Identification of a Novel Human Papillomavirus by Metagenomic Analysis of Samples from Patients with Febrile Respiratory Illness

As part of a virus discovery investigation using a metagenomic approach, a highly divergent novel Human papillomavirus type was identified in pooled convenience nasal/oropharyngeal swab samples collected from patients with febrile respiratory illness. Phylogenetic analysis of the whole genome and the L1 gene reveals that the new HPV identified in this study clusters with previously described gamma papillomaviruses, sharing only 61.1% (whole genome) and 63.1% (L1) sequence identity with its closest relative in the Papillomavirus episteme (PAVE) database. This new virus was named HPV_SD2 pending official classification. The complete genome of HPV-SD2 is 7,299 bp long (36.3% G/C) and contains 7 open reading frames (L2, L1, E6, E7, E1, E2 and E4) and a non-coding long control region (LCR) between L1 and E6. The metagenomic procedures, coupled with the bioinformatic methods described herein are well suited to detect small circular genomes such as those of human papillomaviruses.     

The Mitochondrial Genome of Paramphistomum cervi (Digenea), the First Representative for the Family Paramphistomidae

We determined the complete mitochondrial DNA (mtDNA) sequence of a fluke, Paramphistomum cervi (Digenea: Paramphistomidae). This genome (14,014 bp) is slightly larger than that of Clonorchis sinensis (13,875 bp), but smaller than those of other digenean species. The mt genome of P. cervi contains 12 protein-coding genes, 22 transfer RNA genes, 2 ribosomal RNA genes and 2 non-coding regions (NCRs), a complement consistent with those of other digeneans. The arrangement of protein-coding and ribosomal RNA genes in the P. cervi mitochondrial genome is identical to that of other digeneans except for a group of Schistosoma species that exhibit a derived arrangement. The positions of some transfer RNA genes differ. Bayesian phylogenetic analyses, based on concatenated nucleotide sequences and amino-acid sequences of the 12 protein-coding genes, placed P. cervi within the Order Plagiorchiida, but relationships depicted within that order were not quite as expected from previous studies. The complete mtDNA sequence of P. cervi provides important genetic markers for diagnostics, ecological and evolutionary studies of digeneans.     

The Epidemiology,Virology and Clinical Findings of Dengue Virus Infections in a Cohort of Indonesian Adults in Western Java

Background

Dengue has emerged as one of the most important infectious diseases in the last five decades. Evidence indicates the expansion of dengue virus endemic areas and consequently the exponential increase of dengue virus infections across the subtropics. The clinical manifestations of dengue virus infection include sudden fever, rash, headache, myalgia and in more serious cases, spontaneous bleeding. These manifestations occur in children as well as in adults. Defining the epidemiology of dengue in a given area is critical to understanding the disease and devising effective public health strategies.

Methodology/Principal Findings

Here, we report the results from a prospective cohort study of 4380 adults in West Java, Indonesia, from 2000–2004 and 2006–2009. A total of 2167 febrile episodes were documented and dengue virus infections were confirmed by RT-PCR or serology in 268 cases (12.4%). The proportion ranged from 7.6 to 41.8% each year. The overall incidence rate of symptomatic dengue virus infections was 17.3 cases/1,000 person years and between September 2006 and April 2008 asymptomatic infections were 2.6 times more frequent than symptomatic infections. According to the 1997 WHO classification guidelines, there were 210 dengue fever cases, 53 dengue hemorrhagic fever cases (including one dengue shock syndrome case) and five unclassified cases. Evidence for sequential dengue virus infections was seen in six subjects. All four dengue virus serotypes circulated most years. Inapparent dengue virus infections were predominantly associated with DENV-4 infections.

Conclusions/Significance

Dengue virus was responsible for a significant percentage of febrile illnesses in an adult population in West Java, Indonesia, and this percentage varied from year to year. The observed incidence rate during the study period was 43 times higher than the reported national or provincial rates during the same time period. A wide range of clinical severity was observed with most infections resulting in asymptomatic disease. The circulation of all four serotypes of dengue virus was observed in most years of the study.     

