrPSc-specific epitope. The involvement of both antigen-combining sites in the interaction between V5B2 and the most promising monoclonal anti-idiotypic antibody was further supported by molecular docking. CONCLUSION: The results of the present study not only provide an example of the successful production of Ab2 monoclonal antibodies based on a well planned strategy for selection, but should also provide a new experimental approach that is applicable to the field of prion diseases.

De novo transcriptome of the pallial gland of the date mussel (Lithophaga lithophaga).

Lithophaga lithophaga is a rock-boring bivalve with an ability to dissolve carbonate substrata with its siphonal and pallial gland secretions. However, the molecular mechanism that enables this species to bore into calcareous rocks is not yet known. In order to identify genes potentially involved in chemical boring we performed transcriptome sequencing of pallial-gland tissue samples of L. lithophaga. Transcriptome sequencing using an Ion Torrent platform generated 60.563 million clean reads with an average read length of 96 bp. De novo assembly of clean reads produced 62,490 contigs with a mean length of 408 bp. Since the boring mechanism is attributed to calcium-binding proteins, the search focused on transcripts capable of binding this element. In all, 178 genes with calcium-binding ability were found to be expressed in the pallial gland of L. lithophaga. Annexins, calreticulin, phospholipase A2 and V-type proton-ATPases were considered as possible candidate chemical-boring genes due to their known function and involvement in various other biological processes: e.g. ion transport, cellular catabolic process, protein folding. Transcriptome analysis of L. lithophaga revealed a set of candidate genes putatively associated with chemical boring. The selected set of genes will be studied further to verify their expression patterns and their possible involvement in the rate of chemical boring in L. lithophaga.

Identification of an epitope on the recombinant bovine PrP that is able to elicit a prominent immune response in wild-type mice.

The main cause for the development of transmissible spongiform encephalopathies (TSE) is the conformational change of prion protein from the normal cellular isoform (PrP(C)) into the abnormal isoform, named prion (PrP(Sc)). The two isoforms have the same primary structure, and with PrP being highly conserved among different species, no immune response to PrP(Sc) has been observed in infected humans or other mammals so far. The problem of inducing immune response was encountered when producing monoclonal antibodies against PrP, therefore mice lacking a functional Prnp gene were predominantly used for the immunization. In the present paper we report that by immunizing wild-type BALB/c mice with chemically unmodified recombinant bovine PrP a potent humoral immune response was achieved. Furthermore, we were able to isolate the monoclonal antibody (mAb) E12/2 and few other mAbs, all reacting specifically with bovine and human PrP, but not with PrP from several other mammals. The epitope of mAb E12/2 is located at the C-terminal end of helix 1, with His155 being crucial for binding. It has been proven that mAb E12/2 is useful for human and bovine TSE research as well as for diagnostics. Our results show that there are sufficient structural differences between mouse and bovine PrP to provoke a prominent humoral immune response.

Complete Genome Sequence of a Bovine Viral Diarrhea Virus Subgenotype 1e Strain, SLO/2407/2006, Isolated in Slovenia.

Bovine viral diarrhea virus (BVDV) subgenotype 1e was isolated for the first time in Slovenia in 2006. Here, we report the complete genome sequence of BVDV-1e, strain SLO/2407/2006. The published genome will increase our understanding of the molecular characteristics of the BVDV-1e strains circulating in Europe.

Complete Genome Sequence of the Porcine Epidemic Diarrhea Virus Strain SLO/JH-11/2015.

Porcine epidemic diarrhea virus (PEDV) was detected for the first time in Slovenia in January 2015. The complete genome sequence of PEDV strain SLO/JH-11/2015, obtained from a fecal sample of a fattening pig with diarrhea in September 2015, is closely related to recently detected European strains.

Draft Genome Sequences of Dickeya sp. Isolates B16 (NIB Z 2098) and S1 (NIB Z 2099) Causing Soft Rot of Phalaenopsis Orchids.

The genus Dickeya contains bacteria causing soft rot of economically important crops and ornamental plants. Here, we report the draft genome sequences of two Dickeya sp. isolates from rotted leaves of Phalaenopsis orchids.

Epitope mapping of a PrP(Sc)-specific monoclonal antibody: identification of a novel C-terminally truncated prion fragment.

Monoclonal antibodies (mAbs) against prion proteins (PrPs) are indispensable in research and diagnosis of prion diseases, however the majority of these bind both the cellular (PrP(C)) and the disease-associated (PrP(Sc)) isoforms. According to the widely accepted protein-only hypothesis the two isoforms share the same sequence, but differ in their conformation. In the present study we set to determine the critical binding residues of our PrP(Sc)-specific mAbs with the view of discerning which residues play a key role in the conformational transition between PrP(C) and PrP(Sc). Focussing on the V5B2 mAb that provided differential labelling of prion-affected tissue from individuals positive for transmissible spongiform encephalopathies, we performed alanine scanning and phage-display epitope mapping to elucidate the antigenic determinants of this mAb and gain insight into its specificity on a molecular level. We observed that instead of discriminating between the two prion protein isoforms based on conformational differences, V5B2 binds a previously uncharacterized C-terminally truncated form of PrP(Sc) that ends with the residue Y226, which we named PrP226*. The addition of a single C-terminal amino-acid residue completely abolished V5B2 binding, while Western blots using recombinant full-length PrPs and PrPs terminating at Y226 confirmed that the V5B2 mAb discriminates between the two based on their difference in length.

Antibody variable-region sequencing as a method for hybridoma cell-line authentication.

Cross-contamination and misidentification of various cell lines is a widespread problem that can lead to spurious scientific conclusions. DNA fingerprinting is a powerful identification technique, which can be effectively used for the authentication of human cell lines. In contrast to human cancer cell lines, little attention has so far been given to establishing authentication practices for hybridoma cell lines. Since the majority of hybridomas stem from inbred animals, they have high genetic uniformity, which reduces the applicability of DNA fingerprinting. In the present study, we propose antibody variable-region sequencing as a method of choice for hybridoma cell-line authentication. This method focuses on the most diverse characteristic of hybridoma cell lines and thereby achieves a very high discriminatory power. The sequencing of light-chain variable regions has proven to be especially suitable for routine use because of its high success rate. Two other possible authentication methods, random amplified polymorphic DNA analysis and two-dimensional gel electrophoresis, were also examined. Compared to these and other methods that can be used for discrimination between hybridoma cell lines, variable-region sequencing has many advantages, most notably those of a very high discriminatory power, insensitivity to changes in experimental conditions, simple data analysis, and accessibility to most laboratories.