Intra- and inter-generic transfer of pathogenicity island-encoded virulence genes by cos phages

Bacteriophage-mediated horizontal gene transfer is one of the primary driving forces of bacterial evolution. The pac-type phages are generally thought to facilitate most of the phage-mediated gene transfer between closely related bacteria, including that of mobile genetic elements-encoded virulence genes. In this study, we report that staphylococcal cos-type phages transferred the Staphylococcus aureus pathogenicity island SaPIbov5 to non-aureus staphylococcal species and also to different genera. Our results describe the first intra- and intergeneric transfer of a pathogenicity island by a cos phage, and highlight a gene transfer mechanism that may have important implications for pathogen evolution.Classically, transducing phages use the pac site-headful system for DNA packaging. Packaging is initiated on concatemeric post-replicative DNA by terminase cleavage at the sequence-specific pac site, a genome slightly longer than unit length is packaged, and packaging is completed by non-sequence-specific cleavage (reviewed in Rao and Feiss, 2008). Generalized transduction results from the initiation of packaging at pac site homologs in host chromosomal or plasmid DNA, and typically represents ∼1% of the total number of phage particles. In the alternative cos site mechanism packaging is also initiated on concatemeric post-replicative DNA by terminase cleavage at a sequence-specific (cos) site. Here, however, packaging is completed by terminase cleavage at the next cos site, generating a precise monomer with the cohesive termini used for subsequent circularization (Rao and Feiss, 2008). Although cos site homologs may exist in host DNA, it is exceedingly rare that two such sites would be appropriately spaced. Consequently, cos phages, of which lambda is the prototype, do not engage in generalized transduction. For this reason, cos-site phages have been preferred for possible phage therapy, since they would not introduce adventitious host DNA into target organisms.The Staphylococcus aureus pathogenicity islands (SaPIs) are the best-characterized members of the phage-inducible chromosomal island family of mobile genetic elements (MGEs; Novick et al., 2010). SaPIs are ∼15 kb mobile elements that encode virulence factors and are parasitic on specific temperate (helper) phages. Helper phage proteins are required to lift their repression (Tormo-Más et al., 2010, 2013), thereby initiating their excision, circularization and replication. Phage-induced lysis releases vast numbers of infectious SaPI particles, resulting in high frequencies of transfer. Most SaPI helper phages identified to date are pac phages, and many well-studied SaPIs are packaged by the headful mechanism (Ruzin et al., 2001; Ubeda et al., 2007). Recently, we have reported that some SaPIs, of which the prototype is SaPIbov5 (Viana et al., 2010), carry phage cos sequences in their genomes, and can be efficiently packaged and transferred by cos phages to S. aureus strains at high frequencies (Quiles-Puchalt et al., 2014). Here we show that this transfer extends to non-aureus staphylococci and to Listeria monocytogenes.Since the pac phages transfer SaPIs to non-aureus staphylococci and to the Gram-positive pathogen Listeria monocytogenes (Maiques et al., 2007; Chen and Novick, 2009), we reasoned that cos phages might also be capable of intra- and intergeneric transfer. We tested this with SaPIbov5, into which we had previously inserted a tetracycline resistance (tetM) marker to enable selection, and with lysogens of two helper cos phages, φ12 and φSLT, carrying SaPIbov5 (strains JP11010 and JP11194, respectively; Supplementary Table 1). The prophages in these strains were induced with mitomycin C, and the resulting lysates were adjusted to 1 μg ml⁻¹ DNase I and RNase A, filter sterilized (0.2 μm pore), and tested for SaPI transfer with tetracycline selection, as previously described (Ubeda et al., 2008). To test for trans-specific or trans-generic transduction, coagulase-negative staphylococci species and L. monocytogenes strains were used as recipients for SaPIbov5 transfer, respectively, as previously described (Maiques et al., 2007; Chen and Novick, 2009). As shown in Figure 1 and Supplementary Table 2). In contrast, deletion of the SaPIbov5 cos site (strains JP11229 and JP11230) did not affect SaPI replication (Supplementary Figure 1), but completely eliminated SaPIbov5 transfer (Supplementary Table 2). The TerS protein is essential for φ12 and SaPIbov5 DNA packaging, but not for phage-mediated lysis (Quiles-Puchalt et al., 2014). As expected, this mutation abolished SaPIbov5 transfer (Open in a separate windowFigure 1(a) Map of SaPIbov5. Arrows represent the localization and orientation of ORFs greater than 50 amino acids in length. Rectangles represent the position of the ori (in purple) or cos (in red) sites. Positions of different primers described in the text are shown. (b) Amplimers generated for detection of SaPIbov5 in the different recipient strains. Supplementary Table 2 lists the sequence of the different primers used. The element was detected in S. epidermidis JP829 (Se-1), S. epidermidis JP830 (Se-2), L. monocytogenes SK1351 (Lm-1), L. monocytogenes EGDe (Lm-2), S. xylosus C2a (Sx) and S. aureus JP4226 (Sa).

