Virus integration detection bY Paired End Reads) outperforms existing similar tools in speed and accuracy. We analysed whole genome data from childhood acute lymphoblastic leukemia (ALL), which is characterised by genomic rearrangements and usually associated with radiation exposure. This analysis was motivated by the recently reported virus integrations at genomic rearrangement sites and association with chromosomal instability in liver cancer. However, as expected, our analysis of 20 tumour and matched germline genomes from ALL patients finds no significant evidence for integrations by known viruses. Nevertheless, our method eliminates 12,800 false positives per genome (80× coverage) and only our method detects singleton human-phiX174-chimeras caused by optical errors of the Illumina HiSeq platform. This high accuracy is useful for detecting low virus integration levels as well as non-integrated viruses.Several pathogenic viruses such as hepatitis B and human immunodeficiency viruses may integrate into the host genome. These virus/host integrations are detectable using paired-end next generation sequencing. However, the low number of expected true virus integrations may be difficult to distinguish from the noise of many false positive candidates. Here, we propose a novel filtering approach that increases specificity without compromising sensitivity for virus/host chimera detection. Our detection pipeline termed Vy-PER ( This mechanism is exploited for example by HBV232411http://goo.gl/q7SaUZ). Childhood ALL is mostly classifiable by fusion genes2729Two primary drivers for developing virus integration detection methods are the fields of disease therapy and disease etiology. In gene therapy and immunotherapy studies, a major consideration is the non-integration23689121415Virus integrations can be detected using Illumina paired-end sequencing , if one 2336141516In 2012 the German Federal Office for Radiation Protection initiated a multi-center deep sequencing pilot study on childhood ALL  online. The paired tumour and control samples were collected within the International BFM-Study Group (I-BFM-SG) from Austria, France, and Germany. The patients were enrolled by the AIEOP-BFM study group  and the FRALLE study group (France). Informed consent for the use of spare specimens for research was obtained from study individuals, parents or legal guardians. The research project reported here was approved by the Ethics Committee of the Medical Faculty, Christian-Albrechts-University, Kiel, Germany. All methods were carried out in accordance with the approved guidelines. The biological samples and sequence data are not consented for access outside of our consortium.Illumina TruSeq 2 × 101 bp paired end sequencing was performed on Illumina HiSeq 2000 and HiSeq 2500 instruments . In brief, we used 1 μg of DNA per sample, which is fragmented by sonication to lengths of approximately between 250 and 650 bps before end-repair and ligation of Illumina sequencing adapters and molecular barcodes. The resulting libraries are each attached to random positions on a glass carrier plate (flowcell), and sequenced: Put simply, in the “first run”, the positions of all clusters on the glass plate are optically identified, and the first end of each library is sequenced to a length of 101 bps. In the second run, the library is sequenced from the second end to a length of 101 bps. Importantly, the position of each library on the flowcell is optically identified by the instrument and in rare cases a library position may be identified differently in the second run, leading to a chimeric sequencing artefact consisting of two different libraries.Virus integration detection bYPaired End Reads) are freely available at http://www.ikmb.uni-kiel.de/vy-per. The downloads include example scripts for a Linux cluster, human genome hg19 reference, NCBI virus genome references and benchmark data. The Vy-PER scripts are implemented in Python 2.7 with pysam v0.6, R, the Bioconductorhttp://www.rub.de/spezzoo/cm/cm_phobos.htm), and SAMtoolswww.sciengines.com), but for general users, Vy-PER also provides the option of using BLAT on a Linux cluster.All Vy-PER scripts (The bioinformatic workflow is summarised in http://gib-v.genes.nig.ac.jp/) for VirusFinder. The benchmark timings were obtained by running each pipeline on a single 16-core linux computer node with 120 GB RAM, except for Vy-PER which only required a single core and less than 10 GB RAM; all file input/output was performed via a 10 Gbit/s network to a central EMC Isilon X200 storage cluster. We included the Amazon EC2 cloud-based instance of SURPIWe here also compare the virus integration detection by Vy-PER with several recently published pipelines using their default settings or recommendations: the VirusSeq pipelinehttp://www.ikmb.uni-kiel.de/vy-per.Download links to all four example data sets are available at NA12878V is a synthetic data set that we constructed by extracting 10,000 read-pairs from a 1000 Genomes ProjectL526401A liver cancer sample (RNA). As our patient data cannot be publicly released, we here demonstrate the detection of known HBV integrations into liver cancer cell genomes employing publicly available Illumina paired-end (2 × 50 bp) transcriptome data198T and 268T comprise publicly available “cleaned” Illumina paired-end (2 × 90 bp) whole genome tumour data with known HBV integrations, from two liver cancer patients in a study of 88 liver cancer patients198T and 268T sequencing data to test the sensitivity of our method and of other approaches at ultra low virus integration content. The subsets consist of 82,708,061 (198T) and 82,450,511 (268T) read-pairs, respectively. Before the Vy-PER run, alignment to hg19 was performed using BWA and SAMtools, which required 3 hours 22 minutes (198T), and 3 hours 45 minutes (268T), respectively. Then the Vy-PER pipeline was run as described.Using our Vy-PER method, we searched for virus integrations in 10 tumour samples that were sequenced to a minimal coverage of 80×, and in 10 matched normal samples from the same patients that were sequenced to a minimal coverage of 40×. Before final filtering see  online, Childhood acute lymphoblastic leukemia whole genome data from one HiSeq lane. To compare run times between different pipelines , 1185 × phiX, and 2 × M13 chimeras where the human reads were aligned with a mapping quality of at least 20. HHV-3 and phiX were reported as non-integrated viruses by SURPI and VirusFinder, but not by ViralFusionSeq or VirusSeq (VirusSeq . The knoVirusSeq  online.L526401A liver cancer sample (RNA). The known HBV integration loci were reported by Chen and colleaguesTECRL gene. This locus is nevertheless annotated as “TECRL/3-prime”, which the VirusSeq authors have also copied into their publicationLPHN3, is less than half the distance to TECRL. The ViralFusionSeq pipeline reported 10 clipped fusion sequences of which most are duplicates, but did not report their integration loci  which has a tropism for CD4+ T-lymphocyteshttp://www.cs.utexas.edu/users/phylo/research/projects.html) dumbs down its level of detail as necessary, from strain to species or even to higher levels in the phylogeny, when ambiguous reads are encountered. However, the tested other pipelines currently still report viral strain information, which can give users a false sense of security, especially as there are vast homologies between the strains of a species, and we are unsure how correct these virus databases actually are. For example, the less experienced user may well be excited by the carp herpesvirus incorrectly reported in three out of five example data sets by one pipeline, or the human herpesvirus type 5 (instead of M13) incorrectly reported by another pipeline. No doubt each pipeline was developed for a specific type of virus and cancer data set, so it is probably worthwhile for a researcher to run a suspicious data set, i.e. one in which Vy-PER detected a virus, through each of these pipelines and to keep the potential problems in mind. It is for example remarkable that ViralFusionSeq detected viruses in only two of the five example data sets, but nevertheless detected three of the four fusion sequences in the low-coverage data subset of tumour sample 268T. Vy-PER only detected one of these fusions to be a virus integration event into the human host, because it searches only for chimeric paired-ends and not also for fusion sequences. Nevertheless, the scan for chimeras by Vy-PER was more sensitive than the approaches in other pipelines, correctly detecting the known chimeras in all five examples.The results shown here demonstrate that Vy-PER is sensitive, specific and efficient enough to be used as a first pipeline for routinely scanning all NGS data sets for virus integrations. The efficiency of Vy-PER is explained by its filtering design for speedy processing of genomic data that can be furthermore parallelised for multi-lane genomes (map-reduce approach), and – when integrated into a BWA based pipeline – by avoiding the duplication of any expensive whole genome alignment computations. Once viruses of concern have been found in a sample, a more detailed analysis with ViralFusionSeq, VirusFinder and VirusSeq can then be performed. These latter pipelines are still in their early days and fairly complicated to install and understand, but each has its own merits. All of them also perform a search for viruses that did not integrate into the host genome. VirusSeq annotates the integration loci with gene information and computes breakpoints. VirusFinder is supposed to do the same, but our tested version (version 2) did not compute any integrations. ViralFusionSeq reports only the human/virus fusion reads but not the integration locus. Whereas Vy-PER was designed with concise clinical reporting in mind, the other pipelines are more detailed and do not provide a summary graphic or table. Vy-PER uses the NCBI virus genomes which are sufficient for uniquely pinpointing a virus at species level, and the summary tables show whether some or all virus reads were unambiguously assigned to a single virus species . SimilarSurprisingly, Vy-PER is also able to detect non-integrated viruses. Taking the example of phiX (1% Illumina sequencing control library spike-in) and M13 (trace contamination of phiX library), the known presence of these non-integrated viruses is detected as chimeric virus/human read-pair artefacts. In our whole genome leukemia sequencing study, on average 160 phiX/human chimeras were detected per HiSeq lane (0.0001% of read-pairs). The chimeric sequencing artefacts can be interpreted as randomly sampled non-integrated virus sequences. Indeed, on the related topic of bacterial identification from shotgun sequencing, it was recently suggestedAs a matter of speculation, future studies may find that the concept of a human-virus breakpoint at a single base pair is not appropriate for all viruses. On the one hand it is known that retroviruses integrate at a distinct breakpoint when an integrase binds to its preferred nucleotide motifs57In the treatment of HIV infections, cART may be unable to completely eliminate HIV if replication competent proviruses are integrated into long lived or proliferating CD4+ cells. Of particular interest are the integration loci which are preferentially located in genes that promote cell proliferation6Finally, the wealth of incidental metagenomic findings in human NGS data is highly exciting for potential pathogen detection, even when only microscopically low read numbers are available that our approach can detect and assign to a virus genome.How to cite this article: Forster, M. et al. Vy-PER: eliminating false positive detection of virus integration events in next generation sequencing data. Sci. Rep.5, 11534; doi: 10.1038/srep11534 (2015).


