Search

Quality management and independent assessment of high-throughput sequencing-based virus diagnostics have not yet been established as a mandatory approach for ensuring comparable results. The sensitivity and specificity of viral high-throughput sequence data analysis are highly affected by bioinformatics processing using publicly available and custom tools and databases and thus differ widely between individuals and institutions. Here we present the results of the COMPARE [Collaborative Management Platform for Detection and Analyses of (Re-) emerging and Foodborne Outbreaks in Europe] in silico virus proficiency test. An artificial, simulated in silico data set of Illumina HiSeq sequences was provided to 13 different European institutes for bioinformatics analysis to identify viral pathogens in high-throughput sequence data. Comparison of the participants’ analyses shows that the use of different tools, programs, and databases for bioinformatics analyses can impact the correct identification of viral sequences from a simple data set. The identification of slightly mutated and highly divergent virus genomes has been shown to be most challenging. Furthermore, the interpretation of the results, together with a fictitious case report, by the participants showed that in addition to the bioinformatics analysis, the virological evaluation of the results can be important in clinical settings. External quality assessment and proficiency testing should become an important part of validating high-throughput sequencing-based virus diagnostics and could improve the harmonization, comparability, and reproducibility of results. There is a need for the establishment of international proficiency testing, like that established for conventional laboratory tests such as PCR, for bioinformatics pipelines and the interpretation of such results.

This study investigated the co-localization of the Middle East respiratory syndrome coronavirus (MERS-CoV) and its receptor dipeptidyl peptidase-4 (DPP4) by immunohistochemistry (IHC) across respiratory and lymphoid organs of experimentally MERS-CoV infected pigs and llamas. Also, scanning electron microscopy was performed to assess the ciliary integrity of respiratory epithelial cells in both species. In pigs, on day 2 post-inoculation (p.i.), DPP4-MERS-CoV co-localization was detected in medial turbinate epithelium. On day 4 p.i., the virus/receptor co-localized in frontal and medial turbinate epithelial cells in pigs, and epithelial cells distributed unevenly through the whole nasal cavity and in the cervical lymph node in llamas. MERS-CoV viral nucleocapsid was mainly detected in upper respiratory tract sites on days 2 and 4 p.i. in pigs and day 4 p.i. in llamas. No MERS-CoV was detected on day 24 p.i. in any tissue by IHC. While pigs showed severe ciliary loss in the nasal mucosa both on days 2 and 4 p.i. and moderate loss in the trachea on days 4 and 24 p.i., ciliation of respiratory organs in llamas was not significantly affected. Obtained data confirm the role of DPP4 for MERS-CoV entry in respiratory epithelial cells of llamas. Notably, several nasal epithelial cells in pigs were found to express viral antigen but not DPP4, suggesting the possible existence of other molecule/s facilitating virus entry or down regulation of DPP4 upon infection.

In situ hybridization (ISH) is a technique to determine potential correlations between viruses and lesions. The aim of the study was to compare ISH techniques for the detection of various viruses in different tissues. Tested RNA viruses include atypical porcine pestivirus (APPV) in the cerebellum of pigs, equine and bovine hepacivirus (EqHV, BovHepV) in the liver of horses and cattle, respectively, and Schmallenberg virus (SBV) in the cerebrum of goats. Examined DNA viruses comprise canine bocavirus 2 (CBoV-2) in the intestine of dogs, porcine bocavirus (PBoV) in the spinal cord of pigs and porcine circovirus 2 (PCV-2) in cerebrum, lymph node, and lung of pigs. ISH with self-designed digoxigenin-labelled RNA probes

