Genital ulcer disease (GUD) prevalence increases in the first month of antiretroviral treatment (ART), followed by a return to baseline prevalence by month 3. Since most GUD is caused by herpes simplex virus type 2 (HSV-2), we hypothesized that genital HSV detection would follow a similar pattern after treatment initiation.We conducted a prospective cohort study of 122 HSV-2 and HIV-1 co-infected women with advanced HIV disease who initiated ART and were followed closely with collection of genital swab specimens for the first three months of treatment.At baseline, the HSV detection rate was 32%, without significant increase in genital HSV detection noted during the first month or the third month of ART. HIV-1 shedding declined during this period; no association was also noted between HSV and HIV-1 shedding during this period.Because other studies have reported increased HSV detection in women initiating ART and we have previously reported an increase in GUD during early ART, it may be prudent to counsel HIV-1 infected women initiating ART that HSV shedding in the genital tract may continue after ART initiation. Herpes simplex virus (HSV) is a common sexually transmitted infection (STI) among HIV-1 infected persons . With wiWe previously reported a significant increase in GUD prevalence among HIV-1 infected women with advanced disease in the first month of ART, compatible with immune reconstitution inflammatory syndrome, followed by a return to baseline by the end of the third month . Given tIn this study, we analyzed prospectively collected data and tested genital swab samples for the presence of HSV DNA and HIV-1 RNA among women co-infected with HSV-2 and HIV-1. Our objective was to determine the pattern of genital HSV detection in co-infected women in the early months following ART initiation and its relationship to genital HIV-1 shedding.We conducted a prospective study of HSV-2 seropositive women with advanced HIV disease who initiated ART. The women were part of an ongoing cohort study of female sex workers in Mombasa, Kenya.Data were collected from women who participated between March 2004 and September 2007 in a study to evaluate the effect of ART initiation on genital HIV-1 shedding. Women were invited to participate if they were eligible to initiate ART according to WHO and Kenyan national guidelines at that time (CD4 count <200 cells/μL or WHO stage 4 disease). All women initiated a standard treatment regimen of stavudine or zidovudine, lamivudine and nevirapine.Data were collected from two different, non-overlapping populations of participating women. Population 1 comprised a subgroup of 21 women who consented to intensive follow-up in the first month of treatment. After screening and enrollment, follow-up visits with genital HSV swab collection were on days 1, 2, 4, 7, 14, and 28. Data from this population were used in the analysis of genital HSV detection in the first month of ART. Population 2 comprised 102 women who were followed monthly after ART initiation, with collection of swabs for genital HSV detection at baseline and month 3. Data from this population were used to evaluate changes in genital HSV detection and the association between genital HSV and HIV-1 detection at baseline and at month 3 of ART.Visit procedures were standard and identical for both populations. At enrollment and at each subsequent visit, participants underwent a questionnaire-guided, face-to-face interview followed by a standardized physical examination which included a pelvic speculum examination and screening for genital infections. Genital specimens for HSV detection were collected by inserting a Dacron swab into the cervical os, then rolling it firmly along the lateral fornix, vaginal wall, vulva, labial region, and finally the perianal region. Swab samples were placed in 1 mL of PCR digestion buffer and stored at -70°C until shipment to Seattle. Details on the specimen collection procedure for genital HIV-1 detection have been described elsewhere .HSV-2 serology testing was performed on archived frozen specimens using a type-specific, HSV-2 IgG-based ELISA . HSV-2 seropositivity was determined as an index value  >1.1, according to the manufacturer’s instructions . At the T. vaginalis were detected by light microscopy of vaginal saline wet preparations. BV was evaluated by Nugent scoring of a vaginal Gram stain.After Gram staining of cervical secretions, the number of polymorphonuclear leukocytes in three non-adjacent oil immersion fields on microscopy was quantified. Yeast and 10-transformed to approximate normality. Linear mixed effects models with a Poisson link were used to analyze associations with HSV detection in each study population, as such models provide estimates of within-person  effects following treatment initiation and produce results in the form of an incidence rate ratio, which is more easily interpreted than an odds ratio.The primary outcome was the frequency of genital HSV detection at the pre-ART baseline and at each time point after ART initiation. HIV-1 RNA was set at half the lower limit of detection (50 copies/swab) for samples with a result below 100 copies/swab. Because HIV-1 RNA values were not normally distributed, they were log10-tranformed vaginal viral load (VVL) and cervical viral load (CVL) were analyzed as separate predictors of genital HSV detection. Second, these two variables  were combined into one dichotomous variable , which was then analyzed for its association with genital HSV detection.We selected two primary predictors to test: time point and genital HIV-1 shedding. For population 1, each participant contributed one row per time point assessed for HSV shedding . For population 2, participants contributed one row for baseline and one row for the month 3 visit. Time point was assessed using a series of indicator variables. Two different approaches were taken to analyze genital HIV-1 as a predictor of HSV detection. First, logPotential confounders analyzed in bivariable analysis for their association with genital HSV detection at any time point included baseline CD4 count, WHO clinical stage at treatment initiation, hormonal contraception use, menstrual status, douching with soap, and the presence of vaginal infections or cervicitis. Final multivariable analyses were performed with time point, cervical and vaginal viral loads (model 1) or genital HIV-1 detection (model 2), adjusted for any potential confounder that was associated with HSV detection at a p value ≤0.10 in univariate analysis. Finally, Chi squared or Fisher’s exact tests were used to compare binary outcomes  at baseline and month 3.The study was approved by the ethical review committees of the Kenya Medical Research Institute, the University of Washington, and the Fred Hutchinson Cancer Research Center. Prior to obtaining approval, all consent forms, case report forms and other data collection tools were reviewed by the IRBs. All participants gave written informed consent.Baseline demographic and clinical characteristics including median age, years of education, and CD4 count at ART initiation were broadly similar between the two populations . Of 21 w10, with a median of 4.36 log10. One of two women with HerpeSelect index values <3.5 had genital HSV detected at two visits. HSV detection was somewhat less frequent on days 1–4 and somewhat more frequent at day 28 than at baseline  women at baseline and in 10 of 20 (50%) women at day 