Metabolomics-Based Discovery of Small Molecule Biomarkers in Serum Associated with Dengue Virus Infections and Disease Outcomes

Background

Epidemic dengue fever (DF) and dengue hemorrhagic fever/dengue shock syndrome (DHF/DSS) are overwhelming public health capacity for diagnosis and clinical care of dengue patients throughout the tropical and subtropical world. The ability to predict severe dengue disease outcomes (DHF/DSS) using acute phase clinical specimens would be of enormous value to physicians and health care workers for appropriate triaging of patients for clinical management. Advances in the field of metabolomics and analytic software provide new opportunities to identify host small molecule biomarkers (SMBs) in acute phase clinical specimens that differentiate dengue disease outcomes.

Methodology/Principal Findings

Exploratory metabolomic studies were conducted to characterize the serum metabolome of patients who experienced different dengue disease outcomes. Serum samples from dengue patients from Nicaragua and Mexico were retrospectively obtained, and hydrophilic interaction liquid chromatography (HILIC)-mass spectrometry (MS) identified small molecule metabolites that were associated with and statistically differentiated DHF/DSS, DF, and non-dengue (ND) diagnosis groups. In the Nicaraguan samples, 191 metabolites differentiated DF from ND outcomes and 83 differentiated DHF/DSS and DF outcomes. In the Mexican samples, 306 metabolites differentiated DF from ND and 37 differentiated DHF/DSS and DF outcomes. The structural identities of 13 metabolites were confirmed using tandem mass spectrometry (MS/MS). Metabolomic analysis of serum samples from patients diagnosed as DF who progressed to DHF/DSS identified 65 metabolites that predicted dengue disease outcomes. Differential perturbation of the serum metabolome was demonstrated following infection with different DENV serotypes and following primary and secondary DENV infections.

Conclusions/Significance

These results provide proof-of-concept that a metabolomics approach can be used to identify metabolites or SMBs in serum specimens that are associated with distinct DENV infections and disease outcomes. The differentiating metabolites also provide insights into metabolic pathways and pathogenic and immunologic mechanisms associated with dengue disease severity.     

phenoSeeder - A Robot System for Automated Handling and Phenotyping of Individual Seeds