Likewise, it is now feasible to rescue infectious arenaviruses containing predetermined mutations in their genomes to investigate virus-host interactions and mechanisms of pathogenesis. The use of reverse genetics approaches has also allowed the generation of recombinant arenaviruses expressing additional genes of interest. These advances in arenavirus molecular genetics have also facilitated the implementation of novel screens to identify anti-arenaviral drugs, and the development of novel strategies for the generation of arenavirus live-attenuated vaccines. In this review, we will summarize the current knowledge on reporter-expressing, replicating-competent arenaviruses harboring reporter genes in different locations of the viral genome and their use for studying and understanding arenavirus biology and the identification of anti-arenaviral drugs to combat these important human pathogens.Several arenaviruses cause hemorrhagic fever (HF) disease in humans and pose an important public health problem in their endemic regions. To date, no Food and Drug Administration (FDA)-licensed vaccines are available to combat human arenavirus infections, and current anti-arenaviral drug therapy is limited to an off-label use of ribavirin that is only partially effective. The development of arenavirus reverse genetic approaches has provided investigators with a novel and powerful approach for the study of arenavirus biology including virus–host interactions underlying arenavirus induced disease. The use of cell-based minigenome systems has allowed examining the  Arenaviruses cause chronic infections of rodents with a worldwide distribution . AsymptoArenaviridae consists currently of two genera: (1) Mammarenavirus and (2) Reptarenavirus. Reptarenavirus is a new genus that has been established to accommodate the distinct features of recently discovered snake arenaviruses . I. I91]. I50) for A3 was about 100-fold lower than for ribavirin . M. M105]. The characterization of a recombinant Pichinde virus, PICV (strain P18) with a trisegmented RNA genome (rP18tri-G) that expresses GFP from the GPC locus  104], s, s104], The development of arenavirus reverse genetics systems has provided investigators with a novel and powerful experimental approach to study basic aspects of arenavirus biology, including the identification of viral determinants, and their mechanisms of action, which contribute to arenaviral human diseases. The ability to manipulate the genome of arenaviruses has proven to be a superb model system to study virus–host interactions and associated disease. Moreover, generating recombinant arenaviruses with predetermined mutations allows investigators to gain a detailed understanding of arenavirus–host interactions and phenotypic outcomes of virus infection. Furthermore, the generation of r3 or bicistronic arenaviruses expressing appropriate RG, together with the development of specific cell-based assays for each of the different steps of the arenavirus life cycle, is facilitating novel approaches to discover and characterize antiviral drugs against these important human pathogens.


Unfolded protein response (UPR) is a cellular homeostatic response to endoplasmic reticulum (ER) stress. Increasing evidence suggests an intimate relationship between virus and UPR. This research topic collated a number of review articles and original research article, in an attempt to highlight how viruses interact with the host UPR in the establishment of acute, chronic and latent infections.Virus infection represents an arm race between virus and the host. On one hand, the host mobilizes the UPR in an attempt to restrict virus infection. On the other hand, virus subverts or even manipulates the UPR to assist in its own infection. The consequence of this is that the UPR is often skewed during virus infections to either favor virus elimination or virus invasion. Whoever won, the outcome could be pathogenic. The relationship between virus and UPR and its associated autophagy is being addressed in three reviews focusing on RNA viruses, as their life cycles are closely associated with the ER  possesses the smallest genome of human herpesviruses and lacks some genes used by other herpesviruses to manipulate the UPR. The key question is therefore whether VZV UPR induction is merely a host response or a result of viral manipulation. By using a UPR PCR array, John Carpenter and Charles Grose demonstrated VZV differentially induced the UPR to expand the ER to cope with viral glycoprotein synthesis  is part of an UPR functioning to extract unfolded/misfolded proteins from the ER into the cytosol for proteasomal degradation. Not surprisingly, this process is also targeted by virus. Jaquelin Dudley and her group re-captures the ERAD process in details followed by an illustration of how viruses exploit this process , culminating in the release of interferon, which in turn activates the professional virus killer, one of which is RNase L. One of the proximal UPR sensors, inositol-requiring enzyme 1 (IRE1), is evolutionarily related to RNase L. In the review of Sankar Bhattacharyya, he provides a structural and functional comparison between IRE1 and RNase L and comments on a potential anti-viral function of IRE1 by the creation of “danger signals” via the regulated IRE1-dependent decay (RIDD) pathway (Bhattacharyya, It is increasingly popular to use viruses in clinical applications such as gene therapy and oncolytic virotherapy. The use of viral vectors/viruses in the clinics will not be valid without a thorough understanding of virus-host interaction. Giridhara Jayandharan and his group presents a review on the emerging impact of UPR on gene therapy and how the understanding of this will allow us to exploit and improve the use of viral vectors in gene therapy (Sen et al., To date we are still at the sprouting stage of understanding this virus-host interaction. We hope that this selection of articles will provide a foundation to spark more interest in this research area. This will not only lead to a deeper understanding of virus infection and pathogenesis but will also unravel novel anti-viral mechanisms. Eventually it will help to unlock novel anti-viral targets and may also impact on optimizing the use of viruses in the clinics.The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The impact of administrative costs on the distribution of terminal wealth is approximated using a simple formula applicable to many investment situations. We show that the reduction in median returns attributable to administrative fees is usually at least twice the amount of the administrative costs charged for most investment funds, when considering a risk-adjustment correction over a reasonably long-term time horizon. The example we present covers a number of standard cases and can be applied to passive investments, mutual funds, and hedge funds. Our results show investors the potential losses they face in performance due to administrative costs.  The administrative costs of saving and pension schemes have received increasing attention in recent years, at a time when a massive shift has been witnessed from actively to passively managed funds—often exchange-traded funds (ETFs)—that incur lower costs. The reason is obvious. Most studies of the performance of actively managed funds are unable to demonstrate their higher performance –3. MutuaIn this paper we show that the impact of administrative costs on terminal wealth can be substantial. We also propose a simple method for comparing median returns in the presence and absence of such costs, holding risk constant, and report that, for certain investment strategies, administrative fees can at times be twice as high as returns. Median values as opposed to expectations are used because the instrument we employ is the value-at-risk measure .While a good historical performance has been reported for a few actively managed funds, it is by no means straightforward to prove that this performance is anything but a random phenomenon . LetWhen calculating the consequences of extra costs, a standard approach is used to employ a deterministic prediction of expected loss on returns, which is the difference between the expected returns on terminal wealth with and without the extra administrative costs. This is often sufficient to demonstrate the impact of administrative fees. A typical calculation, for example, might show that an additional one percent cost will mean that a quarter of a saver's pension disappears. This paper shows that while such calculations are highly thought as provoking, they may well be overly conservative, given that the saver may well incur costs greater than one percent. The difference between the expected returns on terminal wealth without administrative costs and with them is an estimate of only part of the impact, because extra costs influence the whole distribution of terminal wealth, not only first-order moments or location measures. This paper further examines this difference and provides a simple analytic expression for calculating the risk-adjusted impact of administrative costs on the median returns of terminal wealth. A deterministic prediction such as the difference between the expected or median returns of terminal wealth in the presence and absence of additional administrative costs fails to take the saver's risk aversion into account. It is tacitly assumed that the saver takes the same level of risk with or without these extra costs. Yet, we know that this is not the whole story. If a saver loses a quarter of his pension, then he will tend to be more risk averse with his remaining money; he will not want to lose much more. As such, a pension saver paying one percent in additional fees will tend to approach risky investments with more care than a saver who is not being charged these extra costs—this insight runs contrary to most descriptions in the literature. Another way of understanding this behaviour is in terms of a lost opportunity: the saver loses the opportunity to take risks with the one percent extra costs that he is apparently not prepared to lose. Such risky positions, however, give better median returns than less risky positions. As a result, the lost opportunity hits hard. We tacitly assume that greater risk implies better median returns and that greater risk implies that our risk measure reacts in the “right" direction. We use a simple quantile as our downside risk measure and we make the above assumptions explicit in our parameter space.Many authors have studied optimal consumption-investment problems –13. PerfMany authors examine performance but do not focus on administrative costs –19. OtheWhen studying mutual fund administrative fees, Khorana et al.  find thaFor lifetime investors, the importance of performance and transparency of pension schemes is widely recognized –31. Smalr and stock index (St) following a geometric Brownian motion:μ is the expected return, σ is the volatility, and W is a standard Brownian motion.We assume a simple price process. Consider a Black-Scholes financial market, that is, a market with a constant risk-free rate of interest X denote a wealth process and assume that wealth is invested at a constant proportion α in stocks and the rest at the risk-free rate. Assume further that administration costs are deducted continuously from the wealth process with a rate of ν proportional to the amount invested in risky assets. We therefore consider administrative costs  to be given by αν. The dynamics of X are thus given byLet X0 ≡ 1. More realistic conditions, including more general price processes with variance jumps or distributions with heavier tails, might be considered in the future. It could be argued that the stock price dynamics in our models are too simple to capture long-term effects. However, our aim is to study the model presented here first and, at a later date, to check the robustness of our results in more sophisticated settings. Additionally, wealth distribution may include the present value of pensions and future social security benefits together with some other forms of wealth. However, pension wealth is not perfectly fungible and some other household assets may also have liquidity constrains. Besides, differential tax treatment may also be applicable. Our approach does not consider this more general framework.The wealth process is a geometric Brownian motion with the well-known representation: edit 1 U is a standard normal variate, it follows that the γ-percentile, qγ,t, in the distribution of Xt is given bydγ denotes the γ-percentile of a standard normal distribution.Since Specifically, the median is given bym = exp(ρt). From  is equal to a constant minus the administrative costs αν.We define the (geometric) rate of returnρ + r)t], is always increasing in α. This reflects the fact that for high equity exposures the wealth distribution is highly skewed, the majority of the mass being close to zero and a long right tail.The expected value of Given the skewness of the wealth distribution, here we prefer to compare medians rather than means.