Metschnikowia bicuspidata) capable of infecting a number of host species. Costs were quantified as reductions in host lifespan, total reproduction, and mean clutch size as a function of pathogen exposure (resistance cost) or infection (infection cost). We provide empirical support for infection costs and modest support for resistance costs for five Daphnia host species. Specifically, only one host species examined incurred a significant cost of resistance. This species was the least susceptible to infection, suggesting the possibility that host susceptibility to infection is associated with the detectability and size of resistance cost. Host age at the time of pathogen exposure did not influence the magnitude of resistance or infection cost. Lastly, resistant hosts had fitness values intermediate between unexposed control hosts and infected hosts. Although not statistically significant, this could suggest that pathogen exposure does come at some marginal cost. Taken together, our findings suggest that infection is costly, resistance costs may simply be difficult to detect, and the magnitude of resistance cost may vary among host species as a result of host life history or susceptibility.Pathogen infection is typically costly to hosts, resulting in reduced fitness. However, pathogen exposure may also come at a cost even if the host does not become infected. These fitness reductions, referred to as “resistance costs”, are inducible physiological costs expressed as a result of a trade‐off between resistance to a pathogen and aspects of host fitness . Here, we examine resistance and infection costs of a generalist fungal pathogen ( Daphnia magna) parasitized by a bacterial pathogen  species for the presence of resistance and infection costs to a virulent fungal pathogen (Metschnikowia bicuspidata). Resistance and infection costs were measured in terms of three host fitness measures: total reproductive output, mean clutch size, and lifespan. We found a statistically significant resistance cost  in only one host species, D. pulicaria, which is the least susceptible host species. Second, we found nearly universal costs of infection. However, there were no significant differences between exposed‐uninfected and infected host individuals. Taken together, we found limited support for significant costs of resistance, but qualitative evidence that exposed‐uninfected hosts had fitness values intermediate between infected and control hosts, suggesting that pathogen exposure can reduce host fitness, although the effects may be marginal. These nuanced costs of resistance, while not statistically significant when comparing control to exposed‐uninfected hosts, add an interesting dimension, and a potential avenue for quantifying resistance costs. Specifically, the relative difference between exposed‐uninfected hosts and both control and infected hosts contains information on the cost of resisting or tolerating a pathogen infection.Here, we addressed the impact of pathogen exposure and infection on host fitness using a generalist microparasite of Daphnia species  were maintained in experimental media best suited for host survival , except for D. dentifera, which were maintained in dilute pondwater (50%). Host species clones were laboratory‐reared for many generations before this experiment, but were originally cultured from a small pond in Victoria Bryant State Park (D. ambigua), a Michigan Lake , Ellenton Bay , a small pond in Northern IL (D. mendotae), and Oneida Lake . All host cultures were fed 50 μL of a 2 g/L suspension  of pulverized blue‐green algae (Spirulina sp.), and kept on the laboratory benchtop under constant overhead lighting. Previous exposure of host clones to M. bicuspidata could potentially alter the expression of resistance or infection costs, but the data on previous pathogen exposure were not available for the clones studied here. However, laboratory clones were raised under laboratory conditions for more than 20 generations before their first pathogen exposure, which reduces the possibility of potential legacy effects of pathogen exposure.Monoclonal lines of five M. bicuspidata) was originally isolated from D. dentifera in Michigan lakes (provided by Meghan Duffy). The pathogen was cultured in vivo by exposing D. dentifera to infectious spores and harvesting the spores by homogenizing infected animals in deionized water. Parasite fitness may be altered by host genotype, but no heritable variation exists in the fungal pathogen studied . The host range of the fungus is unknown, but includes a variety of both terrestrial and marine organisms , creating a uniform age gradient for all host species. Animals were placed individually in 50 mL of appropriate media, and either exposed to pathogen (200 pathogen spores/mL) or a slurry of crushed D. dentifera as a control (sham) inoculum. This was performed because the pathogen inoculum was created by crushing infected hosts, and the presence of crushed Daphnia may signal an alarm response from conspecifics.To remove the confounding effects of host age and maternal environment, we sequentially isolated offspring from parthenogenetic females raised in isolation to obtain individuals of known age and maternal environment. Keeping maternal environmental conditions fairly uniform, and randomly placing individuals in experimental groups serves to reduce any effect of maternal environment. Sequential isolation was performed for three generations before hosts were used in experiments, and the resulting offspring of this process were randomly assigned to exposure treatment. Host age may influence within‐host pathogen competition . In this approach, Daphnia hosts were crushed between glass microscope slide and coverslip and examined thoroughly for the presence of pathogen spores, which normally average over 10,000 per infected host  production. Infections are typically unobservable before 7 days postinfection challenge, and mortality typically occurs after 12 days or more. Infection was assessed visually daily from day seven onward and confirmed at death by examining We quantified costs as relative changes in three host fitness measures; total reproduction, mean clutch size, and lifespan. Total reproductive output  and mean clutch size (mean number of offspring per reproduction event) were both measured after the host had been exposed to the pathogen (or control inoculum). Host lifespan was measured as the total number of days from host birth to host death.post hoc tests were used to examine pairwise differences between exposure classes. This analysis allowed for the separate determination of costs of resistance (control compared with exposed‐uninfected host fitness), and costs of infection (control compared with infected host fitness). Further, this approach also enabled us to compare the rank distributions of exposed‐uninfected hosts to infected hosts, thereby providing insight into how costly resistance is compared to infection. All analyses were performed in R , we used Kruskal–Wallis rank tests. Nemenyi  Pohlert .μc−μi¯ = 18.6 neonates), mean clutch size (μc−μi¯ = 1.1 fewer neonates per clutch), and lifespan (μc−μi¯ = 6.8 days). The consistent finding of infection costs was not found for D. dentifera, which was excluded from the analyses as a result of enhanced mortality early in the experiment .Host fitness, measured as total reproduction, mean clutch size, and lifespan, was systematically reduced as a function of pathogen infection Table , suggestDaphnia–microparasite system is not costly. However, significant resistance costs were observed for D. laevis with respect to lifespan, and in all fitness measures for D. pulicaria , clutch size (μc−μr¯ = 0.08 fewer neonates per clutch), and lifespan (μc−μr¯ = 6.9 days).Meanwhile, exposure to pathogen without infection did not cause a significant reduction in host fitness for a majority of the host species and fitness measure combinations Table , suggestia Table . This hoia Table . While nD. laevis hosts when costs were measured in terms of lifespan or mean clutch size .Host age was strongly and positively related to host fitness measures, as older hosts at the time of pathogen exposure produced more offspring, had larger mean clutch sizes, and had longer lifespans relative to hosts that were younger at the time of pathogen exposure. However, we found little evidence for variation in resistance or infection costs as a function of host age at the time of pathogen exposure, although this relationship was significantly positive in Daphnia host species. We provide evidence that fungal pathogen infections come at a fitness cost to all susceptible host species, but that the fitness consequences of pathogen exposure were more nuanced. Specifically, significant resistance costs were only observed in D. pulicaria, a completely resistant host species. However, exposed‐uninfected (resistant) hosts had fitness values intermediate between control hosts and infected hosts. This suggests that pathogen resistance still comes at a price, although this difference is insignificant based on our limited sample size. Neither resistance nor infection costs varied as a function of host age at the time of pathogen exposure, although previous studies have found an age‐dependent cost in Daphnia parasitized by a castrating bacterial pathogen . However, consistent evidence for resistance costs remains sparse, both in laboratory , which was significant when costs were measured in terms of change in host lifespan between control and exposed‐uninfected hosts. Potentially the most obvious explanation for this relationship is that less susceptible species are less susceptible because they are able to mount an effective, though costly, behavioral or immunological response. Behaviorally, hosts could reduce feeding, which would reduce pathogen transmission, but would also reduce fitness through resource limitation. This behavioral response could also explain previous findings in natural systems, in which populations of D. dentifera exhibited a negative relationship between pathogen transmission rate and host birth rate . The mean lifespan of hosts, regardless of pathogen exposure, was <30 days, likely a result of experimental conditions . A final explanation could be the effects of pathogen dose or environmental conditions (apart from resources as described above). This explanation could explain not just the lack of detected age dependence, but also potentially the lack of detectability of resistance costs in invertebrate systems.Host age has been hypothesized to influence the size of the host response to pathogen exposure. This has been shown previously in a castrating bacterial pathogen of Daphnia, including the use of an immutable trait to quantify cost, and a limited understanding of invertebrate immunology , and host life history may provide a more detailed understanding of when a host response to pathogen exposure can be costly. By examining multiple host species, we provide little evidence for resistance costs in None declared.Table S1. Mean and standard error for fitness measures  for control, exposed‐uninfected, and infected individuals.Figure S1. Distribution of lifespan by host species.Figure S2. Costs of resistance and infection to a generalist fungal pathogen.Figure S3. Resistance costs along a gradient of host age at pathogen exposure.Figure S4. Infection costs along a gradient of host age at pathogen exposure.Figure S5. Resistance costs scale with host susceptibility.Figure S6. Resistance costs scale with host susceptibility, but not when D. dentifera is included.Click here for additional data file. Click here for additional data file. Click here for additional data file.