28. Overall, genital HSV was detected at 45 of 142 (31.7%) visits in the first month of ART. At each visit, genital HSV detection ranged between 19% and 50%. When detected, HSV DNA levels ranged from 2.20 to 7.53 logbaseline .3 at initiation of treatment was associated with increased genital HSV detection in the first month of ART (p = 0.01). In multivariable analysis with the a priori inclusion of log10 vaginal and cervical HIV-1 viral load and of baseline CD4 count and bacterial vaginosis based on p values, there was still no significant change from baseline in HSV detection at any time point during the first month of ART, including at day 28 .Associations with HSV detection at any of the seven timepoints during the first month of ART are presented in 10, with a median of 4.69 log10. All three women with HerpeSelect index values <3.5 had genital HSV detected on at least one visit. a priori inclusion of log10 vaginal and cervical HIV-1 viral load and baseline CD4 count (based on results in Population 1), there was no significant change from baseline to month 3 in genital HSV detection .Genital HSV was detected in 32 of 100 women (32.0%) at baseline, in 36 of 95 women (37.9%) at month 3, and overall during 68 of 195 (34.9%) of study visits by population 2. When detected, HSV DNA levels ranged from 2.27 to 7.62 logAt baseline, 100 women (99%) in population 2 had genital HIV-1 detected compared to 32 (32.0%) who had genital HSV detected. At month 3, genital HIV-1 detection reduced by more than half , in contrast to a slight increase in genital HSV detection . There was no association between genital HSV and HIV-1 detection at either day 0 or month 3 . Baseline genital HSV detection had a borderline association with genital HSV detection at month 3 (p = 0.06), but no association with genital HIV-1 detection at month 3 (p = 0.69). A visible ulcer on physical examination was associated with genital HSV detection (p = 0.012), but not with genital HIV-1 detection (p = 0.76).In this study, we describe genital HSV detection in HIV-1-and HSV-2-co-infected women at several time points during the first three months of ART, along with its association with genital HIV-1 shedding. Genital shedding of HSV was very frequent, occurring at up to 50% of visits at some time points and overall on 31.7 and 34.9% of visits for the two populations respectively. The prevalence of genital HSV detection decreased from baseline during the first 4 days of ART initiation, then was somewhat higher than baseline at day 28 and month 3, but these changes in prevalence did not reach statistical significance. Genital HIV-1 shedding at ART initiation was also not associated with genital HSV detection during this period. In general, genital HSV shedding persisted at levels similar to baseline in the setting of rapidly declining genital HIV-1 RNA levels.Data on the effect of ART on genital HSV shedding are inconclusive. Four studies have reported a significant effect of ART in reducing clinical reactivations associated with HSV, but no significant effect on genital HSV shedding –12. One This latter study, by Tobian et al , shares We found no association between genital HSV and genital HIV-1 detection at any time point, in contrast to studies of women not taking ART , 16. In Our study had several strengths. Participants were followed frequently in the first month in population 1, which enabled us to collect closely timed samples and detect changes in both genital HSV and genital HIV-1 from as early as the first day of treatment. Genital samples for HSV were also collected from an extensive area of the external genitalia, corresponding to where HSV-2 reactivates. Our study was limited by the small sample size of population 1. Because this study is exploratory, we did not correct for multiple comparisons. Finally, the study population included women with advanced immunosuppression, who may not be representative of the general population of women in Kenya or in other locations for this and other reasons.In conclusion, we found that genital HSV detection was extremely prevalent both at baseline and at 3 months after ART initiation, without appreciable changes. Genital HSV detection was not associated with genital HIV-1 detection at any time point, nor did it predict future genital HIV-1 detection during the first three months of ART. Because other studies have reported increased HSV detection in women initiating ART and we and others have reported an increase in GUD during early ART, it may be prudent to counsel HIV-1 infected women initiating ART that HSV shedding in the genital tract may continue after ART initiation. However, our results suggest that HSV shedding does not substantially impact genital HIV-1 shedding in women who initiate ART.


The objective of our study was to estimate direct healthcare costs incurred by a population-based sample of people with psychological distress or depression. We used the 2002 Canadian Community Health Survey on Mental Health and Well Being and categorized individuals as having psychological distress using the Kessler-6, major depressive disorder (MDD) using DSM-IV criteria and a comparison group of participants without MDD or psychological distress. Costs in 2013 USD were estimated by linking individuals to health administrative databases and following them until March 31, 2013. Our sample consisted of 9,965 individuals, of whom 651 and 409 had psychological distress and MDD, respectively. Although the age-and-sex adjusted per-capita costs were similarly high among the psychologically distressed  and those with MDD  compared to the comparison group , the population-wide excess costs for psychological distress ($441 million) were more than twice that for MDD ($210 million) as there was a greater number of people with psychological distress than depression. We found substantial healthcare costs associated with psychological distress and depression, suggesting that psychological distress and MDD have a high cost burden and there may be public health intervention opportunities to relieve distress. Further research examining how individuals with these conditions use the healthcare system may provide insight into the allocation of limited healthcare resources while maintaining high quality care. Depression is an important public health concern that impacts approximately 350 million people worldwide  and is tPrevious costing studies for depression have focused on patients in primary care or psychiatric care settings rather than population-based samples, which may be more representative of all people with depression, particularly those who do not seek health services –11. ManyThe primary objective of this study was therefore to determine the direct healthcare costs associated with psychological distress and major depression in Ontario. The secondary objectives were to understand how these costs vary by healthcare sector and what proportion of total costs were for mental health and addictions (MHA) versus non-MHA related healthcare services.We used the Ontario sample of the Canadian Community Health Survey on Mental Health and Well Being, cycle 1.2 (CCHS 1.2) conducted by Statistics Canada in 2002 to select our study cohort. The CCHS 1.2 is a nationally representative community mental health survey of individuals aged 15 years or older living in private dwellings. The survey excluded individuals living on Indian Reserves and on Crown Lands, institutional