α,  μ,  σ,  r,  t and the quantile level is γ. We restrict our parameter space to those parameters for which the median increases and the γ-percentile decreases when exposure to the risky asset increases.Our model parameters are Xt. Thus, we assume that any risk-averse saver will only increase their exposure to the risky assets if the median return increases. We consider this restriction natural. Besides this property, we only consider nonnegative administrative costs (ν). All details are provided in The reason why we restrict our parameter space to this combination is to ensure that our assumptions are realistic. An increase in the median return is a necessary reward when downside risk increases in the distribution of r = 4%, μ = 8%, and σ = 20%. We also assume that α = 50% is invested in stocks and that ν = 0, so there are no administrative costs.For illustrative purposes, we present an example that we discuss in detail in the sections that follow. We use the capital market parameters γ = 10%,  5%,  1% and 0.1%. We plot ln/t so we have a monotonic transformation of the quantile .scale of  divided qγ,t)/t is negative in the initial period. Values of ln/t measure the loss in the lowest quantiles and capture the value-at-risk for the worst possible scenarios of the wealth process. After forty years, ln/t is clearly above zero, indicating that initial wealth has increased even for a situation that is at the 5% level of the downside tail.In our example, all the conditions of the parameter space are satisfied for a time horizon of less than a hundred years.We are interested in studying the effect of administrative costs on the median rate of return when fixing the level of risk in the wealth distribution.  The level of risk in the wealth distribution can be measured by a (low) percentile in the distribution, that is, a value-at-risk (VaR) measure.ν = 0, and calculate a risk measure for the distribution of wealth at time t. Then, we determine the equity exposure in the presence of administrative costs, ν > 0, that corresponds to the same risk as the given equity exposure with no administrative costs. Hence, we seek to find the reduction in equity exposure, Δν, such that the γ-percentile of the wealth distribution with equity exposure α − Δν and administrative costs ν, qγ, matches the γ-percentile of the wealth distribution with equity exposure α and no administrative costs qγ.Our procedure for analysing the impact of administrative costs is based on a constant risk factor. Thus, we examine the case for which we have no administrative fees, ν, such thatThus, we seek ΔA = σ2/2,  edit 1 C = να.According to , we obtaB2 − 4AC ≥ 0. So, there is a solution for Δν, and it is given by edit 1 It immediately follows from t as a function of α and the corresponding quantile when there are administrative costs. The graph shows that the level of investment in stocks α = 50% shifts to the left if we consider γ = 10% and ν = 0.1%. This means that when administrative costs exist, an investment equivalent in terms of our risk measure to one that has 50% invested in stocks and no administrative costs has a lower proportion invested in stocks, Δν = 2.38%, for t = 40.ρν be the rate of return with administrative costs ν and exposure α − Δν. It follows immediately from  for Xt. However, note that Δν depends on t. As a consequence, the reduction in the rate of return attributable to administrative costs does not necessarily tend to zero as t increases. In certain specific cases, it might be constant, but in general it is increasing in t.In , as dγρ − ρν on t or its rate of convergence to a constant as a function of t.In order to understand expression  we need ν. We derive a second-order Taylor expansion of Δν at ν = 0. Since by definition Δ0 = 0 we haveOur aim is to examine the behaviour of the reduction in the rate of return as a function of ν yieldsν = 0 and rearranging the terms, we obtainDifferentiating  with resν = 0 we obtainSimilarly, by differentiating  twice anUsing  and afteν can be seen on the reduction in the rate of return.From these expressions, the first- and second-order effects of β is positive and strictly larger than 1. The term  edit 1 dγ is negative, because γ < 50%) than −1.Note that our chosen parameter space insures that ρ − ρν isβ, which is a multiplier, to obtain a simple, easy-to-use, first-order approximation of the impact of administrative costs (αν) on the lost rate of return. Moreover, we note that β tends to one as t decreases, but depending on the parameters and in the medium horizon of t, β can be quite large, as becomes evident in the illustrations presented in the next section. So, when measuring the impact of administrative costs in terms of the multiplier β, which is larger than 1, we see that it can grow rapidly. (The second-order effect is in the same direction.) According to  and is increasing with the percentile γ, because when γ increases then dγ increases. Note again that since we concentrate on downside risk, we take γ < 50%, and then dγ is negative. The opportunity cost also depends on the particular combination of stock exposure α and volatility σ2. Additionally, we also see the interrelations between the opportunity costs Cop and the investment period. Thus, the most interesting result is that the opportunity cost Cop also increases with the investment period t and can be extremely large when t increases. Note that, according to t equals [σdγ/(μ−r−ασ2)]2 and then the opportunity cost tends to infinity. Recall that dγ is negative.We turn now to concentrate on It is our belief that the size of the opportunity cost has perhaps been underestimated in the literature to date on the grounds that it may dilute over time. We have shown that the opportunity cost depends on the period of investment. This particular result is of critical importance to pension savers, who make long-term decisions. We show in the illustrations that follow in reasonably long-term horizons of forty years the opportunity cost effect can be quite substantial. Our result provides a possible explanation as to why too often most funds present a poor performance, especially for long-term investment horizons.t = 40 and we use as a risk measure the 10%-percentile; that is, γ = 10% and thereby dγ = −1.28. With α = 50% invested in stocks and no administrative costs the rate of return is ρ = 5.5%. Assuming administrative costs of ν = 0.1%, recall that Δν = 2.38% and then we find that ρ − ρν = 0.1%, and so the reduction in the rate of return is 0.1%.In our example, we consider an investment horizon of β = 1.9744. Thus, the first-order rate of return is reduced by an amount that is equivalent to about twice the administrative costs (αν = 0.05%). We examined the precision of the first-order approximation in our example, the details of which are provided in Using the Taylor expansion we haveβ, as a function of time. Second, we present the dependence of the opportunity costs on the proportion invested in stocks. β as a function of time, shows that while the impact increases, it is not very great in the first few years of the investment; however, the reduction in the rate of return increases substantially in the long term. Below we consider the two main reasons why administrative fees constitute a burden to the investor. First, we analyze the impact of administrative costs on the reduction in the rate of return, In this section we consider three possible standard scenarios for generic investment products. We assume different scenarios governing the mean and variance in market returns.  The model and methodology used for the approximations of the impact of administrative costs are the same for each scenario.σ = 0.16) in the returns. If volatility is only moderate, the mean level of stock investment return is usually also quite low. We assume very low administrative fees in this case. Next, we examine a situation in which expected returns are higher, as are administrative costs. We distinguish three possible medium volatility scenarios (σ = 0.25) in this case, as described in σ = 0.75). Here volatility is much higher than in the previous scenarios and expected returns are also higher, even though the proportion invested in stocks in this example is considerably smaller than that in the previous two cases. Administrative costs in this high volatility scenario are also higher than the fees charged in the low and medium volatility scenarios.Parameter values for the selected scenarios are presented in β in  is roughly twice (β ≈ 2) the amount of these costs in the t = 40 year horizon for the low volatility and the medium volatility scenarios. For optimistic medium volatility investments in the high volatility scenario considered here, the reduction in the rate of return is much higher than twice the amount of administrative costs.Our aim is to assess the impact of administrative costs in terms of the reduction in the rate of return and to do this we measure the multiplier β in . The lasβ as a function of time and in β as a function of α. To simplify the presentation, we select a maximum time horizon of one hundred years. The size of β shows the enormous burden of administrative costs due to the cost of opportunity.In the next section we discuss the evolution in β as a function of time and β as a function of α.In the first scenario, we consider low volatility as described in β is above 4, so the opportunity cost increases more than threefold in a time horizon of 40 years. β keeps growing with time. It might be argued that, in our choice of parameters for a standard, low volatility investment of low expected returns, administrative costs are also low and so the opportunity cost is not relevant. However, this conclusion is drawn on relative terms and the costs can be much more significant in absolute terms or in real wealth.For this case we observe that the opportunity cost due to the presence of administrative costs is well above 1 when the proportion invested in stocks is low. β as a function of time and β as a function of α. We first consider a pessimistic scenario in which the expected returns on investment are low, yet higher than in the case of low volatility. The impact of administrative costs is expected to be similar to that in the low volatility investment case, because even if volatility is now higher, the mean return is also higher. We observe that the impact of administrative costs (β) in the medium volatility case is now lower than it was in the low volatility case. The impact in terms of the return lost due to administrative costs is roughly the same in the breakeven case as in that of the low volatility investment. But the optimistic scenario gives a much worse result than that of the low volatility situation.We also considered a scenario characterized by medium volatility and medium administrative fees. Figures α becomes larger than the values shown, we find ourselves outside the parameter space and cannot calculate our risk-adjusted measure of lost return.It should be noted in α has a larger range but that the impact of administrative costs is also much higher here than in the pessimistic case, especially if α is low. In the most optimistic medium volatility scenario considered here, β due to time persistence.As we proceed to the high volatility scenario, Figures β as a function of time and β as a function of α in the high volatility scenarios. In t = 40 and a quantile probability equal to 10%, we see that for a high volatility investment we obtain the highest value of β, namely, 4.487 for the optimistic case. In Figures β tends to infinity as t approaches 80 and 66, respectively, which means that the impact of administrative costs on the reduction in the rate of return is extreme at that boundary.Long-term investments with high volatility typically also have high expected returns and, in general, incur high administrative fees. Figures α, that is, the proportion invested in stocks, in order to find the risk-adjusted lost return due to administrative costs. Again our results indicate that the impact of administrative costs, even if the excess returns (μ − r) are large, is substantial when the volatility is high.In high volatility scenarios, the parameter space is fairly restrictive for the values of Here we have highlighted the substantial impact administrative costs can have on investment plans with long-term horizons. On the understanding that an analysis limited to expected returns cannot provide a risk-adjusted evaluation of the rate of return that is lost as a result of administrative costs, we propose a one-step method to approximate the size of their impact. We illustrate our method with examples of realistic, standard fund types and, in many plausible scenarios, we often find the multiplier to be well above one, indicating that the impact of administrative fees is much greater than their actual size.Our findings regarding the difference in the rate of return when introducing moderate administrative costs are much greater than expected; and it is our belief that this accounts for the poor performance of many common fund types. If administrative costs are fixed and are not dependent on performance, then they can be so highly detrimental to the long-term return that the investor is trapped in a clearly suboptimal investment strategy.The simple approximation of the risk-adjusted impact can be used to show investors the importance of administrative costs and is a relevant contribution of this study in its own right. The only requirements to make this estimation are the time horizon, the volatility estimates, the excess returns, and the proportion of wealth invested in stocks.Finally, in highly realistic scenarios, we have shown that the impact of administrative costs in terms of the approximate lost rate of return is often twice the amount of these administrative fees. This is particularly disturbing for those investing in pensions. We believe that our contribution can provide greater insights into the analysis of administrative costs and change perspectives regarding the information supplied by intermediaries when communicating to investors the possible impact of administrative fees. Underestimating the effects on lost returns is potentially very high, especially if investors erroneously believe the lost rate of return to be exactly equal to the administrative costs.