Total costs associated with care for older people nearing the end of life and the cost variations related with end of life care decisions are not well documented in the literature. Healthcare utilisation and associated health care costs for a group of older Australians who entered Transition Care following an acute hospital admission were calculated. Costs were differentiated according to a number of health care decisions and outcomes including advance directives (ADs).Study participants were drawn from the Coaching Older Adults and Carers to have their preferences Heard (COACH) trial funded by the Australian National Health and Medical Research Council. Data collected included total health care costs, the type of (and when) ADs were completed and the place of death. Two-step endogenous treatment-regression models were employed to test the relationship between costs and a number of variables including completion of ADs.The trial recruited 230 older adults with mean age 84 years. At the end of the trial, 53 had died and 80 had completed ADs. Total healthcare costs were higher for younger participants and those who had died. No statistically significant association was found between costs and completion of ADs.For our frail study population, the completion of ADs did not have an effect on health care utilisation and costs. Further research is needed to substantiate these findings in larger and more diverse clinical cohorts of older people.ACTRN12607000638437).This study was registered on 13/12/2007 with the Australian New Zealand Clinical Trial Registry ( Australia has a rapidly ageing population with 14 % being over the age of 65 in 2012  . Older pMany older people nearing the end of life may be unable to make health or other personal decisions due to factors that may inhibit their ability to do so including impaired cognition and decision-making capacity . Substit. And while some generic end-of-life-care literature [A number of international studies have documented the relationship between ADs and healthcare costs –38. And terature , 39 suggterature , 36–38 oterature , 33–35. terature  for instterature , based oterature , drawingterature  suggesteterature , 40–42. This study was approved by the Research Ethics Committee at the Repatriation General Hospital in Adelaide  and written informed consent was obtained from all participants.Data for this secondary study were obtained from the Coaching Older Adults and Carers to have their preferences Heard (COACH) randomised controlled trial, the methodological details of which have been reported elsewhere . BrieflyParticipants allocated to the intervention group received a coaching intervention that comprised four additional components: a question prompt list used a week before a geriatric consultation in order to enhance patient participation during the consultation; geriatric consultations with a geriatrician and nurse covering several topics included on a checklist; a written summary of every consultation; and a follow-up telephone call two to three weeks post discharge . The queThere are several different types of legal and non-legal ADs including the GPCP  and EPG  that were documented in this study. The GPCP documents a person’s preferred medical treatment to be pursued once they have been designated palliative while the EPG is a document which appoints and instructs a substitute decision-maker (e.g. friend or family member) to make decisions concerning the person’s health, residential and lifestyle care should the person be unable to make these decisions . There wThese measures were collected at baseline, 3 and 12 months and included QoL as measured by the EuroQoL EQ-5D 3 level (EQ-5D 3 L) instrument  and funcCosts were analysed from a health sector perspective. Within the COACH trial, Medicare costs for out-of-hospital visits were estimated using the Medicare Benefits Scheme (MBS) public subsidy (the Medicare benefit) paid for each of these items. Medicare costs for out-of-hospital visits included costs for tests, treatments and investigations; medical consultations and costs for primary care and general practice visits (Other). For in-patient hospital admissions, data on the diagnosis related group (DRG) associated with each admission as well as the length of stay were collected from hospitals. Using the Australian Refined Groups DRG cost weights , a cost All costs were reported in Australian dollars at 2013/14 unit prices and discounting was not necessary as the follow up period was less than one year .Descriptive statistics (means and standard errors) were generated and simple statistical tests of differences that took into account the distributional nature of the variables in the dataset  were also carried out . Differen = 228). At baseline, individuals had a mean age of 84.1 years and mean EQ-5D 3 L, Barthel and SMMSE scores of 0.46, 59.8 and 23.2, respectively. The baseline GDS-15 mean score was 4.8 while that for BMI was 24.3. The majority of participants in the study sample were female  and, at the end of the 12 months follow up period, the majority remained alive  while nearly half  registered a QoL improvement according to the EQ-5D 3 L (mean EQ-5D 3 L improvement over 12 months of 0.33). Of the 23 % (n = 53) of individuals who had died at the end of the study, almost all died within a hospital  or in a residential aged care or respite facility . For some decedents, the place of death was also the first discharge destination from TC. For instance, the first discharge destination post-TC for 12 (39 %) of the decedents who died in hospital was readmission to hospital while 10 (53 %) of those who died in a residential aged care or respite facility were first discharged to a residential aged care facility  and almost equal numbers completing either an EPG  or an MD . Eighteen of the 53 decedents (34 %) had completed ADs  for the COACH trial of 230 older adults, who were followed up to 12 months, was reached . Two indp = 0.06) and younger (p = 0.09). There were no statistically significant relationships between the type of AD completed and any other variables.Compared to those who did not complete ADs   but the The National Framework for Advance Care Directives in Australia  reports The higher overall and component  costs for males compared to females may be explained by the longer in-hospital length of stay for the former group (five days longer on average). Total costs for decedents  were higher (nearly twice as high) than those for survivors and these results were also borne out by those from international studies: a study reported that costs for US decedents in 2004 were over six times higher than those for survivors  while thThe use of endogenous treatment-regression models showed no statistically significant relationship between completing ADs and costs, whilst controlling for the joint effect of other variables. The regression results also show that when costs were split up into different cost components, none of these costs showed a statistically significant relationship with competing ADs. In line with previous research , 40–42, This study had some limitations. The total follow up period of 12 months was relatively short so the longer-term relationship between ADs, costs and other variables is not known. In addition, we did not have information on whether medical care after completion of ADs was consistent with the preferences expressed within these ADs, which would have given a much more accurate picture of healthcare utilisation and associated costs. A longer follow-up period and/or model-based extrapolation would be helpful in this regard and should be considered in future research. There was a proportion of missing data relating to resource use and other patient variables. However, robust statistical imputation methods were employed to deal with these missing data and the results from the imputed data and complete case analysis are broadly similar. Costs for residential aged care and other support services for this population are likely to be substantial but these were not included as robust data on these costs were not collected as part of the trial. There is also large variation around the mean estimates, particularly relating to the cost data, which is to be expected because this study has a relatively small sample size. Consequently, a larger cohort is needed to substantiate these preliminary findings. Finally, Advance Care Planning (ACP) has been gaining prominence in Australia due to a recent shift from documentation towards the process of ACP . AdvanceThis study has documented, and confirmed, the substantial pharmaceutical, hospital and other costs associated with caring for older people nearing the end of life. However no significant association was found between health care utilisation costs and ADs. If one of the rationales for the introduction of ADs is improving the end of life experience for older people and their families whilst being cost neutral with regards to healthcare expenditures on older people, then this study provides some evidence to support this proposition. However, further research is needed to substantiate these findings in larger and more diverse clinical cohorts of frail older people.