residents, full-time members of the Canadian Forces, and residents of certain remote regions  .Using individual encrypted health card numbers, we anonymously linked survey respondents to multiple health administrative databases housed at the Institute for Clinical Evaluative Sciences (ICES) . We exclth edition (DSM-IV) systems [The CCHS 1.2 used the version of the World Health Organization (WHO) World Mental Health Composite International Diagnostic Interview (WMH-CIDI)  designed systems .In our study, the major depressive disorder (MDD) group included respondents who met the criteria for past 12-month MDD . Among tCosts for hospitalizations, ED visits, and outpatient visits were captured within health administrative databases . All bilFor each individual, we estimated costs incurred from the survey interview date (2002) to the earliest of death, loss of healthcare eligibility or March 31, 2013 (end of study period), such that maximum follow-up was approximately 11 years. All costs were divided by the total length of follow-up to estimate annualized costs and inflated to 2013 values. These were then presented in United States Dollars (USD) using the OECD defined Canadian Dollar (CAD)-USD Purchasing Power Parities averaged over the study time period (1 CAD = 0.82 USD) . We estiFor outcomes with non-zero costs , we used a generalized linear model with a gamma distribution and a log link. For outcomes that included both zero and non-zero outcomes , we used a two-part model, where the first part models whether costs occurred using a probit model and the second part models the level of costs among those with non-zero costs, conditional on incurring costs using a generalized linear model with a gamma distribution and a log link . The choResults from the two-part models are shown as predicted per-capita costs and per-capita cost differences using marginal standardization to estimate the predicted costs . Per-capWe estimated unadjusted, age- and sex-adjusted, and multivariable-adjusted costs. Multivariable-adjusted models included covariates measured at baseline that are thought to be associated with costs and mental health. Most covariates were derived from the survey responses including: age, sex, marital status (married/common law vs other), residence , ethnicity (white vs non-white), immigrant status (immigrant vs non-immigrant) , currentTo estimate the weighted number of individuals within each group we summed up the sample weights of all respondents within each group. To calculate the age- and sex-adjusted population-wide costs or population-wide excess costs, we multiplied the weighted sample size within each group by the age- and sex-adjusted per-capita cost or per-capita excess costs within each study group.From a total of 10,662 initial survey respondents, we excluded 356 respondents with other serious mental illnesses and 145 individuals without healthcare eligibility at baseline and a year prior to baseline, and 6 individuals withdata errors . There was a total of 190 (1.7%) missing observations which were excluded from the analysis. Our final sample consisted of 9,965 individuals: 651 with psychological distress, 409 with MDD and 8,905 comparison group individuals . The weiOur findings were consistent across unadjusted, age- and sex-adjusted, and multivariate adjusted models; below, we focus on age- and sex-adjusted results.The annual per-capita total costs were similar between the psychological distress  and MDD  groups and both had higher costs than the comparison group  . The psyAt the population level, 599,047 were psychologically distressed, 361,389 had MDD, and 8,260,184 individuals had neither condition. The age-and-sex adjusted annual absolute healthcare costs were $2.0 billion for the psychologically distressed population and $1.2 billion for the MDD population . This reTo our knowledge, this study is the first population-based cohort study to estimate the direct healthcare costs associated with individuals with psychological distress who do not meet the criteria for depression, compared to those with MDD and the comparison group with neither condition. Our study found that individuals with psychological distress and MDD incurred greater direct healthcare costs per capita than the comparison group, with significantly large excess outpatient and ED costs. Although on a per-capita basis, individuals with elevated distress have similar costs to those with depression, their annual population-wide costs were almost twice the cost burden associated with depression. Despite similar costs, only 4% of the costs incurred by those with psychological distress are associated with MHA-related health services compared to 14% among individuals with depression, suggesting that individuals with psychological distress may not be identified or treated for their mental health condition. We acknowledge that some of the non-MHA costs are due to higher chronic conditions, although at baseline none of the major chronic diseases were significantly more prevalent among individuals with psychological distress than in the comparison group and adjustment for chronic conditions resulted in minimal changes to the results. Our findings emphasize the need to better understand psychological distress and reasons for these elevated healthcare costs.Very few previous studies have investigated the association between psychological distress and healthcare costs ,15,35,36A primary finding of our study was that individuals with psychological distress cost the healthcare system a similar amount on a per-capita basis as those with MDD. Our findings are similar to results from a US study, which showed no statistically significant differences in costs between elderly patients with subthreshold depressive symptoms, a similar condition to psychological distress, and those with DSM-IV depressive disorders . HoweverAnother key finding is the dose-response gradient in mental health costs across the study groups. In a study by Kleine-Budde et al. , mean anOur study showed that the per-capita excess costs of psychological distress and MDD were $897 and $709 respectively. A systematic review by Luppa et al.  demonstrThis study has several strengths. First, unlike previous studies ,36, the This study also has some limitations that need to be considered. First, the CCHS excluded certain vulnerable populations , some ofIn this population-based study, we found that people with psychological distress and major depression incurred similar per-capita direct healthcare costs and these costs were greater than those without either condition. These two groups differed in the way the totals were reached, with depressed individuals incurring higher mental health and addiction-related costs and psychologically distressed individuals incurring higher non-mental health and addiction costs. Given the high prevalence of psychological distress, at the population level, this group incurred almost twice the healthcare costs compared to individuals with major depression, despite not meeting the clinical cut-off for MDD. The elevated rate of contact with the healthcare system among patients with psychological distress provides an opportunity for them to be identified and receive mental healthcare. Further research is needed to examine whether identifying and treating psychological distress will result in short- or long-term cost savings.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.