Table 1

Intra- and intergeneric SaPIbov5 transfer^a

Huntington's disease: Dancing in a dish

In a recent landmark paper, the Huntington''s disease (HD) iPSC Consortium reports on the establishment and characterization of a panel of iPSC lines from HD patients, and more importantly, the successful modeling of HD in vitro. In the same issue of Cell Stem Cell, An et al. reports on the successful targeted gene correction of HD in human iPSCs. Both advances are exciting, provide new resources for current and future HD research, and uncover new challenges to better understand and, most importantly, treat this devastating disease in the near future.Modeling human diseases using induced pluripotent stem cells (iPSCs) has created novel opportunities for both mechanistic studies as well as for the discovery of new disease therapies. Combined with advanced gene correction technology, human iPSCs hold great promise to provide patient-specific and mutation-free cells for potential cell replacement therapy. Huntington''s disease (HD) is an autosomal dominant neurodegenerative disorder, which causes motor dysfunction, psychiatric disturbances and cognitive impairment¹. HD is caused by an expanded cystosine adenine guanine (CAG) tri-nucleotide repeat encoding polyglutamine in the first exon of the Huntingtin (HTT) gene. To date, there is no effective therapy for preventing the onset or slowdown of this disorder. Preliminary clinical trials using fetal neural grafts had shown long-lasting functional benefits in patients². Though only effective in limited cases, these results suggest that cell-based therapy could be a potential treatment if a reliable and consistent cell source is available. For this purpose, an alternative cell source to overcome the logistical and biological hurdles of this disease had been actively explored in the past decade. With recent advancement in human iPSCs technology, HD patient-specific iPSCs coupled with an efficient directed cell differentiation protocol offers hope for an unlimited supply of autologous cells. Since HD is a monogenic disease, with a very well-established correlation between the number of CAG repeats and the age of disease onset, it provides an ideal target for iPSC-based gene correction that will allow for the production of disease-free cells for potential autologous cell therapy, and at the same time provide a much needed, valuable platform to further study the pathogenesis of the disease^3,4.This is in fact what has been recently accomplished in two reports published in Cell Stem Cell^5,6. The HD iPSC Consortium reports on the generation of HD patient-specific iPSC lines that showed CAG-repeat-expansion-associated phenotypes⁵. The study from An et al.⁶ reports on the successful targeted correction of expanded CAG repeat in HD patient iPSCs and the reversion of disease phenotypes.In the study reported from the HD iPSC Consortium, the authors generated 14 iPSC lines from HD patients and controls (listed in Open in a separate window

A new HCV mouse model on the block

The investigation of virus-induced liver disease and hepatocellular carcinoma needs small animal models modeling hepatitis C virus (HCV) infection and liver disease biology. A recent study published in Cell Research reports a novel mouse model which is permissive for chronic HCV infection and shows chronic liver injury including inflammation, steatosis and fibrosis.Chronic hepatitis C virus (HCV) infection is a major cause of liver disease worldwide. The development of direct-acting antivirals has revolutionized treatment by offering cure¹. However, several hurdles remain. High costs limit treatment access in the majority of patients. Infection is often diagnosed at a late stage when advanced liver disease and cancer are established. Cure in advanced liver disease does not eliminate the risk of hepatocellular carcinoma (HCC). Re-infection remains possible and a vaccine is not available².To better understand the pathogenesis of virus-induced liver disease and HCC, a small animal model permissive for HCV infection and modeling liver disease biology is needed³. HCV infection is limited to humans and chimpanzees, predominantly due to distinct host-dependency factors and innate antiviral immune responses precluding cross-infection of other species⁴. Research efforts have focused on humanizing mice permissive to HCV infection. This has led to the development of conceptually three different types of mouse models.The human liver chimeric mouse is based on immune- and hepato-deficient mice repopulated with human hepatocytes. While the uPA-SCID⁵ and FRG⁶ models are extremely useful to study the viral life cycle and antivirals, the lack of an adaptive immune system and liver disease precludes the use for the study of liver disease biology and vaccine evaluation (7 based on modified Rag-2^−/− mice, activation of the overexpressed FK506-binding protein and caspase-8 fusion protein in the liver induces death of mouse hepatocytes and facilitates engraftment of human hepatocyte progenitor and CD34⁺ haematopoetic stem cells. While infected mice exhibit liver inflammation and fibrosis, this model appears to be limited with detection of virus only in the liver (8. Sustained and robust HCV infection for 90 days was achieved by crossing the 4hEF mice with mice knocked out for STAT1⁹. Furthermore, HCV infection in these mice elicited antiviral cellular and humoral immune responses. Although the animals were not reported to develop liver disease, this robust model represents a major breakthrough since it allows for studying HCV-induced immune responses and the preclinical evaluation of vaccine candidates in a small animal model (

Imprinted gene expression in hybrids: perturbed mechanisms and evolutionary implications