Women considering female genital cosmetic surgery (FGCS) are likely to use the internet as a key source of information during the decision-making process. The aim of this systematic review was to determine what is known about the role of the internet in the promotion and normalisation of female genital cosmetic surgery and to identify areas for future research.Eight social science, medical, and communication databases and Google Scholar were searched for peer-reviewed papers published in English. Results from all papers were analysed to identify recurring and unique themes.pathologisation of genital diversity; female genital appearance as important to wellbeing; characteristics of women’s genitals are important for sex life; female body as degenerative and improvable through surgery; and FGCS as safe, easy, and effective. A significant gap was identified in the literature: the ways in which user-generated content might function to perpetuate, challenge, or subvert the normative discourses prevalent in online pornography and surgical websites.Five papers met inclusion criteria. Three of the papers reported investigations of website content of FGCS providers, a fourth compared motivations for labiaplasty publicised on provider websites with those disclosed by women in online communities, and the fifth analysed visual depictions of female genitalia in online pornography. Analysis yielded five significant and interrelated patterns of representation, each functioning to promote and normalise the practice of FGCS: Further research is needed to contribute to knowledge of the role played by the internet in the promotion and normalisation of female genital cosmetic surgery. There is increasing evidence that women are pursuing surgical modification of the vulva for cosmetic reasons. Popular surgical procedures include, but are not limited to, reduction of the labia minora and clitoral hood, tightening of the vagina, ‘plumping’ of the labia majora, liposuction of the mons pubis, and ‘G-spot’ collagen injections , 2. In AThe apparent popularity of female genital cosmetic surgery (FGCS) procedures in recent years has triggered a flurry of critical academic engagement in the topic. Previous empirical research has shed light on a range of aspects relevant to the phenomenon, from factors that may influence the decision of individual women to seek FGCS  to mainsThere is evidence that women’s external genitalia are highly diverse . HoweverWhereas traditional media have long been understood to influence female beauty ideals  and attiPapers were eligible for inclusion if they were published before September 2014, in English, in peer-reviewed journals, and reported empirical research  about online representations of female genital appearance or female genital cosmetic surgery.social media” OR “social network*” OR internet OR youtube OR twitter OR tumblr OR facebook OR “online communities” OR “bulletin board” OR web* OR wiki OR blog* OR email; in conjunction with: genital* OR labia* OR vulv* OR vagin* OR clitor* OR hymenop*. Where applicable, the relevant subject terms for each database were also included. Google Scholar was searched using the terms “female genital*”, labiaplasty, internet. Reference lists of located articles were hand-searched for other potentially relevant titles.Eight social science, medical, and communication databases  were individually searched using the terms “Following the removal of duplicates, records were assessed for suitability in accordance with the PRISMA guidelines , first bGiven that all eligible papers were likely to report analysis of texts or images, we sought standardised criteria, tools, or frameworks to use in assessing their quality, but found nothing designed for the purpose. Two authors (HM and KM) independently assessed the quality of each study using Kmet’s  assessmeResults reported in all papers were analysed thematically. As themes were identified in each paper, a structure of themes and sub-themes was developed. Each paper was reassessed against the developing thematic structure to ensure the best fit and to establish relationships among themes and concepts. In addition to the results reported and conclusions drawn by the authors of each paper, reviewers used all identified themes in analysing the content of any included data excerpts, in order to achieve the most comprehensive account of the literature.The search strategy yielded five eligible papers, each reporting on a single study. Details of the selection process are outlined in Fig. Four of the five studies were conducted by researchers based in high-income Western countries: the Netherlands , 31, theLiao and colleagues  report rTables pathologisation of genital diversity (identified in 5 papers), female genital appearance as important for wellbeing (4 papers), characteristics of women’s genitals as important for sex life (4 papers), the female body as degenerative and improvable through surgery (3 papers), and FGCS as safe, easy, and effective (3 papers).Five interrelated themes were identified. These were: All studies found that vulval diversity is pathologised in cyberspace, with the concomitant promotion of a homogenised “clean slit” , 16 vulvThree papers reported FGCS surgical provider websites employing a range of disparaging language to signify which genital characteristics or features ought to be considered undesirable. For instance, pubic fat is described as “unsightly”, vaginas as potentially “loose”, and large labia as “protuberant”, “oversized”, “awkward”, and “unappealing” –34. In aFindings from four studies support the conclusion that vulval diversity is pathologised by the presentation of certain characteristics as unnatural or diseased. Two studies identified FGCS provider websites using medical terminology to construct protruding labia minora, amongst other genital characteristics, as pathological and thus requiring surgical correction , 33. A tThe pathologisation of vulval diversity does not occur solely through language. The findings reported in all five papers indicate that online visual representations of female genitalia can be a powerful indicator of socially desirable and undesirable genital characteristics. Four studies reported the presence of before-and-after labiaplasty image galleries on FGCS provider websites, with the frequency ranging from 25 to 60 % of the websites in each sample , 32–34. N = 98 images) to be significantly less protuberant and less varied than the range found in the female population. The authors also analysed a sample of images drawn from feminist-oriented publications (one website and two books) (N = 126 images) which they considered to be a closer reflection of the range of variation amongst women [Howarth and colleagues  found thst women . HoweverResults from four studies indicate a strong discursive connection between female genital appearance and wellbeing. This connection was often drawn by commercial FGCS providers in order to promote their services but was also supported and reinforced by women’s own accounts of their motivations and experiences. Four papers identified FGCS provider websites drawing an unquestioned connection between female genital appearance and psychological or emotional wellbeing or distress , 32–34. Zwier’s  analysisUsing the techniques of critical discourse analysis, Moran and Lee  interrogZwier’s  analysisNotably, two of the websites quoted in Ashong and Batta’s  study suFindings from four papers indicate that FGCS provider websites commonly claim associations between pre-operative genitalia and sexual dysfunction, with the dysfunction described as primarily psychological , 32–34. Although it might be inferred that a woman’s sexual satisfaction is being prioritised, three studies found that FGCS provider websites promoted surgery as the solution to a male partner’s sexual dissatisfaction –34. In sHaving established that a “tight” vagina and a “tidy”, “youthful” vulva is represented online as the (Western) ideal, three papers reported online content that contributes to a cultural representation of a tenuous female body susceptible to degeneration, particularly through childbirth and ageing, and improvable or restorable through surgery –34. AgeiSeveral surgery clinics are reported as promoting “Mommy Makeover” packages, in which vaginal tightening and labia reduction surgeries are bundled together with liposuction, tummy tucks, and breast augmentations , 33. TheIt was reported in papers from three studies that FGCS provider websites claimed benefits and asserted the safety of various FGCS procedures, without providing evidence –34. LiaoTwo papers reported on the success rates published on surgery provider websites, finding that, where cited, these were in the range of 90 – 95 % , 33. LiaIt was found in three studies that providers used medical terminology to confer credibility on FGCS in general and to legitimate specific surgical techniques and procedures –34. HalfAuthors of three papers expressed concern about risk information on FGCS provider websites, arguing that potential risks or adverse outcomes of surgery are minimised or omitted entirely from website content –34. LiaoInformation about potential surgical complications was almost exclusively in the context of warnings against “botched jobs” elsewhere, such as, “we have seen many unfortunate examples of terrible scarred uneven results of labiaplasty from other physicians” . AccompaThis systematic review examined findings from five studies exploring online content relating to female genital appearance or female genital cosmetic surgery. Four of the five studies analysed the content of FGCS provider websites, and the consistency of their findings reveal the ways in which providers of female genital cosmetic surgery are promoting and normalising the practice of FGCS online. The most prominent theme, the pathologisation of vulval diversity, was also found in an analysis of online pornography. The results of this review suggest adverse implications for the women accessing these sites and reveal sociocultural attitudes to female genitalia and FGCS.On female genital cosmetic surgery websites, the female body is pathologised by the medicalization and denigration of the aesthetic appearance of certain vulval features. Despite research indicating no significant association between pregnancy, childbirth, or natural ageing and vulval measurements , FGCS prThese websites perpetuate a persistent and unquestioned assumption that genital variation, beyond a restricted ideal, results in psychological and sexual dysfunction. We do not seek to discount the emotional distress and decreased quality of life experienced by some women because of internalised ‘self-loathing’ associated with the appearance of their genitalia. Indeed, the majority of women posting in online communities cite this as a motivation for surgery . HoweverFurther, the construction of certain natural genital variations as objectively abnormal and undesirable enables ‘corrective’ surgery to be positioned as the logical and empowering solution for affected women. Not only is surgery posited as the best solution to distress about one’s genitals, it is also promoted as an expression of personal agency and empowerment, a representation that is highly consistent with consumerist, neo-liberal, and post-feminist discourse more generally , 37. ThiIn line with Braun’s  critiqueA primary critique of FGCS is the extent to which these procedures are being performed in the absence of any clinical data on safety or long-term effectiveness . This reThis body of literature, albeit small, draws attention to significant and interrelated patterns of online representation that function to pathologise natural genital variation and promote and normalise a homogenous, surgically altered ideal vulva to Western women. Although these studies have been able to discern and highlight particular patterns of representation, it should be acknowledged that such representations are open to interpretation, which will largely be influenced by individual viewer characteristics. Studies suggest that online content, including pornography and surgical websites, influences women’s consideration and acceptance of FGCS , 27. FurThe majority of studies in this review analysed solely ‘non-social’ media: surgical websites and online pornography. However, the internet is not all direct marketing and pornography. Rather, the key feature or benefit of the internet is its capacity for diverse many-to-many communication opportunities. We speculate that this capacity may give people using social media the power to challenge dominant representations and discourses in cyberspace. Nevertheless, the sole study of espoused motivations for labiaplasty in online communities demonstrates that many women appear to have internalised derogatory patriarchal connotations of the vulva to an extent that adversely affects their lives and wellbeing . In orde