RNA silencing is a conserved mechanism that utilizes small RNAs (sRNAs) to direct the regulation of gene expression at the transcriptional or post-transcriptional level. Plants utilizing RNA silencing machinery to defend pathogen infection was first identified in plant–virus interaction and later was observed in distinct plant–pathogen interactions. RNA silencing is not only responsible for suppressing RNA accumulation and movement of virus and viroid, but also facilitates plant immune responses against bacterial, oomycete, and fungal infection. Interestingly, even the same plant sRNA can perform different roles when encounters with different pathogens. On the other side, pathogens counteract by generating sRNAs that directly regulate pathogen gene expression to increase virulence or target host genes to facilitate pathogen infection. Here, we summarize the current knowledge of the characterization and biogenesis of host- and pathogen-derived sRNAs, as well as the different RNA silencing machineries that plants utilize to defend against different pathogens. The functions of these sRNAs in defense and counter-defense and their mechanisms for regulation during different plant–pathogen interactions are also discussed. Small RNAs (sRNAs) are 20–30 nucleotide (nt)-long non-coding RNA molecules, which are widely present in eukaryotic organisms. It is well established that sRNAs are involved in the regulation of gene expression through a process generally termed RNA silencing. RNA silencing contributes to almost all eukaryotic cellular processes, including preventing the invasion of viruses or transgenes, inhibiting the movement of transposable elements, and regulating developmental and physiological processes .trans-acting siRNAs (ta-siRNAs), heterochromatic siRNAs (hc-siRNAs), natural antisense transcript-derived siRNAs (nat-siRNAs), and long siRNAs (lsiRNAs). Proteins, such as Dicer-like proteins (DCLs), HYPONASTIC LEAVES 1 (HYL1), HUA ENHANCER 1 (HEN1), and Serrate (SE) are involved in sRNA biogenesis pathways (Plant sRNAs are divided into two major classes: microRNAs (miRNAs) and small interfering RNAs (siRNAs). Most miRNAs are 21–24 nt in length and derived from RNAs with imperfectly base-paired hairpin structures , while spathways . Some sipathways . After ppathways . In planpathways .One of the important functions of RNA silencing is to suppress the infection of pathogens. The RNA silencing machinery of host plants can directly target the genomic RNA and transcripts of viruses, viroids, and virus satellites to suppress their RNA accumulation. However, plants are also susceptible to other pathogens, such as bacteria, fungi, oomycetes, and nemotodes, which unlike viruses, do not replicate or expose their genome in host cells during any part of the infection process. To defeat these pathogens, plants have evolved complicated defense systems, including PAMP-triggered immunity (PTI) and effector-triggered immunity (ETI) . When suNeurospora crassa .Although a potent immune system is necessary for plants to survive pathogen infections, it also deprives the limited resources available for plant growth and development. Although more studies need to be done, a constitutively active immune system in plants may result in reduced growth and seed yield . Thus, pFigure 1A). miR393 is the first miRNA shown to function in anti-bacterial defense. The accumulation of miR393 is up-regulated upon the treatment of the conserved N-terminal part of flagellin, flg22, or the infection of bacterial pathogen Pseudomonas syringae pv. tomato (Pst) DC3000. miR393 enhances host resistance to Pst DC3000 by negatively regulating the expression of F-box auxin receptors, including Transport Inhibitor Response 1 (TIR1), Auxin signaling F-Box proteins 2 (AFB2), and 3 (AFB3)  . Further drought . In addi defense . Further defense . Over-exesponses . Furtheresponses . Thus, mesponses . Anothernfection .Pst DC3000 effector protein AvrRpt2, enhances ETI by suppressing the expression of pentatricopeptide repeats (PPR) protein-like gene (PPRL) and preventing the negative effect of PPRL on the resistance pathway mediated by RPS2, a resistance gene that specifically recognizes effector AvrRpt2 . Magnaportbe oryzae is a rice blast fungus that causes rice blast disease. The accumulation of rice miRNA528/miR1879 is down-regulated by treatment with a M. oryzae elicitor, resulting in up-regulation of their target genes that control oxidative stress  gene and its overexpression results in enhanced resistance to M. oryzae infection  is a powdery mildew fungus that causes devastating disease in wheat, barley, and other plants. Eight different miRNAs, miR159, miR164, miR167, miR171, miR444, miR408, miR1129, and miR1138, that regulate three different defense response processes are significantly induced at the early, but not the late, stage of Bgt infection. Thus, these miRNAs may play a key role in HR at the onset of disease (Blumeria graminis f. sp. hordei (Bgh) (Mildew resistance locus a (Mla), encoding a group of CC-NBS-LRR proteins that respond to Bgh, are targeted by the miRNA family miR9863. miR9863 was shown to guide the cleavage of Mla1 transcripts in barley, and down-regulate the accumulation of MLA1 protein in the Nicotiana benthamiana expression system. In addition, miR9863 can trigger the biogenesis of 21-nt phased siRNAs (phasiRNAs) and further repress the expression of Mla1. Over-expression of miR9863 specifically attenuates Mla1-mediated cell death and disease resistance  . Mildew sistance . miR482 pression . The silpression . Phytophimmunity . Similarnfestans . It is cPotato virus X (PVX) infection. sRNAs complementary to the sense transcript of the transgene and the positive strand of PVX were discovered, indicating that sRNAs participate in PTGS transgene silencing and viral defense  was first identified in both transgenes processing and  defense . Further defense .Figure 3A). Nearly equal amounts of positive and negative strand vsiRNAs without positional bias were derived from Cucumber yellows closterovirus (CuYV), Turnip mosaic potyvirus (TuMV), CMV, Watermelon mosaic virus (WMV), PVX, and Tomato yellow leaf curl virus (TYLCV), all positive ssRNA viruses from different families (Rice stripe virus (RSV), an ambisense virus with four genomic ssRNAs (Cymbidium ringspot virus (CymRSV) are generated from the positive strand (Tobacco mosaic virus (TMV), Tobacco rattle virus (TRV), and Pepper mild mottle virus (PMMoV), in which some positive strand vsiRNAs can account for 97% of total vsiRNAs (Cauliflower mosaic virus (CaMV), a virus from which the commonly used constitutive 35S promoter is derived. Although up to 82% of vsiRNAs are generated from the leader region, these exhibit no strand bias (Tomato yellow leaf curl China virus (TYLCCNV) is a Geminiviridae that has an ssDNA genome. Although the vsiRNAs derived from TYLCCNV display site bias, they map nearly equally to the positive and negative genomes (Viruses contain either single-stranded RNA (ssRNA), double-stranded RNA (dsRNA), ssDNA, or dsDNA genomes . During families . It was c ssRNAs . Howevere strand . Similar vsiRNAs . There aand bias . Tomato  genomes . Thus, bPotato spindle tuber viroid (PSTVd) infection results in full methylation of the PSTVd cDNA sequence that is inserted into the tobacco genome  (Citrus exocortis viroid (CEVd) replicates in the nucleus and mainly generates 5′-phosphorylated and 3′-methylated vdsiRNAs with positive polarity. Most CEVd vdsiRNAs are located within the right-end domain, suggesting that structured RNA is the main substrate of DCL enzymes (Avocado sunblotch viroid (ASBVd), Peach latent mosaic viroid (PLMVd), and Chrysanthemum chlorotic mottle viroid (CChMVd) are three viroids that replicate in the chloroplast. CChMVd and PLMVd generate vdsiRNAs from both polarities  . Viroid-gure 4A) . Citrus  enzymes . Avocadolarities . ASBVd alarities . Thus, bn plants . The facArabidopsis encodes four DCL proteins that generate different sRNAs: DCL1 processes hairpin pri-miRNAs and pre-miRNAs into 21-nt miRNAs; DCL3, DCL4, and DCL2 process long dsRNAs into 24-nt hc-siRNAs, 21-nt siRNAs, and 22-nt siRNAs, respectively. For RNA viruses and viroids, the perfectly paired dsRNA intermediate replication form and the hairpin structure of the single genomic RNA are predominant dsRNA inducers. Indeed, DCL4, DCL2, and DCL3 process ssRNA viruses  into 21-nt, 22-nt and 24-nt vsiRNAs, respectively (Hop stunt viroid (HSVd)- and Hop latent viroid (HLVd)-infected plants. However, very little is known about the biogenesis of vdsiRNAs. 