Middle East respiratory syndrome (MERS) represents an important respiratory disease accompanied by lethal outcome in one-third of human patients. Recent data indicate that dromedaries represent an important source of infection, although information regarding viral cell tropism and pathogenesis is sparse. In the current study, tissues of eight dromedaries receiving inoculation of MERS-Coronavirus (MERS-CoV) after recombinant Modified-Vaccinia-Virus-Ankara (MVA-S)-vaccination (n = 4), MVA-vaccination (mock vaccination, n = 2) and PBS application (mock vaccination, n = 2), respectively, were investigated. Tissues were analyzed by histology, immunohistochemistry, immunofluorescence, and scanning electron microscopy. MERS-CoV infection in mock-vaccinated dromedaries revealed high numbers of MERS-CoV-nucleocapsid positive cells, T cells, and macrophages within nasal turbinates and trachea at day four post infection. Double immunolabeling demonstrated cytokeratin (CK) 18 expressing epithelial cells to be the prevailing target cell of MERS-CoV, while CK5/6 and CK14 expressing cells did not co-localize with virus. In addition, virus was occasionally detected in macrophages. The acute disease was further accompanied by ciliary loss along with a lack of dipeptidyl peptidase 4 (DPP4), known to mediate virus entry. DPP4 was mainly expressed by human lymphocytes and dromedary monocytes, but overall the expression level was lower in dromedaries. The present study underlines significant species-specific manifestations of MERS and highlights ciliary loss as an important finding in dromedaries. The obtained results promote a better understanding of coronavirus infections, which pose major health challenges.

Rabies is a lethal disease in humans and animals, killing approximately 60,000 people every year. Currently, there is no treatment available, except post-exposure prophylaxis (PEP) that can be administered whenever exposure to a rabid animal took place. Here we describe the beneficial effects of a combination treatment initiated at

Rabies virus infects almost all mammals resulting in lethal disease. To date there is no treatment available for symptomatic rabies and there is an urgent need to develop treatment strategies that would prolong survival, thereby providing a window of opportunity for the host to mount a protective immune response. We hypothesized that both virus and excessive immune response contribute to disease and that interfering with both is necessary to prevent lethal disease. Here, we have inhibited the pro-inflammatory response associated with pyroptosis and showed that inhibition of CASP-1 had a beneficial effect on survival time. Our results confirm that some inflammatory responses may be involved in the pathogenesis of severe disease and the results suggest that effective intervention includes inhibition of virus and host response.

Anthropogenic landscape changes contributed to the reduction of availability of habitats to wild animals. Hence, the presence of wild terrestrial carnivores in urban and peri-urban sites has increased considerably over the years implying an increased risk of interspecies spillover of infectious diseases and the transmission of zoonoses. The present study provides a detailed characterisation of the health status of the red fox (Vulpes vulpes), stone marten (Martes foina) and raccoon dog (Nyctereutes procyonoides) in their natural rural and periurban habitats in Schleswig-Holstein, Germany between November 2013 and January 2016 with focus on zoonoses and infectious diseases that are potentially threatening to other wildlife or domestic animal species. 79 red foxes, 17 stone martens and 10 raccoon dogs were collected from traps or hunts. In order to detect morphological changes and potential infectious diseases, necropsy and pathohistological work-up was performed. Additionally, in selected animals immunohistochemistry (influenza A virus, parvovirus, feline leukemia virus, Borna disease virus, tick-borne encephalitis, canine adenovirus, Neospora caninum, Toxoplasma gondii and Listeria monocytogenes), next-generation sequencing, polymerase chain reaction (fox circovirus) and serum-neutralisation analysis (canine distemper virus) were performed. Furthermore, all animals were screened for fox rabies virus (immunofluorescence), canine distemper virus (immunohistochemistry) and Aujeszky's disease (virus cultivation). The most important findings included encephalitis (n = 16) and pneumonia (n =20). None of the investigations revealed a specific cause for the observed morphological alterations except for one animal with an elevated serum titer of 1:160 for canine distemper. Animals displayed macroscopically and/or histopathologically detectable infections with parasites, including Taenia sp., Toxocara sp. and Alaria alata. In summary, wildlife predators carry zoonotic parasit