The first Virus Genomics and Evolution Conference was held at the Wellcome Genome Campus in Hinxton, UK, 8–10 June 2016. In recognition of the increasingly important role that genomics plays in virology and public health epidemiology, the Wellcome Genome Campus hosted the inaugural conference on Virus Genomics and Evolution in June 2016. This multidisciplinary conference brought together public health researchers who are generating viral genomic data from clinical samples and evolutionary biologists and bioinformaticians who are developing methods to interrogate these data.The meeting highlighted the broad temporal and spatial scales over which viral genetic data are having an impact, from understanding the trajectory of resistance mutations during a single infection to understanding the spatial expansion of viral lineages during large epidemics. Several talks featured memorable accounts of the use of genomics in practical settings, from West African field hospitals to European courtrooms. Interestingly, there was little discussion of sequencing technologies themselves; rather the focus was on their practical, actionable applications.In this report, we highlight how genomics is being used to explore viral origins and diversity and to understand transmission and pathogenesis. Additionally, we describe how real-time generation and analysis of viral genomic data are transforming outbreak surveillance and management.Ian Lipkin  launched the meeting with tales of microbe-hunting across many species and diseases, emphasizing that detection of a virus genome in a sample is simply the first step in the process of linking an agent to disease. Viral discovery is fraught with false positives, and potential associations should be explored via multiple avenues, including serology and host response.The scale of the viral discovery problem was highlighted by Eddie Holmes , whose virus-hunting in diverse species—from unusual arthropods to sharks—shows that we have “barely scratched the surface of viral diversity”. Using metatranscriptomics, Holmes’ team demonstrated exceptional levels of virus diversity and revealed the dynamic nature of viral genome size and organization. A novel approach for viral discovery, using small interfering RNA (siRNA) as a marker of host response, was presented by Darren Obbard . Uncharacterized or underappreciated viral diversity is equally apparent at smaller taxonomic scales, as illustrated by the discovery of new viruses related to human hepatitis A virus  and by the re-emergence of human noroviruses from unsampled reservoir populations .Perhaps the strongest theme of the meeting was the use of viral sequencing for surveillance and diagnostics in public health. The surge of interest in this area is no doubt due to the recent West African Ebola virus (EBOV) epidemic, which provided the backdrop for several talks. Kristian Anderson  explained how the Broad Institute’s Lassa Fever genomics infrastructure in Sierra Leone was quickly repurposed for Ebola virus sequencing and gave examples of epidemiological insights drawn from genomics during the outbreak. He cautioned that real-time genomics must lead to real-time action. Specifically, he reported that genomic data could have influenced public health conversations early in the outbreak; sequencing indicated that ongoing EBOV transmission was not due to multiple spillovers from an animal reservoir, yet messages about bushmeat consumption probably gave the opposite impression. The work presented by Kristian also demonstrated the utility of clinical metagenomics in an outbreak, revealing the associations between co-infection with malaria and Ebola mortality. Gustavo Palacios , Frederick, MD) described EBOV genomics in Liberia, where sequencing revealed the roles played by viral persistence in immune-privileged sites in transmission from survivors as the outbreak waned.A glimpse of the future of surveillance was afforded by Gytis Dudas’  meta-analysis of around 1600 EBOV genomes sequenced across the two-year outbreak. His phylogeographic analysis showed how EBOV lineages crossed from one country to another, avoided extinction by spreading within countries, and were magnified in urban areas. Access to a subset of these data during the outbreak resulted in border closures that limited the epidemic, demonstrating that genomic information, acquired in real time, could help to contain future epidemics. Ian Goodfellow  set up an EBOV sequencing center in Sierra Leone and described how the attendant logistical challenges of such a project, from sourcing nitrogen canisters to managing samples, must not be underestimated. Ian demonstrated the benefits of sharing, whether it be of open-access data via platforms like virological.org and nextflu.org or of the sequencing equipment itself, which was eventually rehomed at the University of Makeni. Hinting at a subject that might feature at the next edition of the conference, Oliver Pybus  introduced the ZiBRA project, which is using portable nanopore sequencing in the field in Brazil to explore the dynamics of the Zika virus epidemic.The use of viral genomics as a tool to characterize transmission is not restricted to emerging epidemics, having been applied widely to established human pathogens. Examples presented at the meeting included reconstruction of a measles virus outbreak in Canada , community transmission of respiratory syncytial virus in Kenya , hospital outbreaks that were sequenced for infection control as part of the ICONIC (InfeCtion respONse through vIrus genomiCs) project , and the use of HIV and hepatitis C virus (HCV) phylogenies to establish or disprove criminal transmission in court . Annemie explained why inferring the direction of transmission is not equivalent to inferring direct transmission and, through a series of real-world examples, she established the conditions that must be met to argue for transmission.At a larger epidemic scale, Marion Koopmans  discussed global norovirus dynamics as captured by the Noronet surveillance platform, including the recent emergence of recombinant strains that are undergoing rapid diversification and the association of excess mortality with new variants. Talks from Philippe Lemey  and Kirstyn Brunker  demonstrated how other data sources can be merged with viral genomes to help us to understand epidemic dynamics, with examples showing how predictors such as spatial distance and case counts affected the Ebola outbreak, and how human-mediated connectivity and spatial heterogeneity influence rabies virus transmission in Tanzania.The meeting also reflected a growing area of research in which virus genomics is used to guide the design of effective antiviral drug interventions. Despite the approval of new—but expensive—direct-acting antiviral drugs for HCV infection, we still do not know what determines whether treatment will be successful against the diverse range of HCV genotypes. As Ellie Barnes and M. Azim Ansari  explained, large-scale sequencing of both HCV and human genomes, followed by genome-wide association studies (GWAS), is beginning to tease apart the host and virus genetic components of treatment response.Sequencing can also be used to observe the effects of drugs on viral genomes directly. Daniel Goldhill  showed evidence supporting the hypothesis that the experimental compound favipiravir acts to increase the mutation rate of influenza A viruses. Viral genomics is also being used to plan and evaluate large-scale treatment programs. In the final keynote address, Christophe Fraser  showed how viral phylogenetics is being used to identify the source of new HIV infections in the PopART community-randomized trial of HIV testing and treatment, which involves around 1.2 million people in sub-Saharan Africa. He described how the huge scale of such a project requires the development of new, efficient methods of evolutionary analysis, as well as rigorous thinking around sample size and power calculations for tests based on phylogenetic and molecular clock models.The importance of virus and host co-evolution was appreciated by the audience. Sunetra Gupta’s  dynamic models of the adaptive immune system indicated that decreasing levels of CD4+ T cells determined HIV viral loads during the chronic phase of infection, which, in turn, determined the speed of disease progression. Sarah Cobey  showed how influenza vaccines influence mutations in host B cells, and Andrea Tanzer  asked whether, since Zika virus has been circulating for decades, there are any genetic changes that sparked the current outbreak in the Americas? By comparing the structure and folding kinetics of flaviviral RNA elements involved in host response and virulence, she identified specific mutations leading to RNA structural alterations and evidence for selective pressure by compensatory mutations.Although the conference focused on genomics and evolution—topics that might be considered more theoretical than applied—it was notable that many speakers talked as much about real people, some of whom have suffered or died from viral disease, as they did about data, theories, and technology. The frequency and scale of viral outbreaks in recent years has emphasized the urgency of research in virus genomics. Further, it has prompted efforts to shift academic culture towards closer collaborations between clinicians, biologists, mathematicians, and computer scientists and towards the real-time, open release and analysis of data, with the shared aim of securing the health of our world’s population. The next meeting is provisionally scheduled for June 2018.EBOV, West African Ebola virus; HCV, hepatitis C virus