Causative agents for more than 30 percent of respiratory infections remain unidentified, suggesting that unknown respiratory pathogens might be involved. In this study, antibody capture VIDISCA-454 (virus discovery cDNA-AFLP combined with Roche 454 high-throughput sequencing) resulted in the discovery of a novel type of rhinovirus C (RV-C). The virus has an RNA genome of at least 7054 nt and carries the characteristics of rhinovirus C species. The gene encoding viral protein 1, which is used for typing, has only 81% nucleotide sequence identity with the closest known RV-C type, and, therefore, the virus represents the first member of a novel type, named RV-C54. Viral respiratory infections cause serious health problems in human populations. Many agents can infect and replicate in the respiratory tract, however, causative agents for 30 percent of respiratory infections remain unidentified ,2,3, sugTo overcome these limitations in virus discovery, we recently developed a modification of the VIDISCA-454 method by adding an antibody capture step. This method uses host antibodies elicited by an infection, obtained from convalescent serum one month after the respiratory illness. These antibodies are used to enrich the causative viral agent from the respiratory material that was collected during the acute phase of the infection. The advantage of the antibody capture approach is multi-fold: (1) it decreases the background rRNA increasing the relative percentage of virus sequence reads (2) it identifies viruses that induced an immune response in the patient, which increases the chance to find a pathogenic virus; and (3) it allows the identification of new viruses that have very low identity to known viruses . In thisEnterovirus genus within the Picornaviridae family [. Rhinoviruses were first discovered in the 1950s and have been linked to the majority of upper respiratory tract infections in humans. Furthermore, RV-C infection is found in about half of all rhinovirus infections in young children [Picornaviridae Study Group [Rhinoviruses are non-enveloped viruses with a single-stranded positive-sense RNA genome of approximately 7200 nt. Their genome encodes a single open reading frame (ORF) encoding a polyprotein that is cleaved into four structural proteins  and seven non-structural proteins  . VP1-4 fe family  and three family . Rhinovichildren . Despitechildren , human pchildren . Accordichildren  with thedy Group .Mycoplasma pneumoniae, Chlamydophila pneumoniae, Bordetella pertussis, Legionella pneumophila, Streptococcus pneumoniae, and Haemophilus spp.The respiratory sample was collected in 2009 during the GRACE study ,24 from The ethics review committee in Barcelona (Spain) Comitè ètic d'investigació clínica Hospital Clínic de Barcelona approved the study.g) and 150 µL of the supernatant was mixed with 50 µL of a mixture containing Dynabeads protein A, Dynabeads protein G, and BcMag protein L magnetic beads . After 20 min incubation, 10 µL of convalescent serum was added to the mixture. After a subsequent 20 min incubation with continuous shaking at room temperature, samples were washed six times with PBS using a magnetic particle concentrator. Universal transport medium with TURBO™ DNase  was added to the antibody-antigen complex and samples were incubated at 37 °C for 30 min. The complexes were lysed with Boom-lysis buffer L6 [The respiratory sample was centrifuged . Subsequently, nucleic acids were extracted by the Boom extraction method [6 copies/µL, clonally amplified in an emulsion PCR according to the suppliers’ protocol , and sequenced on a GS FLX Titanium PicoTiterPlate (70 × 75) with the GS FLX Titanium XLR 70 Sequencing kit . Adaptor sequences and rRNA sequences were trimmed and removed from the obtained sequence reads.VIDISCA-454 was performed on the sample (input) and after antibody-capture. The pretreatment to enrich for capsid protected nucleic acids in the input was performed as previously described . In shorn method , with eln method . The nucn method . Second Xcompare2 pipeline (source available on request). The Xcompare2 script creates a custom BLAST nucleotide database [To identify sequences enriched by antibody capture, reads obtained from the input sample (input dataset) and from the post capture sample (enriched dataset) were compared to each other using the Python (version 2.7.8) based database  comprisedatabase ,29 with http://blast.ncbI.nlm.nih.gov/Blast.cgi) tool [Sequences were compared with all available sequences in the non-redundant GenBank database  via the gi) tool . The folgi) tool . The folFull length sequencing of the rhinovirus C genome was performed via the VIDISCA procedure for enrichment of particle-protected nucleic acid isolation and conversion of RNA into double stranded DNA (described above), followed by Illumina MiSeq deep sequencing, exactly as described . The comPhylogenetic analyses  were conducted using MEGA, version 6 . A thousNetPicoRNA program  was usedWithin the GRACE study lower respiratory tract infections are examined for known viruses and bacteria . Some ofSince convalescent serum was available, antibody capture VIDISCA-454 was performed  in ordercompare2 pipeline . The script creates and compares two unique fragment libraries for both input and antibody-captured datasets to calculate the Enrichment Indices. Two consensus sequences only found in the captured sequence library showed some identity to rhinovirus C. This enrichment indicates that antibodies from the patient had captured the virus, strengthening the assumption that rhinovirus C was the cause of the lower respiratory tract infection.To identify sequences enriched by antibody capture, reads obtained from the input sample (input dataset) and from the post capture sample (enriched dataset) were compared to each other using the XThe shared nucleotide identity of the VIDISCA sequences with known rhinovirus C sequences was on average 73%. Since only full genome sequences can reveal whether it truly represents a new rhinovirus type or a recombinant virus, the complete genome sequence was determined with the ViSeq method, that uses Illumina MiSeq sequencing and has the capacity to reveal a full-length virus genome sequence within one run . The fulAnalysis of the rhinovirus genome secondary structure by Mfold revealed that the 607 nt long 5' UTR contains a strong secondary structure at position 1 to 87 A, which A comparison of the VP1 encoding region revealed that the new virus shares 81% nucleotide identity with its closest relative RV-C29. Phylogenetic analysis was performed based on the VP1 and VP4/VP2 regions using available sequences in the GenBank and the inferred phylogenic trees are shown in Attempts to culture the virus on differentiated airway epithelial cells (HAE) failed. As the virus was missed in diagnostics, the primers and probe  sequences of the real time PCR for rhinoviruses were examined in more detail. There are two mismatches in one of the primer binding site used for rhinovirus PCR, which probably caused the negative result in the RV diagnostic test .In this study a new type of rhinovirus (RV-C54) was identified in a patient with respiratory symptoms. The virus was discovered using antibody capture VIDISCA-454, a culture- and sequence-independent virus discovery technique applicable to detect both RNA and DNA viruses. We have previously shown that the method facilitates virus identification by enriching viral material with convalescent autologous patient antibodies prior to deep sequencing . The reqin vitro, and RV-C typing, thus, far has been based on phylogenetic analysis with the VP1, VP4/VP2 and the 5' UTR coding region [The full genome sequence of the novel virus was used to classify the virus. RV-C strains are not easily cultivable g region . Accordig region , and 12%g region . Since Rg region .Rhinovirus species is genetic recombination [One of the important causes for diversification of bination . This caIn conclusion, our results highlight the strength of the antibody capture method to identify novel viruses that have elicited an immune response in the host.