21-nt, 22-nt, and 24-nt vsiRNAs also accumulate in plants infected with Cabbage Leaf Curl Virus , Beet curly top virus (BCTV), and Pepper golden mosaic virus (PepGMV), which are all ssDNA viruses, and CaMV, a dsDNA virus  treatment and TMV infection in tobacco and Arabidopsis, and a mutation in RDR1 permits efficient multiplication of ssRNA viruses  showed that RDR1 suppresses RNA silencing mediated by RDR6 and enhances viral infection in transgenic plants . During the counter-defense response, these pathogen sRNAs facilitate infection by adjusting pathogen gene expression to increase virulence.During plant–microbial pathogen interaction, host miRNAs and siRNAs play a role in modulating host immunity while some sRNAs derived from pathogens can decrease host defense or increase pathogen virulence. Fungi, oomycetes, bacteria, viruses, viroids, and satellite RNAs all produce sRNAs that are either similar to or distinct from plant sRNAs   . Althougirulence . Moreove1 mutant . The sRN-nt long . The 21--nt long . sRNAs girulence . Some sRirulence . The bioproteins . PiAGO1-proteins . In addiilencing .Figure 1B)   that regulate the expression of target mRNAs through imperfect base-pairing of short regions (10- to 25-nt) (gure 1B) . There i of TLR2 . The Caslarensis . Howeveria (Xcv) . The expia (Xcv) . The 115r plants . Howeverlmonella . Hfp-depmotility .Figure 4B). Over-expression of PSTVd hairpin RNA, which produces sRNAs, results in similar phenotypes as PSTVd infection, suggesting that PSTVd may cause disease symptoms by sRNA-mediated silencing (Nicotiana soluble inorganic pyrophosphatase (siPPase) gene and leads to a PSTVd infection phenotype  and triggers signal transduction that eventually leads to viroid disease symptoms  targets and slices the SolWD40 gene, the function of which is unknown  (N. tabacum with a silencing-resistant version of Chll greatly reduces the Y-Sat symptoms (N. benthamiana (S-adenosyl homocysteine hydrolase (SAHH) and rgs-CaM, which will be discussed later  . More angure 3B) . The haigure 3B) . Y-sat psymptoms . satsiR-symptoms . Howeverthamiana . TCV is thamiana . Thus, sthamiana . DNA β sthamiana . The ßC1ed later . Thus, vFigure 3B). Deep sequencing and bioinformatics studies indicate that 16 TMV vsiRNAs potentially target Arabidopsis genes. Two of these vsiRNAs target and slice transcripts of a polyadenylation specificity factor and an unknown protein similar to translocon-associated protein alpha. The slicing of these two genes only happens upon TMV infection, revealing that they are real vsiRNA targets (Zea mays genes are predicted targets of vsiRNAs encoded by Sugarcane mosaic virus (SCMV). Some vsiRNA targets that contribute to biotic/abiotic stress responses and ribosome biogenesis are down-regulated upon SCMV infection  and Hibiscus chlorotic ringspot virus (HCRSV) suggest the existence of virus-encoded miRNAs that may target plant genes, but their detailed functions remain unknown  in both plants. Among these sRNAs, 73 Bc-sRNAs are able to target host genes in both Arabidopsis and S. lycopersicum. These Bc-sRNAs are processed by fungi DCLs and loaded into a host AGO1 protein to slice host targets. A mutation in Arabidopsis AGO1 reduces the susceptibility of the plant to B. cinerea, and a mutation in B. cinerea DCLs decreases fungi pathogenicity. Multiple Bc-sRNA target genes were identified, including Arabidopsis mitogen-activated protein kinase genes MPK1 and MPK2, a cell wall-associated kinase (WAK), a peroxiredoxin (PRXIIF), and the tomato MPK-kinase kinase 4 (MAPKKK4). Suppression of these genes increases the disease susceptibility of the plant  decreases the methylation of transgenic and endogenous loci by an unknown function; betasatellite βC1 inhibits the activity of SAHH (RNA silencing inhibits the infection, replication, and movement of same viruses at different steps. Thus, pathogens also encode RNA silencing suppressors to decrease the accumulation of sRNAs or inhibit the function of sRNAs . Many VS of SAHH .Pst DC3000 infection, the AvrPtoB effector specifically represses the induction of miR393 at the transcriptional level. AvrPto also reduces miR393 accumulation. However, the accumulation of pri-miR393 is not changed in transgenic plants, which indicates that AvrPto may post-transcriptionally down-regulate the processing of miR393 (P. sojae encodes two RNA silencing suppressors: PSR1 down-regulates the accumulation of both host miRNAs and siRNAs, while PSR2 specifically decreases the accumulation of host siRNAs (Phytophthora and viruses. PSR1 interacts with PINP1, a RNA helicase that regulates the accumulation of both miRNAs and siRNAs. The over-expression of PSR1 or the down-regulation of PINP1 impairs the localization of the DCL1 protein complex (P. infestans can also suppress RNA silencing and enhance the plant susceptibility to Phytophthora . Thus, the RNA silencing suppressors encoded by oomycetes might be a general counter-defense mechanism. It will be interesting to see whether fungi also deliver effector proteins to inhibit host resistance.Some bacteria and oomycetes also deliver effector proteins into host cells to suppress RNA silencing. Although miR393 is induced upon f miR393 . Oomycett siRNAs . Both of complex . Anotherophthora . Thus, tThere is an increasing amount of evidence that shows communication occurs between plants and different pathogens via sRNAs. The importance of sRNAs in regulating plant immunity and pathogen virulence allows scientists to utilize and manipulate RNA silencing machinery to improve plant immunity, impair pathogen virulence, and thus increase crop production. RNAi technology has been employed to manipulate plant metabolites, develop plants with improved resistance to environment stresses, and engineer plants to defend against pathogen infections . In planJH wrote the introduction, the summary of sRNA in plant and virus. MY wrote the summary of sRNA in bacteria and fungi. Both authors contributed to the figures and figure legend. XZ and LL supervised and complemented the writing.