To examine the efficiency of FA inactivation, we used Vaccinia virus, Humanadenovirus and Murine norovirus as models and treated them with FA under various conditions. Critical parameters for the inactivation efficiency were the temperature, the duration of the FA treatment, and the resistance of the virus in question. Our results show that FA inactivation at low temperature (4 °C) bears a high risk of incomplete inactivation. Higher temperatures (25 °C) are more efficient, although they still require rather long incubation times to fully inactivate a complex and highly robust virus like Vaccinia. A protocol, which applied 2% buffered FA for 60 min and a temperature–shift from 25 to 37 °C after 30 min was efficient for the complete inactivation of all test viruses, and therefore has the potential to improve both biosafety and speed of diagnostic electron microscopy.Formaldehyde (FA) fixation of infectious samples is a well-established protocol in diagnostic electron microscopy of viruses. However, published experimental data that demonstrate virus inactivation by these fixation procedures are lacking. Usually, fixation is performed immediately before the sample preparation for microscopy. The fixation procedure should transform viruses in a non–infectious but nonetheless structurally intact form in order to allow a proper diagnosis based on morphology. FA provides an essential advantage in comparison to other disinfectants, because it preserves the ultrastructure of biological material without interfering significantly with the preparation ( TExposure of lab personnel and the public by accidentally released viruses during handling of virus–infected biological material must be avoided by efficient and feasible safety precautions. Since most electron microscopes are operated at biosafety level two and below, proper sample inactivation is necessary to guarantee biosafety ,12,13. FVaccinia virus), a standard test virus in disinfection studies (Human adenovirus) and a more robust non–enveloped virus (Murine norovirus). Virus infectivity was determined before and after treatments by standardized methods (plaque or TCID50 assay). Structural integrity of inactivated viruses was checked by negative staining EM. As a main result of our study, we provide a rapid (1 h) inactivation protocol using 2% buffered FA, which completely inactivates concentrated virus suspensions.We therefore started to evaluate the inactivation efficiency of FA–fixation by using three different model viruses known for their environmental stability: a complex enveloped virus , and two non–enveloped viruses, Murine norovirus  and Human adenovirus type 5 . Three different poxvirus strains were used as additional positive control in each plaque assay: Cowpox virus  or Vaccinia virus . Poxviruses were propagated in Hep2 cells , and plaque assays were performed using Vero C1008 cells . Murine norovirus was proliferated in RAW 264.7 cells (ATCC TIB–71) and Human adenovirus in A549 cells (ATCC CCL–185). Tissue culture infective dose 50 (TCID50) assays were conducted using the same cells, respectively.Three different viruses were used for testing the inactivation efficiency. One complex, enveloped virus, eGri07/1 ), CowpoxVaccinia virus (MOI 0.5) were cultivated in Dulbecco’s Modified Eagle Medium (D–MEM), including 1% L-glutamine, (Gibco/Invitrogen) and 5% fetal bovine serum (PAA) for four days at 37 °C and 5% CO2. Cells with media of several culture flasks (175 cm2) were frozen/thawed thrice, scraped and pooled. Cell debris was sedimented by low speed centrifugation at 724 g for 10 min, and the supernatant was ultracentrifuged at 20,000 rpm for 1.5 h  at 4 °C. The pellets were resuspended in D–MEM and stored at −80 °C until use as the stock solution for inactivation experiments. Virus titers were determined by plaque assay and ranged between 1.2 × 108 and 1.3 × 109 plaque forming units (PFU) per milliliter.Hep2 cells infected with Human adenovirus type 5 and maintained in D–MEM including 1% L-glutamine, 1% non–essential amino acid cell culture supplement , 0.11% NaHCO3 and 2% fetal calf serum for 3–5 days at 37 °C and 5% CO2. Cells were scraped and centrifuged at 4696 g for 15 min. The pellets were frozen/thawed thrice and centrifuged again at 4696 g for 15 min. Supernatant was frozen at −80 °C and stored until usage as the stock solution for inactivation experiments. The virus titer was determined by tissue culture infective dose 50 (TCID50) assay and ranged between 1 × 109 and 1 × 1010 TCID50/mL.A549 cells were infected with Murine norovirus was propagated in Raw 264.7 cells, which were cultivated in Minimum Essential Medium (MEM) with Hank’s balanced salts  and HEPES buffer, including 1% L-glutamine , 1% sodium pyruvate , 1% non–essential amino acid cell culture supplement , 0.15% NaHCO3 and 3% fetal calf serum  for 3–5 days at 37 °C and 5% CO2. Cells were scraped and centrifuged at 4696 g for 15 min. The pellets were frozen/thawed thrice and centrifuged again at 4696 g for 15 min. Supernatant was stored at −80 °C until usage as the stock solution for inactivation experiments. The virus titer of the virus preparations ranged between 1 × 107 and 1 × 108 TCID50/mL.Vaccinia virus, a defined volume of the FA stock solution was added to samples . Immediately after the end of incubation time . In the case of Human adenovirus and Murine norovirus, the PFA stock solution was diluted in HEPES buffer to a concentration of 2.2%, and nine units of the diluted PFA were mixed with one unit of the virus stock solution according to the DVV/RKI Guideline [Formaldehyde inactivation solution was prepared from a frozen stock solution of 20% paraformaldehyde  in 0.5 M HEPES buffer (pH 7.2), which was heated to 60–70 °C for 20 min to shift the equilibrium towards non-polymerized FA. In the case of uideline . i.e., no visible aggregates could be detected in the suspensions. For every FA-treated test suspension a 24–well plate  was prepared with 200 µL per well of a Vero cell suspension (1.2–1.5 × 106 cells/mL) supplemented with 5% streptomycin-penicillin  followed by 200 µL of the virus suspension at three dilutions . The dilutions were generated in duplicates and distributed in four different wells . After four days of incubation, cells were fixed with 3.7% formalin  for 30 min, stained with amido black 10B , washed with water, and plaques were counted manually. Untreated virus suspensions  was calculated relative to the virus titer estimated for the untreated virus suspension [Plaque assays were performed according to . Prior tnt wells . In our to 1:108 . Virus tspension . To incr2. Rows 9 to 11 were left blank to avoid contamination of the negative control (100 µL of virus-free medium), which was placed in row 12. The test suspension was sequentially diluted tenfold (from 1:10 to 1:108) in medium (900 µL + 100 µL test suspension), supplemented with 0.5% (w/v) L-histidine to neutralize free aldehyde and 2% (Human adenovirus) or 3% (Murine norovirus) fetal calf serum. All dilutions were tested eightfold in a single 96–well plate. Untreated virus suspensions were tested in the same manner. Cytopathic effect (CPE) was evaluated after seven days for Human adenovirus and after five days in case of Murine norovirus using an inverted microscope. The virus titer was determined using the method of Spearman [Tissue culture infective dose 50 (TCID50) was performed according to ,29. CellSpearman  and KaerSpearman  and exprVaccinia virus). The grids were examined using a JEM–2100 transmission electron microscope (JEOL Corp.) operated at 200 KV. Micrographs were recorded with a Veleta CCD camera (Olympus Soft Imaging Solutions) at a resolution of 2048 × 2048 pixels.Negative staining transmission electron microscopy of the virus stock solutions and test suspensions was performed according to . A drop Vaccinia virus, Murine norovirus and Humanadenovirus suspensions with 2% buffered FA at 4 and 25 °C for different periods. The experiments demonstrated that short incubation times  at 4 °C are not sufficient to completely suppress infectivity of high concentrations (above 107 PFU/mL) of Murine norovirus and Vaccinia virus  were applied. Complete inactivation of Vaccinia virus infectivity was achieved by an incubation for 24 h at 25 °C  and/or temperatures above 25 °C with the most resistant virus of our test viruses,eriments . Other sspension . The effat 37 °C .Vaccinia virus as a model and a slightly modified plaque assay  . Samplesi.e., 25 °C) and rather long incubation time (more than six hours) to completely inactivate complex viruses such as the Vaccinia virus. Although temperature dependency of FA inactivation is known, it came as a surprise that even an exposure of Vaccinia virus to 2% FA for several days at 4 °C did not fully inactivate the virus. The non–enveloped viruses tested were much more susceptible to FA treatment at low temperature than the enveloped Vaccinia virus, but showed also differences in their reactivity, indicating slow reactivity of the FA at 4 °C. In summary, we concluded that inactivation of viruses at 4 °C should be avoided. Inactivation at 25 °C was much more efficient but still needed several hours for a complete inactivation of concentrated suspensions of the complex Vaccinia virus. A significant reduction in incubation time to reach complete inactivation was found with a further increase of temperature to 37 °C after a pre-treatment at 25 °C (temperature–shift protocol). With a total inactivation time of 60 min, the temperature-shift protocol becomes useful for emergency diagnostics. It seems likely that further modifications of the protocol, like increasing temperature to higher levels than 37 °C, could shorten the complete inactivation even further. Increasing the FA concentration  could also accelerate inactivation [Our results show that the inactivation of virus suspensions by FA depends on temperature, incubation time and the type of virus. This result and the influence of other factors, like pH and FA concentration on inactivation efficiency, have been documented by other studies before  ativation . However(1)The inactivation of viruses by FA has been evaluated by using three different test viruses, and results showed that the inactivation efficiency is highly dependent on the virus type, as expected . While (2)The inactivation tests have been conducted using non–purified cell culture supernatants. Although these samples contain biological macromolecules besides virus particles released by the cells in culture, concentration of such material is rather low in comparison to some diagnostic samples, e.g., serum or stool. Therefore, additional experiments, using defined loads of protein or other biological material , must be performed to determine the possible interference of the inactivation by high concentrations of other biological material.(3)9 PFU/mL of the highly stable poxviruses. Although the situation of diagnosing a sample with higher virus load seems rather unlikely, it should be investigated whether the proposed inactivation protocols are efficient at even higher concentrations or not.Concentration of viruses may also be a critical factor for inactivation efficiency. To produce high concentrations and sufficient amount of active test viruses is difficult and laborious. In our tests, we used concentrations up to 10(4)Vaccinia virus suspensions which became apparent only at higher dilutions in the plaque test. Higher dilution could have promoted disintegration of aggregates and release of infectious particles. Further experiments must consider these variables and should provide an estimate regarding their effects.Our results showed a slight variability in the inactivation efficiency, even under ideal experimental conditions. The poxvirus suspension used for the reproduction experiments  was more(5)For an application of the suggested inactivation protocols in emergency diagnostics, the inactivation efficiency of the protocols must be tested also for bacteria, since a pre-screening of the samples for bacteria is not possible in any case. From the published data on FA inactivation, at least a significant reduction of vegetative bacteria can be expected ,40. BactIt was not our goal to study the virucidal properties of FA in detail, but rather to develop an inactivation protocol that is useful in practice. The temperature-shift protocol provides appropriate speed and safety especially for application in emergencies. Inactivation at 25 °C may also be used for diagnostic samples if incubation time can be extended to 24 h. However, the following concerns remain and need further investigation:In summary, the application of the inactivation protocols as suggested in this paper bears a residual risk of incomplete inactivation, which cannot be quantified so far. Since the negative staining procedure and electron microscopy itself seem not to inactivate virus samples , a resid