Flotation of small solid objects and liquid droplets on water is critical to natural and industrial activities. This paper reports the floating mechanism of liquid marbles, or liquid droplets coated with hydrophobic microparticles. We used X-ray computed tomography (XCT) to acquire cross-sectional images of the floating liquid marble and interface between the different phases. We then analysed the shape of the liquid marble and the angles at the three-phase contact line (TPCL). We found that the small floating liquid marbles follow the mechanism governing the flotation of solid objects in terms of surface tension forces. However, the contact angles formed and deformation of the liquid marble resemble that of a sessile liquid droplet on a thin, elastic solid. For small liquid marbles, the contact angle varies with volume due to the deformability of the interface. It is well known that a small object can float on a liquid medium that has a lower density. This phenomenon is critical to a number of both naturally occurring and man-made processes, from water-walking and meniscus-climbing of insects and spiders1246789Numerous studies have been focusing on systems that involve a solid straddling between two fluids910121415161719202122Determining the apparent contact angle of a sessile liquid marble on a solid substrate can be challenging. The most popular preparation method of liquid marbles is rolling a droplet on a powder bed which coats the droplet with layers of microparticles. These particles may aggregate and give the liquid marble an inhomogeneous surface. In contrast to a sessile liquid droplet, the lack of a clearly defined surface impairs accurate inspection of the edges and the apparent contact angles of a marble. For a floating liquid marble, the situation is even worse as the deformed liquid surface completely obscures the TPCL, making visible light not suitable as illumination source for imaging. We solve this problem by using an X-ray computed tomography (CT) imaging technique to obtain the side view of the floating marble, which was similarly conducted in a previous study of a liquid droplet on a soft solid substrateWe used loose polytetrafluoroethylene (PTFE) powder with a nominal diameter of 1 μm (Sigma-Aldrich®) to form the liquid marble. Distilled water was dispensed onto the PTFE powder bed and rolled around until the droplet was completely covered. A pipette  dispenses the accurate volume of the droplet (with an uncertainty of ±4.3%). The liquid marble was then rolled around on a clean surface to dislodge excess powder from its surface. Next, the liquid marble was gently placed in a clear polystyrene container filled with distilled water using a teaspoon and allowed to float. The container is filled almost to the brim so that the liquid marble can roll off the teaspoon gently onto the water surface without dropping. As the liquid marble is merely coated with a very thin layer of loose PTFE powder, simply dropping the marble onto the water surface could destroy it. Once the marble floated on the surface, water was drawn out from the container using a syringe to reduce the water level and to create a clearance at the top of the container for the lid. The container was sealed with its lid to minimise evaporation throughout the experiment. The container was carefully positioned on the sample stage of the X-ray CT equipment , ρw = 1,000kg/m3 and γaw = 0.072N/m, respectively. PTFE has a density of 2,200 kg/m3. Due to the limitations of the liquid marble preparation and transfer methods, the PTFE packing density and coating thickness cannot be accurately controlled. The inhomogeneity of packing density may induce errors in the effective surface tension of the liquid marble.After positioning the sample, the magnification and X-ray parameters were optimised and the profile image of the floating liquid marble was acquired. The recorded image was subsequently processed to fit its surface profile and to measure the contact angles at the TPCL using ImageJ . All images were exported using the lossless TIFF format with a resolution of 1007 × 1007 pixels. The image acquisition and processing procedures were repeated for marble volumes of 1, 2, 5, 10, 20 and 30 μL. Each liquid marble volume was measured three times. All critical properties are determined at room temperature and atmospheric pressure (25 °C and 1 atm). The density and surface tension of deionized (DI) water are taken as Supplementary Information Figure S1 shows an image of the loose PTFE powder on the liquid marble surface. The image taken with an optical microscope indicates that the porous hydrophobic layer has a large range of particle clusters from a few to more than microns due to the aggregation of the loose particles. The curved and complex bottom surface of the floating marble is not accessible for our current imaging techniques. Figure S1 shows the bottom view of a sessile liquid marble on a glass slide to approximate the submerged layer of the floating liquid marble. The depicted image might not represent the actual coating layer of the floating liquid marble. We hypothesise that the coating thickness is different for the submerged and the unsubmerged portion of the marble. The unsubmerged layer is only exposed to air, so the loose particles could build up forming a thicker layer. In contrast, the submerged layer is sandwiched between the liquids of the marble and the bath making the particles to assemble into a thinner layer. The difference in thickness can be seen in the side view of the raw X-ray image in Supplementary Information Figure S2. The PTFE coating shows up as a fuzzy layer at the edge of the marble due to intensity differences. The free layer is several times thicker than the submerged layer. The difference in thickness of the coating layer could translate into a difference in packing density and consequently different surface tensions. Additionally, the interface between the marble and the liquid bath is heterogeneous, as it contains a mixture of air pockets and PTFE particles which gives rise to a large apparent contact angle26γam, the unsubmerged volume V1, the submerged volume V2 and the contact radius r0 , Fs is the surface tension force. The existence of such surface tension force for an axisymmetric body had been rigorously proven by Keller17In a macroscopic system, the condition of flotation is the balance between the weight Fs. The liquid marble coating causes the liquid marble to behave like a deformable soft solid. The contact line dimensions of the present work are in the millimetre scale. At this scale, effects of line tensions (on the order of 10−10 N)32Consequently, we hypothesise that the vertical component of the air-water surface tension along the TPCL yields a net upward force equal to At the TPCL, the air-water and marble-water interfaces are almost parallel. The air-marble interface branches out and inclines slightly more upward than the air-water interface. This configuration is similar to that of a liquid droplet resting on a thin elastic solidβ. The force balance equation can be written as:As the surface tension of the carrier liquid ρm = ρw, V2 can be eliminated from both sides of the equation. Rewriting V3 in terms of r0 and h leads to a force balance equation with experimentally measurable quantities as:Assuming We use the non-dimensional Bond number Bo as the independent variable for the scaling analysis. The Bond number represents the relative significance of the marble weight to the surface tension force:c is the inverse square of the capillary length, a parameter used for liquid marble surface profile fitting as described in the materials and methods section, r is the radius of the non-deformed spherical liquid marble. Neglecting the weight the powder particle, the density of the marble r2 and V2/3.where α is not constant, because the marble is deformable.The constant contact angle α between a floating solid object and the liquid bath can be determined by straightforward measurements. With the known contact angle α, the meniscus angle β can be determined and a unique solution of the floating position can be found. However, our case of a floating liquid marble is different. The contact angle of a floating liquid marble We start off with the force balance equation: Fw scales with r3, while the surface tension force Fs scales with the contact radius r0 and sin β, therefore r scale as The weight Fs scales with r0sin β, therefore r0 by the non-deformed marble radius leads to the dimensionless contact radius The surface tension force Fs = 2πr0γaw sin β and the residual weight of the marble W = (V − πr02h)ρwg, where γaw, ρw and Fs, is the upward vertical force component that arises from the air-water interface tension. The residual weight W is the excess weight of the liquid marble not balanced by the buoyancy force alone. As depicted in α decreases with increasing Bond number. Unfortunately, the trend is not very clear due to the relatively large errors in the measurement. The shape of the liquid marble asymptotically approaches a perfect sphere with 0 contact radius as Bo approaches 0. Therefore the marble contact angle α approaches 180° with volume and Bond number approaching zero (Bo → 0). The marble contact angle α fits a logarithmic dependence in the form of k1, k2 and k3 are curve fitting constants. Due to the limited amount of data, these constants only serve curve fitting purposes. More experimental data will be required for a functional analysis. The liquid marble contact angle α measured in our case is the apparent effective macroscopic angle. Microscopically, there is no physical contact between the marble liquid and the bath liquid26β as function of Bond number. The data points of meniscus angle β fit a square root function of the Bond number Bo1/2 as predicted with the scaling analysis. Since there is no depression of the water surface with an asymptotic zero-volume marble, the meniscus angle β approaches 0° when the volume or the Bond number approach 0 (Bo → 0).Fs is equal to the residual weight W of the marble regardless of its volume. As predicted from the scaling analysis, the surface tension force Fs scales with Bo3/2.29We investigated the floating mechanism of liquid marbles using X-ray CT microscopy and theoretical analysis of the forces involved. We found that the flotation of small liquid marbles is predominantly affected by surface tension forces. The measurement of the apparent contact angles at the TPCL is not straightforward, due to the heterogeneous interface layer of the liquid marble that affects the effective surface tension. The configuration of the angles at the TPCL is similar to that of a sessile liquid droplet on a thin elastic solid whereby the carrier profile is smooth and continuous. The contact angle of a liquid marble varies with its volume due to the deformable interfaces. We hypothesise that marble contact angle decreases from the asymptotic value of 180 degree in the small Bond number range (Bo < 0.1) and remains almost constant with increasing Bond number. An improved preparation method for liquid marble would allow for future investigation in the sub-microlitre and millilitre scale and more conclusive results. Through scaling laws, we found that for a small, floating marble, the meniscus angle approximately scales with the square root of the Bond number How to cite this article: Ooi, C. H. et al. Floating mechanism of a small liquid marble. Sci. Rep.6, 21777; doi: 10.1038/srep21777 (2016).


Filoviridae family of negative-sense RNA viruses. Since its identification in 1976, sporadic outbreaks have occurred in Central Africa. The 2014 outbreak in West Africa provides evidence that EBOV is emerging into new geographic regions. While previous outbreaks have been confined to small areas, the most recent outbreak is atypically widespread with over 25,000 people infected. Evidence from the limited-sequence studies that have been published suggests that the outbreak resulted from a single reservoir-to-human transmission event and subsequent human-to-human spread [Ebola virus (EBOV) belongs to the Infection is initiated by virions entering dendritic cells, macrophages, and, perhaps, hepatocytes . Virus rThe uniquely shaped filamentous particles made by filoviruses are surrounded by an envelope acquired during virion budding from the plasma membrane . Recent The cell surface interactions of filoviruses differ from other characterized enveloped virus/cell surface receptor interactions in that amino acid residues of EBOV GP are not thought to interact with a cell surface receptor. Instead, these viruses bind to target cells through two types of relatively non-specific receptors: C-type lectins (CLECs) that interact with glycans on EBOV GP and PtdSer receptors that interact with the viral envelope PtdSer . CLECs  is a lysosomal receptor for Lassa virus, suggesting this endosomal mechanism of fusion control may be broader than previously appreciated .Despite the progress over the past five years in understanding EBOV entry, many critical questions remain unanswered. For instance, how does PtdSer become enriched on the outer leaflet of enveloped virus membranes? Do virion interactions with CLECs, TIM proteins, or TAM/Gas6 complexes mediate direct internalization of virions into endosomes? If TIM proteins do directly mediate virus internalization, mechanistically, how is this accomplished since some TIM molecules are not thought to signal? Alternatively, do these receptors solely accumulate virions on the surface of cells? If so, is there a yet unidentified cell-surface receptor that is required for EBOV internalization? If PtdSer receptors do directly mediate EBOV internalization, do the different receptors mediate internalization through the same endosomal pathway and into the same endosomal compartment? For those filoviruses that do not require Cathepsin B and L, what endosomal proteases are responsible for their GP processing? What is the role of NPC1? Does NPC1 binding directly lead to EBOV fusion? If so, why are additional proteolysis events required and what protease(s) in which vesicular compartment mediate this second processing step? Finally, in terms of the big picture, what selective advantages are there for enveloped viruses such as filoviruses to use relatively non-specific, low-affinity mechanisms for internalization? Certainly, a growing body of evidence suggests that there must be advantages. These might include a breadth of tropism that would otherwise not be available to enveloped viruses. Further, using these entry mechanisms protects critical RBD and fusion residues from neutralizing antibodies by limiting extracellular exposure of these GP elements; in the current entry model for filoviruses, these sequences are solely exposed late within the endosomal/lysosomal compartments. A better understanding of these paradigm-shifting findings could provide additional drug targets to complement the current repertoire of Ebola virus antivirals in development. No doubt, the continued pursuit of answers to these questions by a number of groups will provide significant insights into both EBOV and, more broadly, virus biology.