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Both the integrity and reproducibility of experiments using select agents depend in large part on unbiased validation to ensure the correct identity and purity of the species in question. Metagenomic deep sequencing (MDS) provides the required level of validation by allowing for an unbiased and comprehensive assessment of all the microbes in a laboratory stock. in vitro-passaged stocks of six viruses . Each library was dual indexed and pooled for sequencing. A custom bioinformatics pipeline determined the organisms present in each sample in a blinded fashion. Single nucleotide variant (SNV) analysis identified viral isolates. We confirmed that (i) each sample contained the expected microbe, (ii) dual indexing of the samples minimized false assignments of individual sequences, (iii) multiple viral and bacterial contaminants were present, and (iv) SNV analysis of the viral genomes allowed precise identification of the viral isolates. MDS can be multiplexed to allow simultaneous and unbiased interrogation of mixed microbial cultures and (i) confirm pathogen identity, (ii) characterize the extent of genomic variation, (iii) confirm the cell line used for virus propagation, and (iv) assess for contaminating microbes. These assessments ensure the true composition of these high-priority reagents and generate a comprehensive database of microbial genomes studied in each facility. MDS can serve as an integral part of a pathogen-tracking program which in turn will enhance sample security and increase experimental rigor and precision.Laboratories studying high-priority pathogens need comprehensive methods to confirm microbial species and strains while also detecting contamination. Metagenomic deep sequencing (MDS) inventories nucleic acids present in laboratory stocks, providing an unbiased assessment of pathogen identity, the extent of genomic variation, and the presence of contaminants. Double-stranded cDNA MDS libraries were constructed from RNA extracted from IMPORTANCE Both the integrity and reproducibility of experiments using select agents depend in large part on unbiased validation to ensure the correct identity and purity of the species in question. Metagenomic deep sequencing (MDS) provides the required level of validation by allowing for an unbiased and comprehensive assessment of all the microbes in a laboratory stock. Virology laboratories must maintain constant vigilance regarding the provenance of strains in their collections. In addition to confirming that experiments are being performed on the intended virus, unbiased methods that can detect viral coinfections and/or other microbial contaminants are critical for ensuring that experiments are faithfully reporting on the biology of a particular virus and not on a polymicrobial exposure. Stock assurance is exceptionally important when working with viruses which require a high level of biocontainment, given the increased security requirements and higher costs and higher stakes of the research.Bacillus anthracis, influenza virus, and smallpox virus highlight the potential for inadvertent laboratory contamination and the need to improve isolate-tracking methods  and their respective frequencies within a given stock. Molecular subtyping was critical in determining the source of the 2001 bioterrorism-associated anthrax outbreak  mycoplasma-contaminated Ebola virus (EBOV), (ii) mycoplasma-contaminated La Crosse virus (LACV) grown on cells with a known lymphocytic choriomeningitis virus (LCMV) contamination, (iii) vesicular stomatitis virus (VSV) grown on LCMV-contaminated cells, (iv) recombinant human respiratory syncytial virus (rHRSV) expressing enhanced green fluorescent protein (EGFP) from an additional transcription unit (ATU) , (v) recIn many sequencing applications, including MDS, it is critical to be able to distinguish low-level true-positive samples from false positives that result from assignment of sequencing reads to the wrong index (“index bleed-through”). Chimeric molecules produced during amplification steps in library preparation or on the sequencer during cluster generation are particularly susceptible to such misassignment. Creating library molecules with barcode sequences on both ends of the molecule  has been shown to reduce the rate at which this misassignment occurs by requiring concordance between the two barcode sequences .6 read pairs, and the two positive samples contained 65,284 and 203,565 virus-mapping reads . We aligned reads from all 50 pooled samples to a virus genome segment present in the two positive samples  paired-end sequences . The datMycoplasma spp., with 3.65 and 0.02 Mycoplasma reads per viral read, respectively. The rMV sample contained more than 900 nonredundant, paired-end Epstein-Barr virus (EBV) sequences  that were expected to come from the EBV-transformed human B-lymphoblastoid cell line (B-LCL) on which the virus was grown  barcodes, in which each cDNA molecule is uniquely indexed at the time of first-strand synthesis .IN) had 109 SNVs (72 nonsynonymous). A majority of SNVs are rare variants, having a frequency of less than 0.10. All six viruses had nonsynonymous and synonymous SNVs at frequencies of greater than 0.10. Four viruses had nonsynonymous SNVs at a frequency of at least 0.90, while five viruses had synonymous SNVs at a frequency of at least 0.90. All of the viruses contained several SNVs compared to their reference sequences that ranged in frequency, including some consensus-level SNVs, as listed in Homo sapiens-tc/COD/1976/Yambuku-Mayinga, complete genome strain (GenBank accession no. NC_002549), and (ii) Zaire ebolavirus strain Kikwit, complete genome (GenBank accession no. JQ352763). Comparing rates of correctly mapped reads did not reveal isolate identity. The same 2,501,8050 filtered read-pairs mapped at virtually identical percentages, 21.03% to EBOV Mayinga and 21.05% to EBOV Kikwit. However, SNV analysis revealed 223 SNVs in the reads that mapped to EBOV Mayinga with a frequency of ≥0.90, while there was only one SNV with a frequency of ≥0.90 in the reads that mapped to EBOV Kikwit  Zaire ebolavirus isolate Ebola virus/V Kikwit . Thus, wWe describe the successful application of a multiplexed MDS assay and bioinformatics pipelines for comprehensively interrogating the provenance of six virus isolates in a blinded fashion. With regard to multiplexing, the frequency of reads that are assigned an incorrect barcode is a function of the abundance of those reads in the correct barcode library and the degree of multiplexing. So, for high-titer viral stock-derived data sets, where viral reads are abundant, bleed-through occurs at a higher frequency. We demonstrated that dual indexing reduces the misassignment of sequencing reads by 37-fold in a pilot experiment and saw essentially no intersample cross-contamination in our multiplexed assay despite very-high-titer virus isolates. It is important to note that most commercially available dual-indexing kits do not provide this benefit because the reduction in misassignment depends on samples being identified by unique index pairs. Most commercially available kits do not actually provide unique index pairs. Instead, they mix and match indexes to create pairs with repeated index sequences, for instance, by permuting eight i5 indexes with 12 i7 indexes to achieve 96 pairs. We also found that using dual indexing decreased the total number of reads per data set by ~6% due to the removal of unassigned read pairs. However, this is a relatively small tradeoff for the large decrease in misassigned reads.Mycoplasma spp.) and six viruses across the six virus stocks. Four of these viral contaminating sequences  reflected the cells on which the viruses were cultured  as well as the additional, nonmicrobial sequences that can