The enormous diversity of seed traits is an intriguing feature and critical for the overwhelming success of higher plants. In particular, seed mass is generally regarded to be key for seedling development but is mostly approximated by using scanning methods delivering only two-dimensional data, often termed seed size. However, three-dimensional traits, such as the volume or mass of single seeds, are very rarely determined in routine measurements. Here, we introduce a device named phenoSeeder, which enables the handling and phenotyping of individual seeds of very different sizes. The system consists of a pick-and-place robot and a modular setup of sensors that can be versatilely extended. Basic biometric traits detected for individual seeds are two-dimensional data from projections, three-dimensional data from volumetric measures, and mass, from which seed density is also calculated. Each seed is tracked by an identifier and, after phenotyping, can be planted, sorted, or individually stored for further evaluation or processing (e.g. in routine seed-to-plant tracking pipelines). By investigating seeds of Arabidopsis (Arabidopsis thaliana), rapeseed (Brassica napus), and barley (Hordeum vulgare), we observed that, even for apparently round-shaped seeds of rapeseed, correlations between the projected area and the mass of seeds were much weaker than between volume and mass. This indicates that simple projections may not deliver good proxies for seed mass. Although throughput is limited, we expect that automated seed phenotyping on a single-seed basis can contribute valuable information for applications in a wide range of wild or crop species, including seed classification, seed sorting, and assessment of seed quality.Seeds play a major role in keeping continuity between successive generations (Esau, 1977) and are key for the distribution and evolution (Moles et al., 2005) of higher plants. Fertile seeds carry an embryo and may contain nutrient storage tissues in cotyledons, endosperm, and/or perisperm, supporting germination and seedling development at early developmental stages. Although this is true for all seed plants, various traits of seeds, such as size, shape, weight, and chemical composition, can be very different between plant species or accessions. For example, the Arabidopsis (Arabidopsis thaliana) accession Cape Verde Islands was reported to yield on average 40% fewer seeds than Landsberg erecta, but they are almost twice as heavy (Alonso-Blanco et al., 1999). Considering today’s plant species, single-seed mass may vary over a range of 11.5 orders of magnitude (Moles et al., 2005). Seed mass is under strong genetic control, whereas the total number of seeds of a plant is largely affected by the environment (Paul-Victor and Turnbull, 2009). It has been demonstrated that the size, mass, and shape of Arabidopsis seeds may be regulated by brassinosteroid (Jiang et al., 2013), and it was shown recently that seed size in rice (Oryza sativa) can be influenced by the epiallele Epi-rav6 (Zhang et al., 2015). The ability of plants to switch between small and larger seeds may be understood as an adaptation to novel environments (Igea et al., 2016). However, it is still not fully understood whether, or to what extent, the variability of seed traits within plant species or genotypes has an impact on the development and further performance of a plant.When comparing biometric seed data of different dimensions such as length (one-dimensional), projected area (two-dimensional [2D]), or volume and mass (both three-dimensional [3D]), one can argue that mass is the most relevant parameter as a proxy for the amount of reserves a seed provides for the offspring. This might be true even when considering that the type of reserves, such as proteins, carbohydrates, or lipids (Rolletschek et al., 2015), and also different seed tissues, such as seed coat, embryo, or endosperm, may contribute differently to seed mass (Alonso-Blanco et al., 1999). While seed mass and time to germination (radicle protrusion) do not necessarily correlate (Norden et al., 2009), in particular under greenhouse conditions, higher seed mass may be advantageous for seedling establishment under adverse environmental conditions (Moles et al., 2005). For example, shade-tolerant species showed largely higher seed masses than cogeneric species growing in open habitats, indicating that seedlings under low-light conditions need more reserves than under good light (Salisbury, 1974). Seedlings of wild radish (Raphanus raphanistrum) emerged more likely from heavier seeds than from small seeds under field conditions but not in the greenhouse (Stanton, 1984), and for Arabidopsis, seed mass was reported to be higher in populations growing naturally at higher altitudes taken as a proxy for harsher conditions (Montesinos-Navarro et al., 2011).Seed mass can be measured individually (Stanton, 1984), but it is generally collected as an average value of batches of 50 to 1,000 seeds (Jako et al., 2001; Jofuku et al., 2005; Montesinos-Navarro et al., 2011; Tanabata et al., 2012). Alternatively, 2D scans are analyzed to determine parameters such as seed length, width, area, and perimeter length as a measure for seed size (Tanabata et al., 2012). This approach can be implemented in high-throughput facilities to obtain projected areas of seed grains combined with genome-wide association studies (Yang et al., 2014). Although projected seed area can easily be measured with a common office scanner (Herridge et al., 2011; Tanabata et al., 2012; Moore et al., 2013), it is not necessarily a precise or reliable measure of the true seed size because it may depend on the shape (Alonso-Blanco et al., 1999) and the orientation of a seed at scan (see “Results”). These issues also apply when using 2D projections to calculate length-to-width ratios as a simple shape factor (Tanabata et al., 2012). Projected seed area also has been used to calculate seed mass, assuming a fixed relationship between these parameters (de Jong et al., 2011; Herridge et al., 2011). This may hold with sufficient accuracy when averaging a large number of seeds but might be misleading when considering individual seeds.From a physical point of view, volume should be a much better proxy for mass than 2D traits. Although it has been stated that for 65 species analyzed seed masses can be compared easily with seed volumes (Moles et al., 2005), it is not clear how these seed volumes were determined. Volumes can be assessed using advanced methods such as x-ray computed tomography (CT) on fruits (Stuppy et al., 2003) or synchrotron radiation x-ray tomographic microscopy applied in paleobiological studies (e.g. on fruits and seed; Friis et al., 2014). Nuclear magnetic resonance (NMR) methods are used to measure water uptake in kidney beans (Phaseolus vulgaris) and adzuki beans (Vigna angularis; Kikuchi et al., 2006) or to estimate seed weight and content (Borisjuk et al., 2011; Rolletschek et al., 2015) rather than volumes. To our best knowledge, affordable methods to measure seed volumes directly are not achievable so far. For that reason, we have set up a volume-carving method for 3D seed shape reconstruction that is described briefly here and in more detail in a recent publication (Roussel et al., 2016).While traits derived from scanning procedures can easily be assigned to individual seeds (Herridge et al., 2011), further handling and processing of phenotyped single seeds is not as simple, in particular for tiny ones like those of Arabidopsis. The aim of this work was to develop an automated seed-handling system that can analyze single seeds of very different sizes or shapes, from Arabidopsis seeds up to barley (Hordeum vulgare) seeds or even bigger. The phenoSeeder system is designed to pick and place seeds, to achieve basic morphometric traits (one-dimensional and 2D data from projections, 3D reconstruction data, and mass) of each individual seed, and to store all analyzed seed traits in a database. Another goal is to use phenoSeeder for seed-to-plant tracking approaches and to analyze whether, or which, particular seed traits have an impact on plant development and performance under various environmental conditions. We describe the main features of the phenoSeeder technology and present results obtained with seeds of three accessions of Arabidopsis, rapeseed (Brassica napus), and barley, respectively. When analyzing the data, we focused particularly on correlations between projected seed area, seed volume, and seed mass, with the hypothesis that the respective seed volume may better correlate with mass than the projected area.     