Bunyaviridae family is comprised of more than 350 viruses, of which many within the Hantavirus, Orthobunyavirus, Nairovirus, Tospovirus, and Phlebovirus genera are significant human or agricultural pathogens. The viruses within the Orthobunyavirus, Nairovirus, and Phlebovirus genera are transmitted by hematophagous arthropods, such as mosquitoes, midges, flies, and ticks, and their associated arthropods not only serve as vectors but also as virus reservoirs in many cases. This review presents an overview of several important emerging or re-emerging bunyaviruses and describes what is known about bunyavirus-vector interactions based on epidemiological, ultrastructural, and genetic studies of members of this virus family.The  Bunyaviridae is the largest RNA virus family, comprised of more than 350 viruses divided into five genera based on serological, morphological, and biochemical characteristics: Orthobunyavirus, Nairovirus, Phlebovirus, Hantavirus, and Tospovirus with additional unclassified viruses  reassortants in mosquitoes, primarily  mutants ,152. Beaseriatus ,153. They glands , which i of SSHV . A later of SSHV . Cheng e and JCV . For LAC55% TAHV . The sig55% TAHV .Bunyaviridae family as a whole is considered, should be considered a priority for those pathogens detailed in this review and additional emerging viruses as necessary.Research on bunyaviruses has contributed a wealth of basic information about vector-virus interactions to the field of arbovirology, although there are still many areas for which sufficient data has yet to be collected. It is clear that bunyaviruses continue to pose a significant threat to human and agricultural public health. Promising areas for additional research on the viruses covered in this review and other medically important bunyaviruses, such as Oropouche virus, include further characterization of the genetic determinants of virus infection, dissemination and transmission by insect and tick vectors and mechanisms for blocking viral infection or transmission by these vectors. Because many bunyaviruses use the vector as a mode of transmission as well as a reservoir, elimination of many of these viruses will be impossible. Therefore, the development of new or additional human and agricultural vaccines to mitigate disease incidence in endemic areas, which include much of the globe when the


Although viral infection and antiviral defence are ubiquitous, genetic data are currently unavailable from the vast majority of animal phyla — potentially biasing our overall perspective of the coevolutionary process. Rapid adaptive evolution is seen in some insect antiviral genes, consistent with invertebrate-virus ‘arms-race’ coevolution, but equivalent signatures of selection are hard to detect in viruses. We find that, despite the large differences in vertebrate, invertebrate, and plant immune responses, comparison of viral evolution fails to identify any difference among these hosts in the impact of positive selection. The best evidence for invertebrate-virus coevolution is currently provided by large-effect polymorphisms for host resistance and/or viral evasion, as these often appear to have arisen and spread recently, and can be favoured by virus-mediated selection. Current Opinion in Virology 2014, 8:73–78Virus evolutionThis review comes from a themed issue on Michael Brockhurst and Scott HensleyEdited by Issue and the EditorialFor a complete overview see the Available online 24th July 2014http://dx.doi.org/10.1016/j.coviro.2014.07.002http://creativecommons.org/licenses/by/3.0/).1879-6257/© 2014 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY license . Comparison of such ‘footprints of selection’ between immune genes and genes with other functions argues in favour of pathogen-mediated selection in arthropods generally [Drosophila [D. melanogaster and D. simulans, with adaptive amino-acid fixations in this pathway estimated to happen every 10–40 thousand years [Drosophila lineages, while homologous ‘housekeeping’ genes do not show this pattern [Selection by viruses could drive frequent and rapid fixations in invertebrate populations, reducing genetic diversity at the selected loci and elevating divergence between species. Selection on amino-acid sequences, which may be common for antagonistic host–virus interaction, could additionally elevate the rate of non-synonymous substitution P conveys resistance to the vertically-transmitted Drosophila melanogaster Sigma Virus (DMelSV), with the resistant allele reducing viral transmission by ∼90% in females and ∼60% in males [Doc transposable element insertion into CHKov1 followed by a partial duplication and inversion involving CHKov1 and CHKov2. The Doc insertion exists at high frequency (80% in a North American population) and reduces infection rates by ∼50%. The subsequent rearrangement gave rise to a virus-inducible CHKov2 transcript associated with an 80–140 fold decrease in viral titre [pastrel  and Anaphase promoting complex 7  or incomplete/ongoing selective sweeps . A largein males . The resin males , 25. A sal titre . Again, al titre , 27. Resfication ••), althfication ••. Finalhallenge ••.Caenorhabditis elegans maps to a non-functional truncation of Drh-1, one of three dicer-related helicases involved in RNAi [Dicer-2) has also been proposed to underlie some of the genetic variance for resistance to Dengue virus in the mosquito Aedes aegypti [Cydia pomonella have recently evolved resistance to its Granulosis virus, via a single dominant sex-linked allele that blocks viral replication [Penaeus monodon has been mapped to single marker associated with a ∼2000-fold reduction in viral titre [High-frequency large-effect viral resistance polymorphisms have also been reported from other invertebrates. For example, segregating resistance to the Orsay Virus in the nematode  in RNAi •. Here t in RNAi •. Polymo aegypti . In othelication , 33. Simal titre , which oal titre .Drosophila and can select for segregating variants such as pastrel [ref(2)P [Such polymorphisms are consistent with negative frequency dependent selection or with incomplete/ongoing selective sweeps ••, but b pastrel •• and re[ref(2)P .It seems certain that viral evolution occurs in response to invertebrates, if only because hosts always dominate the viral environment. For example, viral adaptation may underlie host-specificity seen in some insect viruses , 37, 38,Aedes aegypti mosquitoes [ref(2)P and DMelSV [ref(2)P resistance arose a few hundred years ago and subsequently spread to become the most common form [ref(2)P-insensitive virus can replace the sensitive virus in a resistant ref(2)P host background — indicating that host resistance may indeed drive viral evolution [D. obscura [Given this, it is interesting to ask whether virus evolution occurs in response to specific host immune mechanisms. Genotype by genotype interactions — with host polymorphism for resistance and viral polymorphism for overcoming that resistance — may be indicative of negative frequency-dependent selection or incomplete on-going selective sweeps in the virus, driven by selection mediated by host resistance. For example, genotype by genotype interactions have been reported between Dengue Virus 1 and squitoes , 44. Thed DMelSV , 45, whemon form , 47. Themon form , 49, andvolution . The rap obscura . However obscura , and candN/dS) in the virus [dN/dS is lower in vector-borne viruses [dN/dS = 0.07 vs 0.17 for vertebrates, 0.10 vs 0.19 for plants; see It is often argued that if host resistance drives the recurrent appearance of novel viral protein variants, then this may elevate the ratio of non-synonymous to synonymous variants  is reduced; first, because fewer viruses tested ‘positive’ for adaptive evolution [dN/dS between vectored and non-vectored viruses was reduced when putatively positively selected sites were excluded [dN detectably above dS. dN–dS value or the number of positively selected codons. However, while invertebrate viruses are not strikingly different from the others, the extremely small sample size (n = 4) precludes any firm conclusions regarding patterns of viral protein evolution in invertebrate hosts.It was suggested that vector-borne vertebrate viruses may display reduced volution , and secexcluded , 66. TakdN > dS test, and the number does not differ significantly between these groups P-DMelSV system, in which resistance and the ability to overcome it have both arisen recently and increased in frequency, and each is known to be selectable by the other [Given the difficulty associated with inferring invertebrate-virus coevolution from historic patterns of protein evolution, the best evidence instead comes from patterns of functional polymorphism. Although the most compelling case is arguably the he other , such lahe other  and somehe other , where lhe other , and sug• of special interest•• of outstanding interestPapers of particular interest, published within the period of review, have been highlighted as:


Here, we report the complete genome sequence of an H6N6 avian influenza virus (AIV) isolated from Sansui Sheldrake ducks in Guizhou Province, China, in 2014. Phylogenetic analysis showed that the H6N6 virus was a reassortant virus derived from three different H6 subtype lineages. The finding of this study will help us understand the epidemiology and the evolutionary characteristics of H6 subtypes of AIV in ducks in southwestern China. Waterfowls are known as a giant genetic reservoir for avian influenza virus (AIV), and to date, all 16 hemagglutinin (HA) and 9 neuraminidase (NA) subtypes of AIV have been isolated from waterfowl species , 3. ReceIn this study, an H6N6 subtype AIV strain, A/duck/Guizhou/013/2014 (H6N6), was isolated from apparently healthy Sansui Sheldrake ducks in Guizhou Province, China, in 2014. All eight gene segments of the H6N6 AIV were sequenced by DNA direct sequencing. Editing and analysis of the sequence data were performed with Lasergene 7.1 and MEGA 6.0.The full lengths of the polymerase basic 2 (PB2), PB1, polymerase acidic (PA), HA, nucleoprotein (NP), NA, matrix (M), and nonstructural (NS) genes were 2,341, 2,341, 2,233, 1,744, 1,565, 1,465, 1,027, and 890 nucleotides, respectively. Phylogenetic analysis showed that the nucleotide sequence identities of the HA and NA genes with that of the H6N6 isolates A/duck/Jiangsu/022/2009 (H6N6) and A/duck/Fujian/8089/2007 (H6N6) were 97% and 98%, respectively. The PB2 and PB1 genes were most closely similar to the foreign isolates A/duck/Yamagata/06/2014 (H6N6) and A/duck/Vietnam/LBM6/2011 (H6N6), with which they share 97% and 98% nucleotide homology, respectively. The other four genes were found to be more similar to those of southern China H6N6 strains (up to 97% to 99% nucleotide homology). In addition, the phylogenetic trees revealed that all eight gene segments of this isolate belonged to the Eurasian avian lineage, but the HA and PB2 genes belonged to the sequence type 2853 (ST2853)-like and HN573-like lineages, respectively, whereas the other genes belonged to ST339-like lineage. These results indicate that the H6N6 virus was a reassortant virus resulting from three different H6 subtype lineages.Amino acid sequence analysis showed that the cleavage site motif between HA1 and HA2 was PQIETR↓G; this is a typical characteristic of low-pathogenicity AIV . SimilarKU762356 to KU762363.The complete genome sequences of A/duck/Guizhou/013/2014 (H6N6) have been deposited in GenBank under the accession numbers