Influenza A viruses are major pathogens for humans, domestic animals, and wildlife, and these viruses occasionally cross the species barrier. In spring 2014, increased mortality of harbor seals (Phoca vitulina), associated with infection with an influenza A(H10N7) virus, was reported in Sweden and Denmark. Within a few months, this virus spread to seals of the coastal waters of Germany and the Netherlands, causing the death of thousands of animals. Genetic analysis of the hemagglutinin (HA) and neuraminidase (NA) genes of this seal influenza A(H10N7) virus revealed that it was most closely related to various avian influenza A(H10N7) viruses. The collection of samples from infected seals during the course of the outbreak provided a unique opportunity to follow the adaptation of the avian virus to its new seal host. Sequence data for samples collected from 41 different seals from four different countries between April 2014 and January 2015 were obtained by Sanger sequencing and next-generation sequencing to describe the molecular epidemiology of the seal influenza A(H10N7) virus. The majority of sequence variation occurred in the HA gene, and some mutations corresponded to amino acid changes not found in H10 viruses isolated from Eurasian birds. Also, sequence variation in the HA gene was greater at the beginning than at the end of the epidemic, when a number of the mutations observed earlier had been fixed. These results imply that when an avian influenza virus jumps the species barrier from birds to seals, amino acid changes in HA may occur rapidly and are important for virus adaptation to its new mammalian host.

Infectious disease metagenomics is driven by the question: "what is causing the disease?" in contrast to classical metagenome studies which are guided by "what is out there?" In case of a novel virus, a first step to eventually establishing etiology can be to recover a full-length viral genome from a metagenomic sample. However, retrieval of a full-length genome of a divergent virus is technically challenging and can be time-consuming and costly. Here we discuss different assembly and fragment linkage strategies such as iterative assembly, motif searches, k-mer frequency profiling, coverage profile binning, and other strategies used to recover genomes of potential viral pathogens in a timely and cost-effective manner.

Viral infections remain a serious global health issue. Metagenomic approaches are increasingly used in the detection of novel viral pathogens but also to generate complete genomes of uncultivated viruses. In silico identification of complete viral genomes from sequence data would allow rapid phylogenetic characterization of these new viruses. Often, however, complete viral genomes are not recovered, but rather several distinct contigs derived from a single entity are, some of which have no sequence homology to any known proteins. De novo assembly of single viruses from a metagenome is challenging, not only because of the lack of a reference genome, but also because of intrapopulation variation and uneven or insufficient coverage. Here we explored different assembly algorithms, remote homology searches, genome-specific sequence motifs, k-mer frequency ranking, and coverage profile binning to detect and obtain viral target genomes from metagenomes. All methods were tested on 454-generated sequencing datasets containing three recently described RNA viruses with a relatively large genome which were divergent to previously known viruses from the viral

The amount of information is growing exponentially with ever-new technologies emerging and is believed to be always at the limit. In contrast, huge resources are obviously available, which are underused in the IT sector, similar as e.g. in the renewable energy sector. This is especially for grid with its fast turnover rates very astonishing considering the barriers for further development put forward by the inability to satisfy the need for such resources. The phenomenon is a typical example of the Inverse Tragedy of the Commons, i.e. resources are underexploited in contrast to the unsustainable and destructing overexploitation in the Classic Tragedy of the Commons. An analysis of IT and the grid sector which attempts to share resources for better usage efficiency, reveals two challenges, which lead to the heart of the paradox: i) From a macro perspective all grid infrastructures involve not only mere technical solutions but also dominantly all of the autopoietic social sub-systems ranging from religion to policy. ii) On the micro level the individual players and their psychology and risk behaviour are of major importance for acting within the macro autopoietic framework. Consequently, the challenges of grid implementation are similar to those of other pressing global issues as e.g. climate protection. This is well described by extending the Human Ecology triangle to a rectangle: invironment-individual-society-environment. By applying this extension of this classical field of interdisciplinary basic and applied research to the grid sector, i.e. by further extension to an e-Human Grid Ecology rational, the Grid Inverse Tragedy of the Commons can be understood and approached regarding the internalization challenge into e-Society and e-Life, from which then guidelines for the day-to-day management can be derived. This is of general importance for many complex fields and thus with similar paradoxes and challenges.