DnaJ/Hsc70 chaperone complexes control the extracellular release of neurodegenerative‐associated proteins

It is now known that proteins associated with neurodegenerative disease can spread throughout the brain in a prionlike manner. However, the mechanisms regulating the trans‐synaptic spread propagation, including the neuronal release of these proteins, remain unknown. The interaction of neurodegenerative disease‐associated proteins with the molecular chaperone Hsc70 is well known, and we hypothesized that much like disaggregation, refolding, degradation, and even normal function, Hsc70 may dictate the extracellular fate of these proteins. Here, we show that several proteins, including TDP‐43, α‐synuclein, and the microtubule‐associated protein tau, can be driven out of the cell by an Hsc70 co‐chaperone, DnaJC5. In fact, DnaJC5 overexpression induced tau release in cells, neurons, and brain tissue, but only when activity of the chaperone Hsc70 was intact and when tau was able to associate with this chaperone. Moreover, release of tau from neurons was reduced in mice lacking the DnaJC5 gene and when the complement of DnaJs in the cell was altered. These results demonstrate that the dynamics of DnaJ/Hsc70 complexes are critically involved in the release of neurodegenerative disease proteins.     

Direct evidence implicates feral cat predation as the primary cause of failure of a mammal reintroduction programme

As evidence mounts that the feral Cat (Felis catus) is a significant threat to endemic Australian biodiversity and impedes reintroduction attempts, uncertainty remains about the impact a residual population of cats following control will have on a mammal reintroduction programme. Also, behavioural interactions between cats and their prey continue to be an area of interest. Within the framework of an ecosystem restoration project, we tested the hypotheses that successful reintroductions of some medium‐sized mammals are possible in locations where feral cats are controlled (but not eradicated) in the absence of European Red Fox (Vulpes vulpes), and that hare‐wallabies that dispersed from their release area are more vulnerable to cat predation compared with those that remain at the release site. We used radiotelemetry to monitor the survivorship and dispersal of 16 Rufous Hare‐wallabies (Lagorchestes hirsutus spp.) and 18 Banded Hare‐wallabies (Lagostrophus fasciatus fasciatus) reintroduced to four sites within Shark Bay, Western Australia. Nearly all foxes were removed and feral cats were subject to ongoing control that kept their indices low relative to prerelease levels. All monitored hare‐wallabies were killed by cats within eight and 10 months following release. Significant predation by feral cats was not immediate: most kills occurred in clusters, with periods of several months where no mortalities occurred. Once a hare‐wallaby was killed, however, predation continued until each population was eliminated. Animals remaining near their release site survived longer than those that dispersed. The aetiology of predation events observed offers new insights into patterns of feral cat behaviour and mammal releases. We propose a hypothesis that these intense per capita predation events may reflect a targeted hunting behaviour in individual feral cats. Even where feral cats are controlled, the outcome from consistent predation events will result in reintroduction failures. Managers considering the reintroduction of medium‐sized mammals in the presence of feral cats should, irrespective of concurrent cat control, consider the low probability of success. We advocate alternative approaches to cat‐baiting alone for the recovery of cat‐vulnerable mammals such as hare‐wallabies.