Gastrointestinal and respiratory diseases are major causes of morbidity for young children, particularly for those children attending child day care centers (DCCs). Although both diseases are presumed to cause considerable societal costs for care and treatment of illness, the extent of these costs, and the difference of these costs between children that do and do not attend such centers, is largely unknown.Estimate the societal costs for care and treatment of episodes of gastroenteritis (GE) and influenza-like illness (ILI) experienced by Dutch children that attend a DCC, compared to children that do not attend a DCC.A web-based monthly survey was conducted among households with children aged 0–48 months from October 2012 to October 2013. Households filled-in a questionnaire on the incidence of GE and ILI episodes experienced by their child during the past 4 weeks, on the costs related to care and treatment of these episodes, and on DCC arrangements. Costs and incidence were adjusted for socioeconomic characteristics including education level, nationality and monthly income of parents, number of children in the household, gender and age of the child and month of survey conduct.Children attending a DCC experienced higher rates of GE (aIRR 1.4 [95%CI: 1.2–1.9]) and ILI (aIRR: 1.4 [95%CI: 1.2–1.6]) compared to children not attending a DCC. The societal costs for care and treatment of an episode of GE and ILI experienced by a DCC-attending child were estimated at €215.45 [€115.69–€315.02] and €196.32 [€161.58–€232.74] respectively, twice as high as for a non-DCC-attending child. The DCC-attributable economic burden of GE and ILI for the Netherlands was estimated at €25 million and €72 million per year.Although children attending a DCC experience only slightly higher rates of GE and ILI compared to children not attending a DCC, the costs involved per episode are substantially higher. Over the last decades, the number of children attending day care centers (DCCs) has increased all over the world The shift from home-care to out-of-home care has had a significant impact on the attending child's health. Indeed, numerous studies convincingly demonstrate that children experience a 2–3 times increased risk of mild/moderate In the Netherlands, both the extent of the societal costs per episode of gastrointestinal and respiratory disease experienced by young children, and whether these costs differ between children that do and do not attend a DCC, are largely unknown. Estimates of these costs, and the role of day care therein, would aid epidemiologists and health economists to assess the desirability and feasibility of intensified avocation of preventive measures in day care from a financial in addition to a clinical point of view. The aim of this study was therefore to (1); estimate the societal costs for care and treatment of a child 0–4 years old experiencing an episode of gastroenteritis (GE) and influenza-like-illness (ILI). (2); compare these costs between children that do and do not attend center-based day care.The Dutch Central Committee on Research involving Human Subjects in Utrecht, The Netherlands, gave permission to conduct this study (protocol number: 13-051/C). Given that no subject-identifiable data were generated and the surveillance activities implied no risk or burden for any individuals, the committee judged that no specific ethical permission was required for individual consent. This study is conducted according to the principles of the Declaration of Helsinki.This study is part of a larger web-based, cross-sectional survey on the contribution of young children in transmission of infectious diseases to their families and vice versa Predefined respiratory symptoms and syndromes in the questionnaire included fever (sudden onset of fever (≥38°C) and/or warm to the touch with suspicion of fever), cough (sudden and frequent occurring tussis), rhinitis, sore throat, frontal headache, retrosternal pain and myalgia. Predefined gastrointestinal symptoms and syndromes included diarrhea , vomiting , abdominal pain, abdominal cramps, nausea, blood in the stool, mucus in the stool. Predefined symptoms and syndromes were used to post-define the case definitions of GE and ILI. Consequently, GE was defined as three or more loose stools per day or diarrhea during at least one day accompanied by ≥2 additional symptoms, or vomiting with ≥2 additional symptoms during the past four weeks In addition, the questionnaire included inquiries about the parent's about utilized health care resources and absenteeism from work for care and treatment of illness experienced by their child. In addition, parents were asked to provide information on the demographic and socioeconomic characteristics of their household, including household income, number of members, work status of parents, DCC arrangements and hygiene habits. Each question required an answer before a parent could proceed to the next question. The questionnaire was sent to us only upon completion of the last question.Per GE and ILI episode, we considered three types of costs related to the use of resources for care and treatment of illness: (1) Direct health care costs (DHC); costs related to doctor consultation, (over-the-counter) medication, hospitalization and laboratory testing. (2) Direct non-health care costs (DNHC); costs related to traveling to and from health care services. (3) Indirect non-health care costs (INHC); costs related to productivity losses due to absence from work of parents to care for their ill child. The costs per resource unit per disease episode were calculated as the number of resource units utilized  multiplied by the costs per resource unit. Together, the three types of costs determined the societal costs of an illness episode The following assumptions were made for travelling costs and productivity losses due to absenteeism from paid work based on previous published work The average yearly incidence rate of GE and ILI was calculated as (365/30) x (average four-weekly incidence rate) x 1000 children, since the period of observation was 1 month and we want to express the incidence rate per 1000 child-years. The child-time at risk, the incidence denominator, was defined as the number of participating households per month.2) and “rural” (0–1500 addresses/km2). The education level was ‘high’ if one or both parents had a university diploma, and low otherwise. The number of additional children was categorized as 0, 1,and >1. Children were considered “Dutch” if both parents were born in the Netherlands), “Other Western” if one of the parents was born in a European country other than the Netherlands, and “Other” in all other instances. The age of the child was expressed as above or equal to/below 24 months of age.The costs due to illness represent a semi-continuous outcome. They are characterized by a point mass at zero (representing illness episodes for which no costs were made), followed by a right-skewed continuous distribution of positive values (representing illness episodes for which costs were made). We therefore applied a two-part regression model consisting of a logistic and a log-linear component using the Stata module tpm for cross-sectional models which are commonly used in cost of illness studies The mean estimated adjusted costs per GE and ILI episode were estimated only for the total societal costs. For these costs, bootstrap analyses with replacement (1000 simulations per run) were conducted to estimate the sampling distributions of estimated adjusted mean costs, providing 95% confidence intervals around cost estimates. For the separate resource unit costs and the DHC, DNHC and INHC subtotals, the mean crude costs were provided. These were calculated by multiplying the mean frequency of resource usage per episode times the costs per resource unit. Separate analyses were performed for households with and without DCC-attending children. The annual costs for the Dutch community were estimated as the mean adjusted costs per illness episode times the excess day-care associated incidence per 1000 child-years times the population size of children aged 0–4 year old in the Netherlands attending DCCs. We used STATA/SE 12.0 StataCorp LP, USA) for all descriptive and multivariate analysis.We applied inverse probability or survey weighting to correct both the disease incidence and cost estimates per episode of GE and ILI for underreporting of households based on their socioeconomic status, urbanization degree and age of the child. This allowed us to extrapolate the incidence and costs estimates to the Dutch community in 2012. Socioeconomic characteristics of the household were derived from the population registries from which households were selected, dichotomized and consequently assigned to each participating households. Inverse probability weighting is widely used in household surveys to correct for the potential biasing impact of nonresponse.Of the 24.000 households approached during the study period, 4727 responded to the survey and 3927 met the eligibility criteria . CompareIn 2012, there were approximately 700.000 children aged 0–48 months eligible to attend center-care in the Netherlands. Of these children, 324.800 (46.4%) were registered for DCC attendance. Using the adjusted excess disease incidence associated with DCC attendance, the adjusted mean costs per disease episode, and the number of children registered for DCC services, we estimated the additional costs related to care and treatment of GE and ILI episodes standardized for the Dutch community in 2012. The additional community costs in 2012 for GE and ILI episodes related to DCC attendance were estimated at €25 million and €72 million, respectively.Using a unique general-population-based approach, our study has quantified the (differences in) GE and ILI incidence and related health care costs and productivity losses among children that do and do not attend a DCC in the Netherlands during October 2012–October 2013. Compared to home-cared children, DCC-attending children experience a slightly higher incidence of gastroenteritis (GE) and influenza-like illness (ILI). Yet the societal costs for care and treatment of an episode of GE and ILI are substantially higher for children attending DCCs. The cost differences are predominantly caused by the higher productivity losses experienced by parents with DCC-attending children for care and treatment of their ill child. Our results suggest that a small reduction in the excess number of disease episodes associated with DCC attendance could lead to a substantial reduction in the societal costs these episodes incur.The GE incidence rates presented in this study are comparable with findings from a Dutch population-based cohort study on the incidence of GE in the Netherlands performed in 1998 Some general remarks are in place to put the estimates presented in this paper in perspective. (1) Although all incidence and cost estimates were corrected for non-response and were adjusted for several socioeconomic factors, the low response rate coupled with a slightly higher participation rate among children from households with a higher socioeconomic background and Dutch nationality might limit the generalizability of our findings. (2) Rates of opting-out, if any, might have differed between households with and without DCC-attending children for those questions that households refused or considered irrelevant to answer - but needed to answer - before being able to proceed to the next question. (3) The societal costs were calculated based on standardized unit costs rather than actual costs, reasoning that doing so would increase the likelihood of parents filling-in the complete questionnaire. This approach naturally assumes that identical costs apply for households of which the child does or does not attend a DCC, when in fact these costs may differ. For example, attending a DCC might translate into episodes of GE and ILI that are not only greater in number, but also in severity Concluding, this study provides indications for the overall economic scale of the community burden of GE and ILI associated with day care attendance in the Netherlands. Its main message is not that DCCs are costly and that we should refrain from sending children to day care. Rather, our results suggest that lowering the disease incidence of major childhood diseases in the DCC setting could potentially lead not only to a reduction of the DCC-related clinical impact, but also to considerable societal cost savings. As such, the incidence and cost estimates presented here are of concern not only to parents, employers and health insurers, but also to public health officials and DCC organizations involved in setting priorities and allocating resources for infection prevention in DCCs.