With ever-new technologies emerging also the amount of information to be stored and processed is growing exponentially and is believed to be always at the limit. In contrast, however, huge resources are available in the IT sector alike e.g. the renewable energy sector, which are often even not at all used. This under-usage bares any rational especially in the IT sector where e.g. virtualisation and grid approaches could be fast implemented due to the great technical and fast turnover opportunities. Here, we describe this obvious paradox for the first time as the Inverse Tragedy of the Commons, in contrast to the Classical Tragedy of the Commons where resources are overexploited. From this perspective the grid IT sector attempting to share resources for better efficiency, reveals two challenges leading to the heart of the paradox: i) From a macro perspective all grid infrastructures involve not only mere technical solutions but also dominantly all of the autopoietic social sub-systems ranging from religion to policy. ii) On the micro level the individual players and their psychology and risk behaviour are of major importance for acting within the macro autopoietic framework. Thus, the challenges of grid implementation are similar to those of e.g. climate protection. This is well described by the classic Human Ecology triangle and our extension to a rectangle: environment-individual-society-environment. Extension of this classical interdisciplinary field of basic and applied research to an e-Human Grid Ecology rational, allows the Inverse Tragedy of the Commons of the grid sector to be understood and approached better and implies obvious guidelines in the day-to-day management for grid and other (networked) resources, which is of importance for many fields with similar paradoxes as in (e-)society.

With ever-new technologies emerging also the amount of information to be stored and processed is growing exponentially and is believed to be always at the limit. In contrast, however, huge resources are available in the IT sector alike e.g. the renewable energy sector, which are often even not at all used. This under-usage bares any rational especially in the IT sector where e.g. virtualisation and grid approaches could be fast implemented due to the great technical and fast turnover opportunities. Here, we describe this obvious paradox for the first time as the Inverse Tragedy of the Commons, in contrast to the Classical Tragedy of the Commons where resources are overexploited. From this perspective the grid IT sector attempting to share resources for better efficiency, reveals two challenges leading to the heart of the paradox: i) From a macro perspective all grid infrastructures involve not only mere technical solutions but also dominantly all of the autopoietic social sub-systems ranging from religion to policy. ii) On the micro level the individual players and their psychology and risk behaviour are of major importance for acting within the macro autopoietic framework. Thus, the challenges of grid implementation are similar to those of e.g. climate protection. This is well described by the classic Human Ecology triangle and our extension to a rectangle: environment-individual-society-environment. Extension of this classical interdisciplinary field of basic and applied research to an e-Human Grid Ecology rational, allows the Inverse Tragedy of the Commons of the grid sector to be understood and approached better and implies obvious guidelines in the day-to-day management for grid and other (networked) resources, which is of importance for many fields with similar paradoxes as in (e-)society.

With ever-new technologies emerging also the amount of information to be stored and processed is growing exponentially and is believed to be always at the limit. In contrast, however, huge resources are available in the IT sector alike e.g. the renewable energy sector, which are often even not at all used. This under-usage bares any rational especially in the IT sector where e.g. virtualisation and grid approaches could be fast implemented due to the great technical and fast turnover opportunities. Here, we describe this obvious paradox for the first time as the Inverse Tragedy of the Commons, in contrast to the Classical Tragedy of the Commons where resources are overexploited. From this perspective the grid IT sector attempting to share resources for better efficiency, reveals two challenges leading to the heart of the paradox: i) From a macro perspective all grid infrastructures involve not only mere technical solutions but also dominantly all of the autopoietic social sub-systems ranging from religion to policy. ii) On the micro level the individual players and their psychology and risk behaviour are of major importance for acting within the macro autopoietic framework. Thus, the challenges of grid implementation are similar to those of e.g. climate protection. This is well described by the classic Human Ecology triangle and our extension to a rectangle: environment-individual-society-environment. Extension of this classical interdisciplinary field of basic and applied research to an e-Human Grid Ecology rational, allows the Inverse Tragedy of the Commons of the grid sector to be understood and approached better and implies obvious guidelines in the day-to-day management for grid and other (networked) resources, which is of importance for many fields with similar paradoxes as in (e-)society.