Diverse mechanisms contribute to the evolution of reproductive barriers, a process that is critical in speciation. Amongst these are alterations in gene products and in gene dosage that affect development and reproductive success in hybrid offspring. Because of its strict parent-of-origin dependence, genomic imprinting is thought to contribute to the aberrant phenotypes observed in interspecies hybrids in mammals and flowering plants, when the abnormalities depend on the directionality of the cross. In different groups of mammals, hybrid incompatibility has indeed been linked to loss of imprinting. Aberrant expression levels have been reported as well, including imprinted genes involved in development and growth. Recent studies in humans emphasize that genetic diversity within a species can readily perturb imprinted gene expression and phenotype as well. Despite novel insights into the underlying mechanisms, the full extent of imprinted gene perturbation still remains to be determined in the different hybrid systems. Here we review imprinted gene expression in intra- and interspecies hybrids and examine the evolutionary scenarios under which imprinting could contribute to hybrid incompatibilities. We discuss effects on development and reproduction and possible evolutionary implications.In many plants and animals, interspecific hybridization events yield offspring that are phenotypically different from either of the parent species. Such hybrids typically display developmental abnormalities and, in animals, often have reduced fertility or complete sterility, particularly in males. Hybrid incompatibilities arise because, although the parental species may be genetically similar, the genomes are still too divergent to sustain normal development, physiology and reproduction when mixed in the hybrid offspring (Wu and Ting, 2004). Extensive research has been performed on genetic incompatibilities in plant and animal hybrids (Ishikawa and Kinoshita, 2009; Johnson, 2010). Key loci have been mapped and characterized in experimental model species, providing important insights into the aberrant phenotypes such as male hybrid sterility (Maheshwari and Barbash, 2011).Phenotypic abnormalities in interspecies hybrids often differ greatly between the reciprocal crosses. The classic example of such an asymmetry is seen in reciprocal crosses between donkeys and horses, where both directions of the cross produce sterile offspring, but the gross phenotype of the progeny (that is, ‘mule'' versus ‘hinny'') depends on the direction of the cross. Horses and donkeys have a different chromosome number, but this cannot explain the differential hybrid phenotypes that depend on the direction of the cross (as the reciprocal crosses have the same autosomal karyotype). More than 50 years ago serum concentrations of a placental hormone were reported to be markedly higher in mule than in hinny conceptuses, suggestive of parental genome-specific gene expression (Allen, 1969).The North-American genus Peromyscus (‘deer mice'') has been studied extensively to explore hybrid incompatibilities in mammals (see Vrana et al., 1998). Also in interspecies hybrids in Mus (house mouse), between the sympatric species M. musculus and M. spretus, morphological differences are apparent between reciprocal hybrids (Zechner et al., 2004). These hybrid effects were observed in crosses between a mixed M. musculus domesticus strain and lab stocks of M. spretus. To be definitive about where the incompatibilities lie between M. musculus and M. spretus (or M. m. castaneus, see below), reciprocal crosses between several different wild-derived stocks (or wild caught animals) of M. musculus and M. spretus populations would be needed.

Table 1

Terminology and abbreviations

The roads and bridges of science. Research infrastructures are key components of Europe's future research, but their funding is not guaranteed

Research infrastructures are a crucial component of modern biological research, but the EU has not yet figured out how to fund and maintain them.The development of recombinant gene technology in the 1970s heralded a new era of application-oriented research for molecular biology, with a huge economic impact. During the decades that have followed, biological research and development have become a major enterprise, with an increasing demand for sophisticated technologies, databases, tissue banks and other tools that range from microscopes and DNA sequencers to bioinformatics services and mutant collections. Biology has followed in the footsteps of physics and astronomy, which share costly instrumentation such as particle accelerators, observatories and satellites. A key difference is that biological research infrastructures are often distributed across several sites and are less costly to establish. Nevertheless, they are expensive to operate and maintain, and need long-term financial support.There is no doubt among scientists that research infrastructures are essential for biomedicine and the life sciencesThe European Union (EU) regards biomedical research as an important component of its future economic and social development as part of its ''Innovation Union'' strategy (EC, 2010), but the necessary creation and operation of research infrastructures is not keeping pace. European biologists have been highlighting the problem for years (van Dyck, 2005), to the effect that some pan-European infrastructures for biomedical research and the life sciences have been created, such as the European Bioinformatics Institute (EBI; Hinxton, UK). The European Commission (EC) also established the European Strategy Forum on Research Infrastructures (ESFRI) in 2002, to define the infrastructures required for international research (ESFRI, 2006, 2011). However, most of the planned projects for the biomedical and life sciences (ESFRI, 2011)

Linking metabolite production to taxonomic identity in environmental samples by (MA)LDI-FISH