Phylogenetic analyses revealed that the viruses and viroids might have originated from Europe, along with the host. Single nucleotide variation studies revealed the quasispecies of RNA viruses. Taken together, this study defines complex viral populations in three different grape tissues from a single vineyard.Viruses are ubiquitous and present in a wide range of settings, from living organisms to various environments. Although viruses are regarded as important pathogens in higher plants, viral populations in specific host plants have not yet been fully examined. This study revealed viral populations in grape berries obtained from a cultivar from a single vineyard using currently available grapevine transcriptomes. Eight viruses and two viroids were identified using 11 grapevine libraries. Virus-associated sequences in each transcriptome ranged from 0.2% (seed) to 8.8% (skin). The amount of viral RNAs and virus copy numbers was quantified, thus revealing the dominant virus or viroid in each individual library. In addition, five viral genomes were successfully assembled  Viruses are ubiquitous and can be found in living organisms, such as bacteria, animals and plants, as well as various environments, such as the sea, volcanoes and airRecent next-generation sequencing (NGS) technology has enabled the identification of known and novel viruses in infected hosts. For instance, the extraction of virus-enriched RNAs followed by NGS can identify several viruses and viroids infecting grapevines4567Herein, an efficient approach is provided for how to reveal viral populations in different grapevine tissues using available grapevine transcriptomes followed by several bioinformatics analyses.de novo transcriptome assembly was performed with raw data from each library using the Trinity programGrapevine yellow speckle viroid 1 (GYSVd1) (119 contigs) and Grapevine pinot gris virus (GPGV) (97 contigs), followed by Hop stunt viroid (HSVd) (81 contigs) and Grapevine leafroll-associated virus 2 (GLRaV2) (77 contigs) , Grapevine rootstock stem lesion-associated virus (GRSLaV), Maize rayado fino virus (MRFV), Oat blue dwarf virus (OBDV) and Potato virus S (PVS)  . Only sm S (PVS) . The dom S (PVS) . GPGV-as S (PVS) . GYSVd1  S (PVS) . Four vi S (PVS) . Additio S (PVS) . In tota S (PVS) .Potato virus Y (PVY) (12.8%) based on viral reads , Grapevine redglobe virus (GRGV) and Grapevine rupestris vein-feathering virus (GRVFV) , HSVd (7.6%) and GYSVd1 (6.5%)  . The ide1 (6.5%) .Using the obtained nearly complete genome sequences for three viruses and two viroids, the phylogenetic relationships between the known respective virus and viroid isolates were analysed . The PVYde novo viral genome assembly, it seems that several variants for each virus exist in the vineyard. Therefore, the SNVs for five viral genomes were examined. Many virus-associated reads covered nearly whole regions of the GRSPaV1, GYSVd1 and HSVd genomes  from the second approach were more reliable due to the relatively long sequence lengths. However, the complete genome sequences for GRGV, GRVFV and GAMaV are not currently available. As a result, eight viruses and two viroids were identified from the grape berries of Tannat grown in a vineyard  with default parameters22de novo assembled transcriptome using the Velvet/Oases assembler, a CD-HIT program with a threshold of 90% was applied to filter redundant contigsAll bioinformatics analyses were performed with a workstation (two six-core CPUs and 256-GB RAM) operated using the Ubuntu 12.04.5 LTS operation system. Two paired-end sequenced FASTQ files for each library were first subjected to de novo assembled contigs were blasted against complete reference sequences of viruses and viroids (http://www.ncbi.nlm.nih.gov/genome/viruses/). For the BLAST search, the MEGABLAST algorithm with a cut-off e-value of 1e-5 was applied. MEGABLAST is much faster than other sequence similarity programs and provides very reliable sequence similarities for the identification of viruses and viroids. For the second approach, the same BLAST search was performed against complete reference sequences of viruses and viroids using a whole grapevine transcriptome, which was assembled using the 11 libraries. For the third approach, the raw data from each library were directly blasted. For this, all the FASTQ files were converted into FASTA files using the FASTX-Toolkit (http://hannonlab.cshl.edu/fastx_toolkit/).To identify the viruses and viroids that infect grapevine berries, three different approaches were used. For the first, De novo assembled viral genome sequences were manually compared with the known reference viral genome sequences. Finally, nearly complete genome sequences for three viruses were obtained, including GRSPaV-1 (accession number KR528585), GPGV (accession number KR528581) and PVY (accession number KR528584), as well as complete genomes for two viroids, including GYSVd1 (accession number KR528582) and HSVd (accession number KR528583).To obtain complete or nearly complete genome sequences for the viruses and viroids, the whole grapevine transcriptome was first blasted against complete reference sequences of viruses and viroids. Among the contigs with high levels of similarity, the longest contig for each virus or viroid was selected. To identify single nucleotide variations (SNVs), all the raw data were aligned on the obtained genome sequences for three viruses, including GRSPaV-1, GPGV and PVY, and two viroids, including GYSVd1 and HSVd, using the Burrows–Wheeler Aligner (BWA) with default parametersde novo-assembled genome and the known genome of the corresponding virus or viroid were aligned by the ClustalW program implemented in MEGA6. A large number of genomes were available for PVY; to reduce the number of PYV genomes for phylogenetic tree construction, ten PVY genomes that were highly matched to the isolate Tannat were used. There were many complete genomes for the GYSVd1 and HSVd isolates. Therefore, the genomes of only two viroids were selected, which were identified from the grapevine. Aligned nucleotide sequences were used to construct a phylogenetic tree using the neighbour-joining method with 1,000 bootstrap replicates.For phylogenetic analyses, phylogenetic trees were constructed for GRSPaV-1, GPGV, PVY, GYSVd1 and HSVd using the MEGA6 programHow to cite this article: Jo, Y. et al.In silico approach to reveal viral populations in grapevine cultivar Tannat using transcriptome data. Sci. Rep.5, 15841; doi: 10.1038/srep15841 (2015).


In response to infection, small RNAs (sRNAs) are produced that associate with Argonaute (AGO)-containing silencing complexes which act to inactivate viral genomes by posttranscriptional gene silencing (PTGS). Deep sequencing was used to compare virus-derived small RNAs (vsRNAs) in cassava genotypes NASE 3, TME 204 and 60444 infected with the positive sense single-stranded RNA (+ssRNA) viruses cassava brown streak virus (CBSV) and Ugandan cassava brown streak virus (UCBSV), the causal agents of cassava brown streak disease (CBSD). An abundance of 21–24 nt vsRNAs was detected and mapped, covering the entire CBSV and UCBSV genomes. The 21 nt vsRNAs were most predominant, followed by the 22 nt class with a slight bias toward sense compared to antisense polarity, and a bias for adenine and uracil bases present at the 5′-terminus. Distribution and frequency of vsRNAs differed between cassava genotypes and viral genomes. In susceptible genotypes TME 204 and 60444, CBSV-derived sRNAs were seen in greater abundance than UCBSV-derived sRNAs. NASE 3, known to be resistant to UCBSV, accumulated negligible UCBSV-derived sRNAs but high populations of CBSV-derived sRNAs. Transcript levels of cassava homologues of AGO2, DCL2 and DCL4, which are central to the gene-silencing complex, were found to be differentially regulated in CBSV- and UCBSV-infected plants across genotypes, suggesting these proteins play a role in antiviral defense. Irrespective of genotype or viral pathogen, maximum populations of vsRNAs mapped to the cytoplasmic inclusion, P1 and P3 protein-encoding regions. Our results indicate disparity between CBSV and UCBSV host-virus interaction mechanisms, and provide insight into the role of virus-induced gene silencing as a mechanism of resistance to CBSD. CBSD isomovirus . The virennadius , and mecennadius  and sap ennadius . The virennadius  causing ennadius  and Kaleennadius , to be hPlant-infecting viruses are known to trigger RNA interference (RNAi) , an innaArabidopsis thaliana and other plant species have revealed that the core components involved in plant small RNA biogenesis and the silencing pathways are encoded by multi-protein families , TME 204 (susceptible to both CBSV and UCBSV), and cultivar 60444 (highly susceptible to both CBSV and UCBSV). NASE 3 and TME 204 are farmer-preferred cassava varieties in Uganda , while 622.1N = 20) were confirmed to be free of CBSD viral pathogens by reverse transcription polymerase chain reaction (RT-PCR)   and esta(RT-PCR) . For eac(RT-PCR) .2.2260/280 ratios from 1.8 to 2.1 was visually checked on 1.5% (w/v) agarose gel. Reverse transcription of RNA to cDNA was performed from 2 μg of total RNA using Superscript III™ First-Strand Synthesis System  following manufacturer recommendations. An aliquot of the RNA was used to prepare small RNA libraries.Systemically infected leaves of cultivar 60444 and TME 204 showing clear CBSD symptoms, and stem sections of NASE 3 showing lesions typical of CBSD infection were collected six weeks after grafting (WAG). Similar tissues were sampled for use as controls from asymptomatic, non-inoculated plants of each genotype. Presence of CBSV and UCBSV was detected by RT-PCR and virus levels estimated by quantitative RT-PCR (qRT-PCR) as described previously . The pla2.3® Multiplex Small RNA Library Prep Set for Illumina® (Sets 1 and 2)  following manufacturer recommendations. Briefly, 3′ adapters were ligated to total RNA (6 μg) followed by hybridization of the reverse transcriptase primer to prevent adapter-dimer formation. Subsequently, 5′ adapters were ligated and the RNA subjected to reverse transcription and PCR amplification. The PCR products were purified using a QIAQuick PCR Purification Kit  according to manufacturer recommendations and quality-checked by electrophoresis on a 6% polyacrylamide gel. Bands of ∼140 bp corresponding to the adapter-ligated small RNA fragments were isolated, crushed in elution buffer and re-precipitated using a mixture of Acrylamide, sodium acetate, and absolute ethanol. The small RNA libraries (cDNA pellet) were re-suspended in TE buffer  and re-checked for size and concentration by electrophoresis in 6% polyacrylamide gel.Small RNA libraries were prepared using the NEBNext2.4The small RNA libraries were sent to the Genome Technology Access Center (GTAC), Washington University in St. Louis, Missouri, USA for further size, quality, and integrity checking by an Agilent 2100 Bioanalyzer . Sequencing was performed by Illumina HiSeq 2,500 using 1 × 50 single-end read protocol. Raw sequence data received from GTAC was de-multiplexed by QIIME . Sequenc2.5Cq) value of each triplicate reaction was used for further calculations by the 2−ΔΔCT method for relative normalized expression analysis using COX mRNA as reference gene, and CBSV or UCBSV positive samples of predetermined virus concentration as calibrator, respectively. The relative expression levels were obtained by comparing CBSV and UCBSV titers in virus-infected plants with uninfected plants.Quantitative RT-PCR (qRT-PCR) was performed to measure the expression levels of DCLs and AGO2 in CBSV- and UCBSV-infected cassava using cytochrome c oxidase (COX) mRNA as the reference gene for