be assembled to characterize aspects of the host cell genome. This suggests that MDS is useful for virus forensic analysis, helping to identify the manner and cell type in which the virus was cultured or recovered.de novo assembly. However, this should be followed with a remapping of all the reads back to the assembled reference and a LoFreq* or similar SNV analysis, as we have demonstrated here. The latter analysis will reveal the presence of variations or even a mixture of strains that would be obscured or lost through an assemble-and-BLAST strategy alone.The SNV analysis identified numerous synonymous and nonsynonymous mutations present in each of the isolates. The presence and frequency of such mutations in longitudinal samples may be used to monitor inevitable virus adaptation rigorously in the laboratory setting and the quality of seed stocks  as well 17–In summary, we show that a single multiplex MDS assay can comprehensively assess (i) virus and isolate identity, (ii) SNVs in virus populations, (iii) the presence of viral coinfection, and (iv) the presence of bacterial contamination and (v) can provide information about the cell line used to propagate the virus.2O. The EBOV stock used for this analysis contained a known mycoplasma contamination.All work with infectious EBOV was performed under biosafety level 4 (BSL-4) conditions at the Integrated Research Facility, Rocky Mountain Laboratories (RML), Division of Intramural Research, NIAID, NIH, Hamilton, MT. Vero E6 cells (ATCC CRL 1586) were infected with EBOV at a multiplicity of infection (MOI) of 1, and virus-containing supernatants were clarified by low-speed centrifugation at 4 days postinfection (p.i.). Total RNA from supernatant (140 µl) was purified using the Qiagen viral RNA minikit, according to the RML standard operating procedures for virus inactivation. Purified RNA was eluted in HKS) stocks, B‑LCL cells were infected at an MOI of 0.01. After 4 days, when viral cytopathic effect was maximal, the stock was subjected to one freeze-thaw cycle to release the highly cell-associated measles virus into the medium. Cell debris was removed by low-speed centrifugation, and the supernatant was stored at −80°C as virus stock.To generate rMV Khartoum, Sudan (rMVRI) stocks, Vero cells expressing the CDV receptor canine CD150 were infected at an MOI of 0.01. After 2 days, when viral cytopathic effect was maximal, the stock was subjected to one freeze-thaw cycle to release cell‑associated virus into the medium. Cell debris was removed by low-speed centrifugation, and the supernatant was stored at −80°C as virus stock.To generate rCDV Rhode Island  strain, BHK cells were infected with VSVIN at an MOI of 0.01. Virus was propagated for 24 h before medium was removed. Cell debris was removed by low-speed centrifugation, and individual aliquots of virus were frozen for future analysis.For growth of the VSV Indiana , and RNA was isolated according to the manufacturer’s protocol. Purified RNA was resuspended in HKP071661.1) actually present in two of the 50 samples  using the Spliced Transcripts Alignment to a Reference (STAR) aligner , a component of the paired-read iterative contig extension (PRICE) assembler , followe aligner . Unalign aligner , 32. Aft aligner .JQ352763; LACV long (L) segment, NC_004108; LACV medium (M) segment, NC_004109; LACV short (S) segment, NC_004110; MV, HM439386; rHRSV, KF640637; VSVIN, J02428. The rCDVRI sequence (unpublished) was supplied by W. Paul Duprex (Boston University). According to the recommended guidelines for the variant caller, PCR-duplicate reads were removed using Picard tools (version 2.2.4). Variants were called using LoFreq* (version 2.1.2)  set to remove any read with less than 95% of nucleotides having a 0.98 probability of being correct (-rqf 95 0.98), any read with less than 90% called nucleotides, and any read that matched to the Illumina adapter sequences . Filteren 2.1.2)  using den 2.1.2) . It usesn 2.1.2) . VariantSRP076690).The 150-nt paired-end sequences are located in the National Center for Biotechnology Information (NCBI) Short Read Archive (SRA accession number


Despite the lack of evidence for symptomatic human infection with Maguari virus (MAGV), its close relation to Cache Valley virus (CVV), which does infect humans, remains a concern. We sequenced the complete genome of a MAGV-like isolate (OBS6657) obtained from a febrile patient in Pucallpa, Ucayali, Peru, in 1998. To facilitate its classification, we generated additional full-length sequences for the MAGV prototype strain, 3 additional MAGV-like isolates, and the closely related CVV (7 strains), Tlacotalpan (1 strain), Playas (3 strains), and Fort Sherman (1 strain) viruses. The OBS6657 isolate is similar to the MAGV prototype, whereas 2 of the other MAGV-like isolates are located on a distinct branch and most likely warrant classification as a separate virus species and 1 is, in fact, a misclassified CVV strain. Our findings provide clear evidence that MAGV can cause human disease. Aedes spp., Anopheles spp., Culex spp., Wyeomyia spp., and Psorophora spp.) in Ecuador, Brazil, Trinidad and Tobago, Colombia, Argentina, and French Guiana (Maguari virus (MAGV) was first isolated from a mixed pool of mosquitoes collected in Utinga forest in Brazil in 1957 and has since been isolated from a variety of mosquito species  reassortant, for which the name Itaya virus has been proposed , spanning 1957–1998 and originating from 3 of the 7 countries from which the virus has been isolated  . IsolateWe extracted viral RNA using the QIAamp viral RNA mini kit  according to the manufacturer’s instructions with reverse transcription PCR (RT-PCR) reactions performed using the Superscript III reverse transcription kit  and the iProof High-Fidelity PCR Kit . We obtained preliminary virus genome sequences of the MAGV BeAr7272 and AG83–1746 strains, as well as PLAV (strain 75V3066), TLAV (strain 61D240), and FSV (strain 86MSP18), using the 454 FLX pyrosequencing technology platform . We constructed libraries using previously described methods  Table 1;Phylogenetic and sequence divergence analyses were conducted on the open reading frames (ORFs) and their corresponding amino acid sequences. These analyses incorporated the sequences for MAGV, CVV, TLAV, PLAV, and FSV determined in this study and additional representative orthobunyavirus sequences available in GenBank  Table 2.Sequences for each ORF or corresponding amino acid sequence were aligned using the MUSCLE algorithm, and the evolutionary history for each tree construction was inferred using the neighbor-joining . Furthermore, an additional recombination analysis was performed on concatenated  genome sequences of all CVV, MAGV, PLAV, TLAV, and FSV isolates where full-length sequences were available. Initial alignment was performed by using the MUSCLE algorithm, as implemented in MEGA5 , the other comprises the AG83-1746 strain and the CbaAr426 strain . Both ofOur divergence analysis comparing the 2 groups formed by the isolates originally identified as MAGV showed 95.7% (N), 84.3%–85.2% (GPC), and 90.1%–90.7% (L) amino acid divergence. The extremely low level of N protein variability seems to be a characteristic of these and related virus groups, and the L protein divergence values are barely above the 10%-aa divergence sometimes suggested as a cutoff for speciation. However, the glycoprotein, which is the most variable of the viral protein products, differs substantially from all