One of the greatest challenges in microbial ecology remains to link the metabolic activity of individual cells to their taxonomic identity and localization within environmental samples. Here we combined mass-spectrometric imaging (MSI) through (matrix-assisted) laser desorption ionization time-of-flight MSI ([MA]LDI-TOF/MSI) with fluorescence in situ hybridization (FISH) to monitor antibiotic production in the defensive symbiosis between beewolf wasps and ‘Streptomyces philanthi'' bacteria. Our results reveal similar distributions of the different symbiont-produced antibiotics across the surface of beewolf cocoons, which colocalize with the producing cell populations. Whereas FISH achieves single-cell resolution, MSI is currently limited to a step size of 20–50 μm in the combined approach because of the destructive effects of high laser intensities that are associated with tighter laser beam focus at higher lateral resolution. However, on the basis of the applicability of (MA)LDI-MSI to a broad range of small molecules, its combination with FISH provides a powerful tool for studying microbial interactions in situ, and further modifications of this technique could allow for linking metabolic profiling to gene expression.Ecological analyses of microbial metabolites have thus far been hampered by the difficulty of localizing and quantifying these compounds in situ and tying their production to subpopulations or even single cells of individual microbial taxa. However, recent advances in mass-spectrometric imaging (MSI) techniques provide excellent tools to monitor metabolic processes and chemical communication in an ecological context (Svatoš, 2010, 2011). For example, matrix-assisted laser desorption/ionization mass spectrometric imaging (MALDI-MSI) has successfully been employed to observe antagonistic interactions between Streptomyces and Bacillus strains in vitro (Yang et al., 2009). The analysis of microbial interactions in situ, however, requires the combination of metabolic profiling with taxonomic identification and localization of the involved microorganisms. Previous studies employing microautoradiography or high-resolution secondary ion mass spectrometry in combination with in situ hybridization have provided insights into the metabolism of individually identified bacterial cells in environmental samples (Orphan et al., 2001; Kindaichi et al., 2004; Behrens et al., 2008; Musat et al., 2008). However, the need for isotopic labeling limits the application of these techniques to a subset of biological questions.Here we combine MSI with fluorescence in situ hybridization (FISH) for simultaneous metabolite profiling and taxonomic identification of bacteria, using the defensive symbiosis between beewolf wasps and Streptomyces bacteria as a model. Beewolves of the genera Philanthus, Trachypus and Philanthinus (Hymenoptera, Crabronidae) cultivate ‘Streptomyces philanthi'' in specialized antennal gland reservoirs (Kaltenpoth et al., 2006; Goettler et al., 2007; Kaltenpoth et al., 2014) and secrete the bacteria into their subterranean brood cells before oviposition (Kaltenpoth et al., 2010a). Later, the larva incorporates the symbionts into the cocoon silk, where the streptomycetes produce a cocktail of at least nine different antibiotics (Kroiss et al., 2010) and thereby protect the larva against pathogenic fungi and bacteria during the long (up to 9 months) and vulnerable phase of hibernation (Kaltenpoth et al., 2005; Koehler et al., 2013). Previous studies using MSI revealed that the antibiotics abound on the outer surface of the cocoon, while they are virtually absent from the inner surface (Kroiss et al., 2010).We used (matrix-assisted) laser desorption ionization time-of-flight MSI ([MA]LDI-TOF/MSI) to visualize the abundance of two different antibiotics (piericidin A1 and B1) and subsequently localized the symbionts producing these compounds on beewolf cocoons using FISH. Pieces of beewolf cocoons were fixed to MALDI target plates without any pre-treatment using double-sided adhesive tape, with the outer cocoon surface facing upward. In order to allow for later alignment of ion-intensity maps and FISH images, the cocoon pieces were surrounded by thin paint markings (Edding751, 1–2 mm tip width, white), applied with the tip of a needle. This marker was chosen because it yielded characteristic signals in (MA)LDI-MS (measured at m/z 322.5±0.5) and also showed fluorescence at 640 nm, the excitation wavelength of the fluorescent dye used for FISH (that is, Cy5). MSI was carried out without any pretreatment of the samples (Hoelscher et al., 2009; Kroiss et al., 2010), or after application of 2,5-dihydroxybenzoic acid matrix by sublimation (Svatoš and Mock, 2013). A MALDI micro MX mass spectrometer (Waters, Milford, MA, USA) equipped with a nitrogen laser (337 nm) was used in the reflectron mode and positive polarity for data acquisition as previously reported (Kroiss et al., 2010). The step size in both x and y directions was set to 50 μm corresponding to 508 dots per inch resolution. Two-dimensional ion-intensity maps were reconstructed using the spectral data for the respective potassium adduct ions of piericidin A1 (PA1, m/z 454±0.5 [M+K]⁺) and piericidin B1 (PB1, m/z 468±0.5 [M+K]⁺) with the BioMAP software (Novartis Institutes for BioMedical Research, Basel, Switzerland). After (MA)LDI imaging, samples were subjected to FISH with the ‘S. philanthi''-specific probe SPT177-Cy5 (Kaltenpoth et al., 2005, 2006) as described previously (Kaltenpoth et al., 2010b). Fluorescence images were recorded on a Zeiss AxioImager Z.1 (Zeiss, Jena, Germany) using both the mosaic and z-stack options for obtaining high-resolution images with increased focusing depth. Overlays of (MA)LDI and FISH images were achieved in Adobe Photoshop CS5 Extended 12.0 by using the pen markings as a guide.