normalization as previously described . The pri33.1N = 8) of genotypes TME 204 and 60444 grafted with CBSV-infected scions started showing typical CBSD symptoms two weeks after grafting, a week earlier than plants grafted with UCBSV-infected scions . UCBSV was not detected by RT-PCR in plants of NASE 3 challenged with UCBSV. These results correlated with the visually assessed disease symptoms and indicated that CBSV was able to infect and accumulate viral RNA in all plants across genotypes, whereas UCBSV infected and accumulated viral RNA in TME 204 and 60444 but not in NASE 3 plants.3.2c. 10,000 to 1.3 million across libraries. The majority of small RNA reads were host derived. CBSV-derived small RNAs from cultivars TME 204, 60444 and NASE 3 challenged with CBSV accounted for up to 7%, 13% and 10%, respectively, of the total clean small RNA reads. Similarly, UCBSV-derived small RNAs in TME 204 and 60444 graft-inoculated with UCBSV accounted for 0.4% and 2.6%, respectively  followed by 22 nt size class (19–37%) for both viruses across the three cassava genotypes C. ProporDifferent proportions of CBSV- and UCBSV-derived small RNAs were also reflected in line graphs obtained from normalized genome coverage data for each virus A–F. The A. thaliana, sorting of small RNAs into Argonaute complexes is directed by the first 5′ nucleotide  proteins and their correlation to the abundance of 21 and 22 nt vsRNAs in CBSV- and UCBSV-infected plants. Differing patterns of expression for these genes were observed across the three cassava genotypes when they were infected with the two viral pathogens. In CBSV-infected TME 204, there was down-regulation of MeDCL2 mRNA and 1.2-fold up-regulation of MeDCL4 mRNA compared to the uninfected control. In UCBSV-infected TME 204, both MeDCL2 and MeDCL4 transcript levels were downregulated. In CBSV-infected 60444, both MeDCL2 and MeDCL4 transcript levels were downregulated, whereas in UCBSV-infected 60444 plants both MeDCL2 and MeDCL4 mRNA levels were upregulated  mRNAs was determined by qRT-PCR. The results showed that MeAGO2 mRNA was upregulated in all three UCBSV-infected cassava genotypes and CBSV-infected TME 204 and NASE 3 plants. However, MeAGO2 expression remained unchanged in CBSV-infected 60444 , 60444 (CBSD susceptible) and NASE 3 (CBSD tolerant)  were chaQuantitative RT-PCR analysis also showed that NASE 3 accumulated 2.7- and 1.4-fold higher CBSV RNA than CBSV-infected plants of 60444 and TME 204, respectively . Similarly, UCBSV-infected 60444 accumulated 1.4-fold more UCBSV RNA than UCBSV-infected TME 204 . Comparable qRT-PCR data was obtained in a previous study using field and glasshouse samples from the same cassava genotypes . Data prA. thaliana showed that the 24 nt siRNAs were most abundant (35%), followed by 21 nt siRNAs (28%), but upon infection with cabbage leaf curl virus (CaLCuV), the host siRNA profile was altered such that the 21 nt class became more abundant (32%) followed by the 24 nt class (28%)  . Also, iss (28%) . Contrast siRNAs .A. thaliana have shown that DCL4, DCL2 and DCL3 generate the 21, 22, and 24 nt vsRNAs , crucifer-infecting strain of tobacco mosaic virus (TMV-Cg), cucumber mosaic virus (CMV), and tobacco rattle virus (TRV) (Nicotiana benthamiana infected with potato virus x (PVX),bamboo mosaic virus (BMV), and pepper mild mottle virus (PMMoV) (Reports in t vsRNAs . In addius (TRV)  and Nico (PMMoV) ; potato  (PMMoV) ; grapevi (PMMoV) ; cotton  (PMMoV) ; and mai (PMMoV) , among o (PMMoV) . Similar (PMMoV) B. The lo (PMMoV) .DCL2 and DCL4 proteins are key components of RNAi pathways and both are required for optimal resistance against viruses. Despite differences in the silencing activity of their small RNA products, DCL2 and DCL4 mostly act redundantly yet hierarchically when present simultaneously . QuantitProcessing of dsRNAs by Dicer proteins would ideally yield an equal amount of sense and antisense vsRNAs, the life span of which depends on selective incorporation into specific AGO proteins . AnalysiA. thaliana infected with TuMV and PVX, Cucumis melo infected with WMV (A. thaliana compared to wild type plants revealed the mutants were highly susceptible to TCV and CMV infection (The preferential use of vsRNAs with A or U residues as compared to C and G residues as the first 5′-end nucleotide has been reported in with WMV , and in with WMV . In contwith WMV , and tomwith WMV . Howeverwith WMV , probablwith WMV . Our reswith WMV A. In addnfection . Besidesnfection . Our resA. thaliana (Host RDRs use viral ssRNA to synthesize dsRNAs, which serve as substrates for DCL-dependent formation of secondary vsRNAs to maintain systemic silencing throughout the plant. In addition, DCL4 and RDR1 were reported as major contributors to the abundant pool of 21 nt TuMV-derived siRNAs in thaliana . HoweverFurthermore, viral suppressor proteins have been shown to employ a multitude of mechanisms . For exaPotyviridae. The use of deep sequencing in this study has provided an insight into the molecular interaction between CBSD causing viruses and cassava. The populations of vsRNAs were abundant, diverse and revealed widespread targeting of viral genomes by machinery of the gene silencing pathway. The overall composition of sRNAs in virus-infected cassava unveiled the action of different Dicer proteins in different cassava genotypes. The findings also provided an insight into the differential susceptibility of host plants to the same virus, which is reflected in the severity of symptoms they induce. Finally the results indicate that CBSV and UCBSV interact differently in the same host genetic background.To our knowledge, this study provides the first high-resolution genome map of vsRNAs for an ipomovirus in the family The authors have no conflict of interest to declare.


To present evidence on the total costs and unit costs of delivering six integrated sexual reproductive health and HIV services in a high and medium HIV prevalence setting, in order to support policy makers and planners scaling up these essential services.A retrospective facility based costing study conducted in 40 non-government organization and public health facilities in Kenya and Swaziland.Economic and financial costs were collected retrospectively for the year 2010/11, from each study site with an aim to estimate the cost per visit of six integrated HIV and SRH services. A full cost analysis using a combination of bottom-up and step-down costing methods was conducted from the health provider’s perspective. The main unit of analysis is the economic unit cost per visit for each service. Costs are converted to 2013 International dollars.The mean cost per visit for the HIV/SRH services ranged from $Int 14.23 (PNC visit) to $Int 74.21 (HIV treatment visit). We found considerable variation in the unit costs per visit across settings with family planning services exhibiting the least variation ($Int 6.71-52.24) and STI treatment and HIV treatment visits exhibiting the highest variation in unit cost ranging from ($Int 5.44-281.85) and ($Int 0.83-314.95), respectively. Unit costs of visits were driven by fixed costs while variability in visit costs across facilities was explained mainly by technology used and service maturity.For all services, variability in unit costs and cost components suggest that potential exists to reduce costs through better use of both human and capital resources, despite the high proportion of expenditure on drugs and medical supplies. Further work is required to explore the key drivers of efficiency and interventions that may facilitate efficiency improvements. The debate concerning how to best organize sexual and reproductive health (SRH) and HIV services is long standing. The integration of SRH and HIV services evolved in the 1990s in response to the rapid rise in the HIV epidemic in sub Saharan Africa, which heightened global concerns about the relative lack of services to address broader SRH problems –7 have cAlthough the benefits of integrating these services in terms of increased access to HIV services and continuity of care in these two settings have been well articulated, there remains a dearth of evidence on the costs of integrated SRH/HIV services –10. In aThis paper therefore presents evidence on the total costs and unit costs per visit of delivering six integrated HIV and SRH services across 40 providers in Kenya and Swaziland. Accurate data on economic costs of delivering integrated HIV and SRH services are essential for a range of applications. First, empirical data on costs of these services can be used for resource requirements, programme planning and budgeting purposes. Second, these data can also be used be used by researchers interested in exploring the cost-effectiveness of HIV/SRH services and contribute to efforts aimed at improving efficiency in service provision.This study was conducted as part of the Integra initiative, a non-randomized trial aimed at evaluating the impact of different models of delivering integrated HIV and SRH services on a range of health and service outcomes . The IntBroadly, the Integra initiative set out to evaluate three different models of integrating HIV and SRH service in both countries. These were: integrated FP model which promoted the integration of HIV and STI services into existing FP services; integrated post natal care (PNC) model which promoted integration of HIV and STI services into PNC/FP services; and integrated HIV/STI services provided within SRH clinics . A summaWith national HIV prevalence rates of 26%  and 7.4%To obtain an understanding of the costs of integrated HIV and SRH services, costing of integrated HIV and SRH services was carried out in 30 health facilities in Kenya and 10 health facilities in Swaziland. The study sites were purposefully chosen from six priority regions with established programming based on previous operational research relationships with the Ministries of Health in Swaziland and Kenya . Study sThe study focused only on the MCH-FP and HIV units and integration of HIV/STI services within the MCH-FP units. HIV services evaluated in this study included treatment of STIs, counselling and testing for HIV (HCT) and HIV treatment , ART and treatment of opportunistic infections). SRH services included FP, PNC and cervical cancer (Ca Cx) screening routinely provided within the MCH-FP units. A detailed summary of the service descriptions is provided in Ethics approval for the Integra study was obtained from the Ethical Committee at LSHTM , from the Population Council Review Board (protocol nos. 443 and 444), from the Kenya Medical Research Institute  and the Swaziland Scientific and Ethics Committee . Written informed consents were obtained for all Integra study activities.A retrospective costing study was undertaken from the health provider perspective using a combination of bottom-up and step-down costing methods . The botPrior to cost data collection, a periodic activity review (PAR) tool was developed to document the nature, range and methods of delivery of HIV and SRH services in each health facility. Specifically, the PAR provided an understanding of how resources are combined to produce integrated HIV and SRH services. The PAR tool included questions on facility characteristics, staffing types and levels, scope and number of services offered, descriptions of client flow, and overall description of the integration of services. The PAR tool was implemented in all SRH/HIV clinics and laboratory and pharmacies (if available onsite) from July 2010—June 2011 and April 2010—March 2011 in Kenya and Swaziland respectively.Following the PARs in each health facility, cost and output indicators were collected retrospectively for the financial year 2010–2011. Cost data were collected for the entire facility in the health centres, public