Aristotelia chilensis. We propose to name the endogenous sequence as Aristotelia chilensis Virus 1 (AcV1). High-throughput sequencing of the genome of this tree uncovered an endogenous viral element, with a size of 7122 bp, corresponding to the complete genome of AcV1. Its sequence contains three open reading frames (ORFs): ORFs 1 and 2 shares 66%–73% amino acid similarity with members of the Caulimoviridae virus family, especially the Petunia vein clearing virus (PVCV), Petuvirus genus. ORF1 encodes a movement protein (MP); ORF2 a Reverse Transcriptase (RT) and a Ribonuclease H (RNase H) domain; and ORF3 showed no amino acid sequence similarity with any other known virus proteins. Analogous to other known endogenous pararetrovirus sequences (EPRVs), AcV1 is integrated in the genome of Maqui Berry and showed low viral transcriptional activity, which was detected by deep sequencing technology (DNA and RNA-seq). Phylogenetic analysis of AcV1 and other pararetroviruses revealed a closer resemblance with Petuvirus. Overall, our data suggests that AcV1 could be a new member of Caulimoviridae family, genus Petuvirus, and the first evidence of this kind of virus in a fruit plant.Here, we report the genome sequence and evidence for transcriptional activity of a virus-like element in the native Chilean berry tree  Similar to all eukaryotic organisms, plant genomes commonly have inserted mobile genetic elements and viruses . New higAristotelia chilensis [Molina] Stuntz) is an endemic Chilean tree. It has a high economic value due to the characteristics of its fruits, which possess a high antioxidant capacity [i.e., strawberries, blueberries and grapes [in vitro and protects human endothelial cells against oxidative stress [Maqui . Comparative genomics revealed a strong phylogenetic relationship of AcV1 with the Caulimoviridae family, genus Petuvirus. Viruses from this genus commonly infect plants and replicate by reverse transcription (RT) [Petunia vein clearing virus (PVCV), which is able to integrate within the host’s Petunia genome, and is classified as an endogenous pararetrovirus (EPRV). It affects plants, causing a variety of different phenotypes, like chlorotic tissues, leaf malformation, vein-clearing symptoms and other affections [We used next generation sequencing (NGS) to obtain the first draft genome sequence of Maqui berry. This initiative allowed us to uncover for the first time the presence of a new virus inserted in the Maqui genome, named here as: ion (RT) . The unifections . BesidesFinally, we decided to perform RNA-seq assays in order to obtain the overall transcriptional activity of the virus in different plant tissues. Our results showed that AcV1 genes have a low transcriptional activity, which might be associated with the absence of an infective phenotype. This study constitutes the first report evidencing the presence of a virus in Maqui berry and an important step towards the molecular characterization of mobile genetic elements in this native Chilean tree.A. chilensis (sample M152). Data analysis of assembled contigs allowed us to identify the initial signs of viral sequences integrated within the genome. Based on sequence similarities searches using BLASTn against the NCBI nucleotide NT database, we identified a contig sequence with high similarity with viral sequences. This contig contains a length of 12,109 nucleotides (nt), a GC content of 38.04% and is composed of 12,032 reads. This initial similarity analysis showed top 10 BLASTn hits with complete pararetrovirus and EPRV sequences , suggesting the presence of a complete virus inserted in Maqui genome, named here as Aristotelia chilensis Virus 1 (AcV1), containing a full size estimated in 7122 bp. In order to check if AcV1 was present in a single copy in Maqui berry, we mapped the complete viral nucleotide sequence against all contigs and scaffolds from Maqui genome. It confirmed that the virus was present once in the genome (query coverage ≥ 50%).Next generation sequencing approaches were used to study the genome of the native Chilean fruit tree Caulimoviruses [Badnaviruses [Tungrovirus [Open reading frame (ORF) predictions using the software Artemis  and BLASoviruses , Badnaviaviruses  and Tunggrovirus .In order to predict the putative functions of the virus ORFs, we used their translated amino acid sequences to perform BLASTp searches. This analysis indicated that ORF1 encodes a Movement protein, whereas ORF2 corresponds to a Reverse transcriptase (RT). On the contrary, ORF3 showed no amino acid sequence similarity with any known virus protein sequence available in the NCBI database (unknown protein). Besides its absence in public databases, together with ORF1 and ORF2, the ORF3 is part of the virus due to its localization between key features available in viral elements: the tRNA binding site in one side, and the poly(A) signal on the other one. Therefore, we conclude that ORF3 is potentially exclusive of AcV1.e-value = 8e-106) [Citrus endogenous pararetrovirus (CitPRV)  [Figwort mosaic virus (FMV)  [Caulimoviridae family’s movement proteins (plant pararetrovirus) [and Carnation etched ring virus (CERV) respectively [Caulimoviridae family and several other virus groups [Our alignment searches revealed that ORF1 has high similarity with the movement proteins of PVCV ( 8e-106) , followe= 4e-94)  and Figw= 5e-06) . A deepe= 5e-06)  showed trovirus) . ORF1 alectively   [e-value = 1e-149) [e-value = 2e-90) [e-value = 8e-83) [e-value = 2e-86) [Caulimoviridae pararetrovirus family and selected retransposon are shown in The same approach was applied to ORF2, in which BLASTp alignments showed higher scores with RT proteins from PVCV  1e-173) , CitPRV  1e-149) , CaMV (e= 2e-90) , CERV (e= 8e-83)  and FMV 3 , CitPRIn addition, ORF2 showed a conserved RNase H domain according to BLASTp analysis. Multiple sequence alignments of this domain showed a 64% identity with RNase H protein from PVCV and 27% from Ty3 retrotransposon . RNase HFlorendoviruses based on the Family of different host plants [S. cerevisiae , 7161 bp long [Petuvirus PVCV, 7205 bp long. Based on previous reports, AcV1 could be classified as a small plant virus [Using a bioinformatics approach we identified that AcV1 is a single-copy endogenous pararetrovirus inserted in Maqui berry genome. The full length of AcV1 was estimated in 7122 bp, similar to other pararetroviruses from the e family A. We fou bp long  and the nt virus ,15,31.A. chilensis. Furthermore, we found a short non-coding sequence, located 80 bp upstream of the ORF1, that has complementarity with the first 14 nucleotides of the 3' strand methionine tRNA, which was previously described in plants  and a GAGA motif  located in the antisense strand, downstream to ORF3. This promoter structure is similar to the downstream promoter sequence (dps) of RTBV, which is known to contribute with its transcriptional activity. The efficacy of this promoter in RTBV is associated with the genomic localization of the GAGA motif, which might be located immediately downstream to the TATA-box . Also, wCaulimoviridae family  was performed  and leaf.ublished ,7,8. HowA. chilensis, the absence of symptoms described to date makes it difficult to identify its real effects in the plant.A total of 43 million high quality reads from all libraries were mapped against the virus genomic region. Of these, 21 mapped to ORF1 (movement protein), 23 to ORF2 (reverse transcriptase), 11 to ORF3 (unknown protein) C. As obsA. chilensis Virus 1 as an endogenous pararetrovirus, part of the Caulimovirus family, and specially related with PVCV from Petunia hybrid [Citrus citrange [Petuvirus. However, additional genome sequences of other viruses and further experimental assays are necessary in order to support this hypothesis.We describe here the first virus inserted in the genome of the Chilean plant Maqui berry, AcV1, using deep sequencing and bioinformatics approaches. We provided evidence of its phylogenetic relationships, genomic structure and the first support of a potential expression activity. Our data, together with the known characteristics of PRV and EPRV viruses, are important evidence that support the classification of a hybrid  and CitPcitrange . This reBanana Strike Virus (eBSV) [Tobacco Vein Clearing Virus (eTVCV) [Petunia vein-clearing virus (ePVCV), among others [Caulimoviridae family, and its presence is also strongly related to plants evolution [endogenous Aristothelia chilensis Virus 1) and classified as a Petuvirus. However, Southern blotting assays and transmission experiments using isolated full-length AcV1 clones could give a definitive support to classify AcV1 as a pararetrovirus.AcV1 sequences were identified inserted in the Maqui berry genome, suggesting an integration event, available in an episomal single copy identified in the contig sequence. These integration events were previously described for endogenous pararetroviruses, such as s (eBSV) , Tobacco (eTVCV)  and the g others ,39,40. Bg others ,42, thatvolution ,39. Base. This small expression in EPRV viruses has been shown associated with an epigenetic control regulated by DNA methylation of integrated EPRV genome regions in Solanaceae healthy plants [The observed AcV1 transcriptional activity (low expression) is an additional signal of the viral presence and activity. The evidenced small transcriptional activity can be due to the fact that the sequenced Maqui plant was asymptomatic under normal growth conditions, and should be verified by PCR/qPCR or Northern blot assays. The low expression level of viral transcripts is probably associated with the absence of symptomatic phenotypey plants . Its de-y plants . Bioinfoy plants .P. hybrida and A. chilensis, were their characteristic dark purple colors, directly associated to anthocyanin production [This is the first described virus sequence and EPRV inserted in Maqui berry Genome. Until now, the only described similarities between these hosts plants, oduction ,43, and oduction ,38. AddiAristotelia chilensis virus 1 (AcV1) (GenBank accession number KJ094314) was identified from the recently sequenced genome of Maqui (Aristotelia chilensis) sample M152, plant #6 [de novo assembly of the draft genome was performed using the MIRA assembler tool [The plant #6 , which wplant #6 . A totaller tool . Viral sler tool . Caulimoviridae family and Ty3 retrotransposon were performed using multiple alignments tool available in CLUSTAL OMEGA [Caulimoviridae family that we used were: CaMV, PVCV, CsVMV, DMV, DMV-10-DR, DMV-10-NZ, CERV, FMV, CitPRV Strawberry vein banding virus (SVBV), Blueberry red ringspot virus (BRRV), Peanut chlorotic streak virus (PCISV), Soybean chlorotic mottle virus (SoyCMV), Commelia yellow mottle virus (CoyMV), RTBV, Sugarcane bacilliform virus (SCBV), CSSV and the retrotransposon Ty3 from S. cerevisiae.For the annotation process, all viral contigs were used as input for the Artemis tool . All ideAL OMEGA . The visAL OMEGA . The repThe VISTA  comparisCaulimoviridae family and Ty3 retrotransposon. This approach was constructed using the Maximum Likelihood method [A phylogenetic tree was built with the multiple alignment of RT under bootstrap analysis (1000 replicates) , using Md method . The lend method . ® Reagent . RNA quality was determined by the quotient of the 28S to 18S ribosomal RNA electropherogram peak using a Bioanalyzer Chip RNA 7500 series II . Four libraries were prepared using the TruSeq RNA Sample Preparation Kit v2 (Illumina) according to the manufacturer’s instructions. First, magnetic beads containing poly-T molecules were used to isolate poly(A) mRNA from 1 μg of total RNA. Then, samples were fragmented by fragmentation buffer and reverse transcribed into cDNA with a PrimeScript™ 1st Strand cDNA Synthesis Kit . Next, end repair and A-base tailing was purified using the QIAquick PCR Purification Kit . Finally, Illumina adapters were ligated to the cDNA fragments. Each library had an insert size of 300 bp sequenced in paired-end reads of 150 bp using a MiSeq Desktop Sequencer.Total RNA from Maqui berry samples was isolated using TRIzolRaw reads were used to identify expression levels of all three genes associated to AcV1. In order to align raw reads against our virus genome, Bowtie2  was empl