(MA)LDI-MSI revealed a patchy distribution of antibiotics across the outer cocoon surface of European beewolves. The two measured antibiotic substances showed very similar distributions (Figures 1a–j), suggesting that both compounds—as well as possibly the other seven antibiotics produced by the symbionts on the beewolf cocoon that could not be measured here because of their low concentrations—are produced by individual bacterial cells or subpopulations of cells. This is supported by MSI with a high-resolution atmospheric pressure scanning microprobe (AP-SMALDI-MSI) of PA1 and PB1 produced by ‘S. philanthi'' on beewolf cocoons and in vitro, which confirmed the colocalization of both antibiotics (Figures 1k–n and Supplementary Figure S1, for experimental procedures see Supplementary Online Material). Thus, different symbiont subpopulations apparently do not specialize in the production of individual compounds, but instead produce a mixture of antibiotics.Open in a separate windowFigure 1(MA)LDI-FISH of antibiotics produced by symbiotic ‘Streptomyces philanthi'' bacteria on a beewolf cocoon (Philanthus triangulum) and in vitro. Ion-intensity maps of (a) the paint marker for alignment of LDI and FISH pictures (m/z 322.5); inset: image of the cocoon piece surrounded by white paint markings on the LDI target plate, (b) piericidin A1 (PA1, m/z 454.5 [M+K]⁺) and (c) piericidin B1 (PB1, m/z 468.5 [M+K]⁺). (d–f) The same maps, overlayed with a FISH micrograph of the cocoon piece. Symbiont cells were labeled with the fluorescent oligonucleotide probe SPT177-Cy5. (g–h) Magnifications of (e, f), respectively, with individual bacterial cells visible. (i–j) MALDI-FISH of (i) PA1 (m/z 454.5 [M+K]⁺) and (j) PB1 (m/z 468.5 [M+K]⁺) on another cocoon piece. (k–n) AP-SMALDI imaging of antibiotics produced by ‘S. philanthi'' in vitro. (k) Light microscopic image of an ‘S. philanthi'' colony, (l) PA1 (m/z 416.27 [M+H]⁺), (m) PB1 (m/z 430.25 [M+H]⁺), (n) overlay of PA1 (green) and PB1 (red).On the cocoon, FISH allowed for the visualization of individual symbiont cells, which were abundant across the entire cocoon surface and often occurred in highest densities along the outer cocoon threads (Supplementary Figure S2). The presence of the matrix had no influence on the efficiency of FISH after MSI (data not shown). The alignment of ion-intensity maps with FISH images revealed high concentrations of antibiotics around some subpopulations of cells, whereas other cell aggregations were surrounded by much lower amounts of antibiotics (Figures 1g–j), highlighting the possibility for cheating in the symbiosis. However, the current limitations in sensitivity and lateral resolution of MALDI-MSI do not permit the visualization of compounds on the single-cell level (~1 μm) and thereby may obscure fine-scale patterns of antibiotic production. This is supported by AP-SMALDI-MSI of beewolf cocoons at two different resolutions (step sizes 20 and 5 μm, Supplementary Figure S1): Whereas the high-resolution measurement revealed high concentrations of antibiotics along the outer cocoon threads, which agrees with the FISH experiments showing a similar pattern of symbiont cell densities (Supplementary Figure S2), this pattern was not as apparent at lower resolutions (Supplementary Figure S1). However, the high laser intensities required for a step size of 5 μm were destructive for the samples; therefore, subsequent FISH experiments could not be performed.The combination of (MA)LDI imaging and FISH provides a powerful tool for tying metabolite profiling to taxonomic identification in environmental samples. However, the laser intensity needs to be carefully adjusted for the desired application to achieve maximum sensitivity and resolution while at the same time conserving the structure of the sample. This problem can be circumvented by using desorption electrospray ionization (DESI) imaging, which we also successfully combined with FISH in preliminary experiments (data not shown). Still, the limitations of both (MA)LDI and DESI in lateral resolution and sensitivity currently prohibit single-cell resolution of metabolic profiling and restrict the technique to mapping the distribution of metabolites on the subpopulation level (Svatoš, 2011). Thus, the exploration of complex environmental samples is at present limited to microbial communities with distinct spatial structure. However, the major strength of MSI-FISH is its broad applicability to a wide range of small molecules as well as proteins (Svatoš, 2010). Therefore, (MA)LDI-FISH and DESI-FISH allow for addressing a multitude of questions in microbial ecology, ranging from interactions in mixed-species biofilms or cross-feeding associations to the chemical basis and dynamics of mutualistic and antagonistic encounters. As several signal enhancement techniques for FISH have been developed (for example, Schönhuber et al., 1997; Zwirglmaier, 2005), modifications of the approach described here could also be employed to tie metabolic profiling by (MA)LDI or DESI imaging to the presence (Moraru et al., 2010) or expression (Pernthaler and Amann, 2004) of particular genes of interest in microbial communities or eukaryotic tissues. Future studies should explore the possibility for using oligonucleotide labels and hybridization protocols that allow for simultaneous MSI of labeled cells and metabolites of interest, which would obviate the necessity for subsequent FISH and thereby circumvent the problems with high laser intensities. Alternatively, new MALDI matrices capable of dissipating the high ultraviolet-laser intensities and thus preventing DNA damage could be developed.