health units and SRH clinics; while in the hospitals, cost data were collected for the MCH-FP/HIV departments only.Costs were classified into two main categories: capital and recurrent costs. Capital costs considered included buildings, equipment and training costs. All capital costs were annualized and discounted at the standard rate of 3% . The facTraining related to HIV/STI and SRH service delivery was identified through interviews with service providers. Training costs were then estimated from training facilitation costs, staff per diems and transport allowances received by the service provider.Recurrent costs included building maintenance (including utility expenses), transport costs, staff salaries drugs, diagnostics and supplies. Utility expenses were obtained from the central administration expenditure records for each facility. Personnel costs including benefits and allowances were estimated for all staff working in the MCH/PHU, HCT, HIV, pharmacy and laboratory departments, on the basis of position and salary levels. Expenditures on the recurrent costs of drugs, diagnostics and supplies were obtained from requisition notes and records within the facilities.Overhead and administrative costs associated with the different HIV/SRH services were allocated using the step-down costing approach. Room space was used to allocate utilities and building maintenance and staff numbers in each unit used to allocate management and administrative costs. Costs of drugs, diagnostics and supplies were allocated to each individual service based on actual resource usage. This was obtained through a combination of staff observations, staff interviews and patient records. In particular, staff time was allocated using a combination of an initial interview, followed by observations, followed by a week of timesheet reporting and then a follow-on interview to confirm allocations.The same costing methods were used in all the 40 study sites. Two researchers collected data across sites and results were quality controlled by a third researcher. All costs were converted to standardized 2013 international dollar (Int$ 2013) using the general purchasing power parity (PPP) index  rather tThe main unit of this analysis is the unit cost per visit for each service, calculated by dividing the total costs for each service by the number of client visits of the respective services. Data on HIV and SRH service utilization was collected from registers and monthly reports at the facility level. In few instances where client registers were missing and service statistics were not kept, estimates of services provided were made through a review of the drug dispensing records and interviews with staff to determine average number of consultation visits made for a particular condition or need.We present total and unit costs by country, facility type and model of integration, in order to illustrate the extent of variation in cost across a range of providers. Costs were further assessed and presented as fixed or variable costs. Variable costs include the costs of drugs, diagnostics and supplies, which vary with changes in output whereas fixed costs include capital, salaries, and other building maintenance costs that did not vary with output levels within the time period of a year.We also observed considerable variation in the relative proportions of visits and costs for the different services by health facility type. In Kenya, all the facility types provided more SRH services; compared to HIV services, with SRH services accounting for between 47% and 86% of visits and their proportion of total costs ranged from 26% to 77% across facility types. In Swaziland, SRH visits accounted for between 12% and 89% of total visits and 4% to 70% of total costs of HIV/SRH services. The public health units and SRH clinics in Swaziland provided more SRH visits compared to the hospital and health centres and in these facilities, costs for these visits together accounted for >50% of HIV and SRH service costs. SRH visits in the hospital and health centres only accounted for 4%– 8% of total HIV/SRH service costs.The mean unit costs per visit by facility type and model type for each service disguises considerable variation found between individual health facilities across the two settings, When analyzed by service type, cervical cancer screening visits had the highest proportion of fixed mean costs accounting for (69–92%) in both countries. In contrast, variable costs are the most expensive component of the mean unit cost per HIV care and treatment visit accounting for the 71% to 84% of the mean unit cost per visit.This is the first study to our knowledge to provide a detailed description of the resources used to deliver integrated HIV and SRH services across different settings. Our findings show that HCT and HIV treatment costs account for a significant proportion of total health service costs in both Kenya and Swaziland. However, there is considerable variation in the unit costs, levels of fixed costs and patterns of input use in the provision of integrated HIV and SRH services between facilities suggesting considerable room to improve efficiency at both the facility and service level.Our analysis showed that even after adjusting for differences in relative prices, there were still large disparities in unit costs between the two countries. Some variation in unit costs across the different services may be associated with site characteristics, although we found few consistent patterns across facility types. For example, in Kenya, the estimated unit costs of FP and PNC visits were consistently higher in the NGO SRH clinics compared to the other health facility types. The higher unit cost for these visits in the NGO clinics may be indicative of provision of more complex FP methods such as intrauterine contraceptive devices, implants and bilateral tubal ligations which require more equipment and staff time.There are also service specific explanations for the differences in costs between facilities. Some of these can be explained by the technology used. For example the wide range in unit costs per family planning and cervical cancer screening visits result from the wide variation in methods provided within the facilities. The underlying data confirm that facilities providing more long term FP methods and pap smears for cervical cancer screening as opposed to visual inspection had higher unit cost per visit.Besides the method mix, most of the variability in costs stemmed from the variability in the level of fixed rather than variable unit costs. The high proportion of fixed costs as a proportion of total unit costs, suggests that there is a mismatch between planning of fixed resources and the demand for services. It should also be noted that, despite the fact that HIV care and treatment has the highest proportional mean unit variable cost per visit, it has one of the highest absolute level of fixed unit cost per visit. From an HIV programme perspective drugs and variables costs may be of most concern to planners. However, given the overall percentage of HIV care and treatment related visits particularly in the higher level facilities in Swaziland costed, HIV care and treatment related fixed costs may be key to the planning of fixed resources at the facility level.Another explanation for service specific variation may also be service maturity . This maMean unit costs estimated in our study for HCT and HIV treatment visits in Kenya and Swaziland did not differ greatly from costs estimated in previous studies within sub Saharan Africa –29 from Some limitations of this analysis should be noted. While the study incorporates high quality micro-costing methods rarely used in low and middle income countries, routine monitoring data was used to estimate the unit costs. Although this was partially validated through comparison with other study instruments from the broader study, it is likely that the use of routine services will bias our results. There may be an incentive for example to over-report visits that may result in lower unit costs; or alternatively reporting may be incomplete, that would result in an over-estimation of unit costs. This reporting may also vary by service. A particular concern is the reporting of STI visits, which were not uniformly recorded across sites resulting in higher unit costs estimated for these services.Secondly, this analysis excludes the above service delivery costs or costs incurred at the administrative level outside the point of service delivery which may comprise an important component of costs, particularly fixed costs . Such coNotwithstanding these limitations, the findings of this study yield important policy and practice implications regarding the optimization of health resources to improve efficiency at the health facility level. First, this study indicates that the current level of efficiency of integrated HIV and SRH services can still be improved. Second, the study findings suggest that on its own, integration does not resolve the issue of cost variation between services, although more work is required to isolate the specific impact of integration efforts. However, given that fixed costs account for a significant proportion of unit costs particularly for SRH services, and vary considerably between sites, integrated delivery of HIV and SRH services still offers the potential for better use of resources. It also suggests that in some settings, the fixed capacity exists to absorb this extra demand, but it is also clear that even when a policy of integration has been adopted this is not always achieved in practice. Further guidance is therefore required for facility managers on staffing services, not solely from a clinical perspective, but also taking into account the staff workloads and local demand for different HIV/SRH services.Finally our findings highlight the complexity of the factors that may influence costs. Many of these issues are hard to address at the national level, yet managers at the facility and district level rarely have access to data on the underlying costs of the inputs and outputs they provide. Despite the fact significant investments have been made in decentralizing health systems, it is still difficult to find the necessary data to conduct and interpret even the simplest costing at the local level; and without this, it is unreasonable to expect that managers integrating services are able to move towards the lowest cost model. Policy makers and planners should therefore focus on strengthening simple information systems that match cost with financial information which would help managers identify local solutions that fully reflect the range of factors driving inefficiencies.This study provides the most complete evidence to date on the unit costs of HIV and SRH services, in a variety of facilities, across a medium and high HIV prevalence setting including a unique description of the cost breakdowns for each visit type. The considerable variation in unit costs of integrated HIV and SRH services found suggests a potential to improve efficiency. Given the large proportion of fixed costs for most of the services, if potential efficiency gains are to be realized, better use of existing human resources at the facility level should be advocated alongside integration policies and generation of demand for services. Finally, while this study has provided an important characterization of the costs of different HIV and SRH services in multiple sites, further research and analysis of these data is required to examine the determinants of costs, including whether the extent of integration has an impact on costs.S1 Fig* Mean cost per visit includes drugs, diagnostics and supplies.(TIF)Click here for additional data file.S1 Table(PDF)Click here for additional data file.