Table 1

Comparison of established methods for linking localization and taxonomic identification of microbes to the production of particular metabolites of interest in environmental samples

EU-LIFE revives funding debate: A group of mid-level life science research institutes is reopening the debate on how to fund research at the EU level calling for a stronger emphasis on excellence

EU-LIFE, which represents 10 European life science research institutes, has reopened the debate about how to fund research at the European level by calling for the budget of the European Research Council to be drastically increased.For more than a decade, European scientists have lobbied policy makers in Brussels to increase European Union (EU) funding for research and to spend the money they do provide more efficiently. This debate eventually led to the establishment of the European Research Council (ERC) in 2007, which provides significant grants and does so on the sole criterion of scientific excellence—something for which the scientific community pushed. As such, there seemed to be consensus about how to judge and fund science at the European level, including in the debate about the EU''s Horizon 2020 funding scheme—the EU''s framework for research and innovation—which will spend €80 billion over the next seven years (2014–2020). The conclusion seemed to be that the ERC should continue to support basic research on the basis of excellence, whereas other parts of the programme would focus on large cooperative projects, improving the competitiveness of Europe and meeting societal challenges such as climate change and public health.But a new body called EU-LIFE—set up in May 2013—has reopened the debate about how to fund science and is campaigning for a greater focus on rewarding excellence, even at the expense of funding projects on the grounds of fairness or to correct imbalances between EU member states. EU-LIFE was founded by 10 institutions including the Centre for Genomic Regulation (CRG; Barcelona, Spain), the Institut Curie (Paris, France) and the Max Delbrück Centre (Berlin, Germany), partly to provide a collective voice for mid-sized research institutes in the life sciences that might lack influence on their own (InstituteAdvanced grantStarting grantProof-of-concept grantTotal ERC grantsTotal ERC funding (million €)Centre for Genomic Regulation (Spain)3911319.0Free University of Brussels (VIB; Belgium)51412033.3Institut Curie (France)711–1834.5Max Delbrück Centre for Molecular Medicine (Germany)44–815Instituto Gulbenkian de Ciência (Portugal)14–57.8Research Centre for Molecular Medicine of the Austrian Academy of Sciences (Austria)12145.1European Institute of Oncology (Italy)31158.7Central European Institute of Technology (Czech Republic)–––––The Netherlands Cancer Institute (Netherlands)64–1019.5Institute for Molecular Medicine Finland (Finland)–––––Open in a separate windowERC, European Research Council.But while claiming to speak for the cause of European research as a whole, EU-LIFE also has a specific remit to speak up for its own members, mostly mid-sized institutions that consider themselves poorly represented in the corridors of EU decision-making. “There are several reasons why we decided to start this initiative,” said Luis Serrano, Director of the Centre for Genomic Biology in Barcelona, Spain, one of the EU-LIFE founders. “First we see that institutes of research do not have a voice in Brussels as a group, unlike universities or international organizations like EMBL. While in many cases our goals will be similar, this is not always the case. Second, we think that there are excellent research institutes in Europe, at the same level as many top places in the USA, that do not have enough visibility due to their size. By coming together and offering similar standards of quality, we want to achieve critical mass and become attractive to PhD and post-doctoral fellows from all over the world who currently mainly go to the USA. Third we think that all EU-LIFE members have specific strengths and know-how on different aspects of the life sciences. By sharing our experiences we think we could improve the quality and competitiveness of all of us.”While few scientists or policy makers would argue with EU-LIFE''s aim to stimulate international collaboration and attract the best young researchers to Europe, not everyone agrees with the organization''s call to do so by distributing more funds via the ERC. Although the ERC is widely regarded as successful in encouraging excellence and ‘curiosity-driven'' research—as opposed to distributing funds purely equitably between member countries—Mark Palmer, director of international strategy at the UK Medical Research Council (MRC), which spent £759.4 million (about €900 million) on research in the financial year 2011/2012, questions whether the ERC should receive even more funding than it does at present: “We support excellence, but if you put all the resources into one sort of mechanism, you lack the visibility for reaching across countries to join together to do research,” he said. “So there is an advantage in having a mixed pot of funding. If you put too much money in the ERC it becomes so distorted that you haven''t got European added value. You might as well have left the money back home and done it through the normal mechanisms.”“If you put too much money in the ERC it becomes so distorted that you haven''t got European added value”The ERC itself felt it was inappropriate to comment on its own budget, but Ernst-Ludwig Winnacker, who served as its secretary general from 2007 to 2009, pointed out that while he agrees in principle with the Commission''s proposal to double the ERC''s budget under Horizon 2020, this will not guarantee that the number of suitable high-quality applicants for funding would double as well. “Let us not forget that we are talking about scientific excellence only,” Winnacker, now General Secretary of the Human Frontier Science Program, said. “I have often asked myself how much excellence of the level expected to get supported by the ERC do we have in Europe. Would we really be able to spend twice the amount of money at the same quality level as now? I doubt it.”Winnacker indicated therefore that the ERC budget should increase at a sustainable level that ensures that the quality of projects funded is maintained. He also highlighted another risk in focusing a growing proportion of funds through the ERC, which is that it might make other agencies envious.“I have often asked myself how much excellence of the level expected to get supported by the ERC do we have in Europe”Palmer, for the MRC, said that he agrees with the current level of proposed funding increase for the ERC, but argued that it is important to preserve other sources of funding that support large-scale programmes involving multiple institutions, especially in the life sciences. In particular, major clinical screening programmes call for huge samples of patients, in some cases from diverse populations, which requires international collaboration, irrespective of the individual excellence of the departments involved. “For example the EPIC [European Prospective Investigation into Cancer and Nutrition] cohort has been going 20 years with over 500,000 people across 10 different countries,” Palmer said. “That diversity is something that you have to do at the European level.” EPIC is the world''s largest study on the relationship between diet and lifestyle factors and chronic diseases: A total of 521,457 healthy adults, mostly aged 35–70, were enrolled in 23 centres in 10 countries between 1993 and 1999, and the study showed with high statistical confidence that a modest change in lifestyle can yield a massive gain in life expectancy [1].There may be broad agreement that large projects in biomedical research require a European-wide approach. The argument, though, boils down to whether or not funds designated for research should be used as a way of building infrastructure or collaborative frameworks alongside excellence, rather than being subordinated to it. This is the belief—and to some extent the remit—of the European Science Foundation (ESF; Strasbourg, France), which has promoted networking and the dissemination of information among research teams whose work is already being funded by other agencies. Now this role has been passed to Science Europe, headquartered in Brussels, while the ESF is focusing on its public communication activities.EU-LIFE will seek to collaborate with both the ESF and Science Europe, according to Michela Bertero, Head of International and Scientific Affairs at CRG. “We are in contact with both initiatives. They operate at a higher science policy level and on a larger scale, and we want to engage with them as research stakeholders,” Bertero said.Yet while the organization agrees with the ESF that science should tackle societal challenges, EU-LIFE disputes that this is best done by grants awarded solely on the basis of large collaborative projects. “Excellence should always be at the forefront for awarding grants,” explained Serrano. “This does not mean that societal and industrial challenges should not be tackled. But if there is no expertise in an area, then instead of funding groups which are not competitive, money should be used to train and hire the right personnel.”By challenging Horizon 2020 to distribute more money on the basis of excellence rather than goals, EU-LIFE seems to have reopened the debate on how research funds should be spent and to what purpose. Others, however, are calling for some research money to be put towards infrastructure in regions with the potential for high-quality science, but which lack resources and laboratories. This has actually been acknowledged and catered for in Horizon 2020, according to Joanna Newman, Director of the UK Higher Education International Unit, a registered charity funded by various public bodies, which coordinates engagement between UK universities and international partners. “Excellence should be the main criterion for awarding research funding,” Newman said. “As this is public money, it would be unfair to the public to fund less excellent projects. However, there is also a responsibility to help other Member States to build research capacity. Horizon 2020 will include a cross-cutting ‘Spreading Excellence and Widening Participation'' programme line to address this, by funding the partnering of institutions and/or researchers with different grades of current research capacity.”One European player even argues that the EU should extend this policy to assist building infrastructure in developing countries. “Developed countries have a responsibility in helping capacity building in the field of research,” said Antoine Grassin, Directeur Général of Campus France, the country''s agency for promoting higher education and international mobility. “From that point of view, it may be very helpful for researchers from developing countries to be able to join the international scientific community, which may require financial help, such as grants.”“…if there is no expertise in an area, then instead of funding groups which are not competitive, money should be used to train and hire the right personnel”In the case of Europe, Newman pointed out that links between the Horizon Framework programme and the Structural Funds to improve infrastructure and research capabilities within regions will be stronger under the 2020 regime from 2014 to 2020 compared with the current Framework Programme 7. But this alignment between the allocation of funds designated for structural purposes and those granted for research purposes is precisely one of EU-LIFE''s main complaints about the Horizon 2020 programme—the resulting allocations are not always based on excellence.Furthermore, Winnacker argued that excellence does not mix well with other societal factors within a single programme, never mind an individual project. “If other parameters are included, politics would immediately interfere,” he said. “The ERC only survives because it has impeccable scientific standards, which politicians do not dare to touch without being ridiculed. There are enough programs in Horizon 2020, and elsewhere, like the structural funds, which can take care of regional and societal issues. These are of course important, but let''s face it, the real ‘disruptive'' innovations which create jobs only come from fundamental research.”According to Lieve Ongena, Science Policy Manager at the Free University of Brussels (VUB; Belgium), one of the EU-LIFE founding members, it is for these sorts of reasons that EU-LIFE wants to divert more funds to the ERC. “It''s clear that the ERC is an absolutely necessary funding source,” she said. “The scientists can bring their own ‘pet'' project without addressing any top down action lines agreed upon by the member states. In addition, the money provides sufficient critical mass for a sufficiently long time line: five years. Above all, the evaluation excellence is the ‘sole'' selection criterion, and thus by definition grantees will help to increase Europe''s competitiveness.” Ongena emphasized that EU-LIFE would draw the attention of decision-makers to the ERC whenever possible. “Ultimately, they hope to convince ERC President Helga Nowotny to increase the budget, which is today only 17% of the speculated Horizon 2020 budget.”… there is a broad consensus that research priorities have changed and that Horizon 2020 necessarily includes a greater societal dimensionThe view that the ERC should become Europe''s dominant funding agency is still open to debate, however, even among institutions committed both to excellence and to supporting research at a European level. The European Molecular Biology Laboratory (EMBL) in Heidelberg obtains funding from 20 member states and its Director General Iain Mattaj argues for the continued existence of multiple funding sources. “While recognizing the very important role of the ERC in European research funding, I find it essential that research continues to be supported by a diversity of mechanisms, both national and European,” he said. “In the case of Horizon 2020, these include funding for Research Infrastructures, Marie Sklodowska Curie (MSC) Actions that fund the training of young research fellows and research in the area of Health. In particular, EMBL has advocated increased funding not only for the ERC but also for MSC Actions and for Research Infrastructures.” However, within these programmes, Mattaj emphasized that excellence should also be the main criterion for awarding grants in every case.Meanwhile EU-LIFE also has a grander vision beyond funding to make Europe more competitive and attractive for research, according to Geert Van Minnebruggen, Integration Manager at VUB. “To keep Europe a competitive and attractive place for top scientists, we should be prepared to offer them similar budget categories as the US and China,” Van Minnebruggen said. “EU-LIFE sees it as one of its major tasks, through dialogue with policy makers, to create awareness of this necessity.”Palmer points out that attracting scientists from outside the EU is not just about money, but also about culture. “With a lab, the culture is pretty well English language now, people publish in English and apply for grants in English. That can be an inhibitor, both for scientists and their partners, in the case of countries where English isn''t the first language,” he said. This issue has been taken on board by EU-LIFE, according to Serrano: “All EU institutes should try to become more international, use English as the main speaking language, ensure competitiveness and external evaluations, recognize merit and support it, favour mobility, and be open to new ideas and initiatives.”Despite disagreements over funding mechanisms and targets, there is a broad consensus that research priorities have changed and that Horizon 2020 necessarily includes a greater societal dimension. “We''re interested now in health and demographic changes and wellbeing challenges, which is very different from how they were funding science under previous frameworks,” Palmer said. “It is very much driven by the economic situation, about citizens as patients, health delivery and how to be sure patients get access to treatment.”Ongena has similar views: “As responsible life scientists, EU-LIFE community members should do everything possible to drive basic and translational research forward and to translate findings into benefits for society,” she said. But she reiterated EU-LIFE''s position that all this should be done on the criterion of excellence only. It seems that the debates from the past decade about how to properly support research are not yet over.     

Evidence for a New Avian Paramyxovirus Serotype 10 Detected in Rockhopper Penguins from the Falkland Islands

The biological, serological, and genomic characterization of a paramyxovirus recently isolated from rockhopper penguins (Eudyptes chrysocome) suggested that this virus represented a new avian paramyxovirus (APMV) group, APMV10. This penguin virus resembled other APMVs by electron microscopy; however, its viral hemagglutination (HA) activity was not inhibited by antisera against any of the nine defined APMV serotypes. In addition, antiserum generated against this penguin virus did not inhibit the HA of representative viruses of the other APMV serotypes. Sequence data produced using random priming methods revealed a genomic structure typical of APMV. Phylogenetic evaluation of coding regions revealed that amino acid sequences of all six proteins were most closely related to APMV2 and APMV8. The calculation of evolutionary distances among proteins and distances at the nucleotide level confirmed that APMV2, APMV8, and the penguin virus all were sufficiently divergent from each other to be considered different serotypes. We propose that this isolate, named APMV10/penguin/Falkland Islands/324/2007, be the prototype virus for APMV10. Because of the known problems associated with serology, such as antiserum cross-reactivity and one-way immunogenicity, in addition to the reliance on the immune response to a single protein, the hemagglutinin-neuraminidase, as the sole base for viral classification, we suggest the need for new classification guidelines that incorporate genome sequence comparisons.Viruses from the Paramyxoviridae family have caused disease in humans and animals for centuries. Over the last 40 years, many paramyxoviruses isolated from animals and people have been newly described (16, 17, 22, 29, 31, 32, 36, 42, 44, 46, 49, 58, 59, 62-64). Viruses from this family are pleomorphic, enveloped, single-stranded, nonsegmented, negative-sense RNA viruses that demonstrate serological cross-reactivity with other paramyxoviruses related to them (30, 46). The subfamily Paramyxovirinae is divided into five genera: Respirovirus, Morbillivirus, Rubulavirus, Henipavirus, and Avulavirus (30). The Avulavirus genus contains nine distinct avian paramyxovirus (APMV) serotypes (Table ​(Table1),1), and information on the discovery of each has been reported elsewhere (4, 6, 7, 9, 12, 34, 41, 50, 51, 60, 68).

TABLE 1.

Characteristics of prototype viruses APMV1 to APMV9 and the penguin virus