other related virus groups and also between these 2 groups. Furthermore, the consistent evidence for separate evolutionary histories inferred by the phylogenies for all 3 proteins indicates that these viruses most likely differ sufficiently to warrant reclassification of the AG83-1746 and CbaAr426 strains as a distinct virus species. In accordance with the prevailing naming conventions for bunyaviruses, which dictate naming based on the location of initial isolation, we use “Córdoba virus” (CODV) and “CODV lineage” herein to refer to viruses belonging to this group and to distinguish them from the canonical MAGV strains.In contrast to these various misidentified viruses, the OBS6657 isolate clearly grouped with the prototype MAGV strain BeAr7272 and showed 100% (N), 97.1% (GPC), and 99.0% (L) amino acid identity, respectively, across each of the three major gene products, despite being isolated >40 years later. Given that this virus was isolated from a patient exhibiting clear evidence of an acute febrile infection, our data provide strong evidence that MAGV infection can cause clinical disease in humans. In light of this evidence, and particularly when considered with serologic data that indicate frequent infection with MAGV-like viruses (2%–64% positive serum samples) . Taken together, the phylogenetic arrangement , malaise, muscle aches, and sore throat. The isolate cross-reacted very closely with CVV, MAGV, and PLAV virus in complement fixation but only with CVV and PLAV by plaque-reduction neutralization test  to the CVV group, and identification of FSV as a previously unrecognized CVV reassortant, is to substantially expand the known geographic range of CVV. On the basis of these data, the range of CVV must now be considered to include not only the United States and northern and central Mexico but probably all of Central America and the northern areas of South America. Furthermore, although both PLAV strains appear to belong to CVV lineage 1, TLAV belongs to CVV lineage 2, as does the CVV donor of the FSV M segment. This finding suggests that both lineages exist in Central America and that lineage 2 has been circulating in that region since at least the 1960s, when the first of these viruses were isolated. This supposition is also supported by the assignment of the MAGV CoAr3363 strain, which was isolated in 1964, to lineage 2. Taken together, these findings suggest that although these CVV lineage 2 strains have only recently emerged in the eastern United States, they might in fact have been circulating in South America for decades and have only recently been introduced into the United States from these regions.Our analysis of the ORF and amino acid data identified several distinct clades representing CVV (lineages 1 and 2) and 2 separate clades of what had been identified as MAGV strains . These cThe extent of this issue is further highlighted by our divergence analysis (In summary, we identified MAGV as the causative agent of a human febrile infection in Peru and showed that the virus associated with this infection is highly similar to the prototype MAGV isolate, suggesting that other viruses of this lineage also might have pathogenic potential in humans. In addition, our in-depth analysis of the closely related CVV group and other similar viruses showed that PLAV and TLAV do not exist as distinct virus species but are misclassified strains of other existing groups and that the human pathogenic FSV is a previously unrecognized reassortant between CVV, itself a known human pathogen, and a new lineage whose members had previously been identified as MAGV but are genetically distinct from the lineage occupied by the prototypical MAGV isolates. On the basis of its location of origin, we suggest the name “Córdoba virus” for this new lineage. Based on the reclassification of these virus isolates, it is clear that the endemic region for CVV is much larger than previously recognized and that CVV and MAGV most likely are responsible for unrecognized febrile infections throughout North Central, and South America. We hope that a better understanding of the genetic relationships between bunyavirus groups, particularly in relation to their relevance for human infection, coupled with an increased availability of reference genome sequence information, such as provided by this study, will help enable future surveillance and diagnostic efforts and increase awareness of the importance of orthobunyaviruses as human pathogens.Source and sequence information for Maguari virus strains sequenced in this study; GenBank accession numbers for reference orthobunyavirus sequences used in the phylogenetic analyses; comparison of phylogenetic analyses of nucleoprotein, glycoprotein and polymerase amino acid sequences; comparison of phylogenetic analyses of nucleoprotein, glycoprotein and polymerase open reading frame nucleotide sequences; divergence analyses for nucleoprotein, glycoprotein, and polymerase sequences.


In Volume 94, Issue 9, September 2016, page 687, the fourth, fifth and sixth sentences of the third paragraph should read: "The description of the average annual administration costs of IFFIm as $115m per year is inaccurate. On average between 2010 and 2014 the true operating costs of IFFIm were $5m per year. The remaining $110m was the average annual financing costs of the bonds, including interest payments, which depend on the terms of each transaction such as issue size and maturity, and not incurred as part of IFFIm’s operating costs."


Betatorquevirus genus. This is the first identification of betatorqueviruses in brain tumors. The viral DNA was in its expected non-integrated circular form, and it is unclear if the viruses contributed to tumor formation. Whether the viruses originated from blood, or the primary skin tumor could not be ascertained. Overall, our results demonstrate the usefulness of viral metagenomics to detect previously unknown exogenous virus in human brain tumors. They further suggest that active viral infections are rare events in brain tumors, but support a follow-up larger scale study to quantify their frequency in different brain tumor subtypes.The role of viral infections in the etiology of brain cancer remains uncertain. Prior studies mostly focused on transcriptome or viral DNA integrated in tumor cells. To investigate for the presence of viral particles, we performed metagenomics sequencing on viral capsid-protected nucleic acids from 12 primary and 8 metastatic human brain tumors. One brain tumor metastasized from a skin melanoma harbored two new human anellovirus species, Torque teno mini virus Emory1 (TTMV Emory1) and Emory2 (TTMV Emory2), while the remaining 19 samples did not reveal any exogenous viral sequences. Their genomes share 63-67% identity with other TTMVs, and phylogenetic clustering supports their classification within the  Cancers largely result from genetic mutations that induce cell transformation. While the role of some viruses in animal carcinogenesis is well established, virus-mediated oncogenesis has only been shown for a small number of human cancers. These oncoviruses include human alpha papillomaviruses (mainly HPV16 and 18), Kaposi's sarcoma-associated herpesvirus (HHV8), Merkel cell polyomavirus, Epstein–Barr virus, Human T-lymphotropic virus type 1, and hepatitis B virus . Human iWhat triggers the genetic mutations that cause primary brain tumors is mostly unknown. In healthy individuals, the brain is considered a privileged organ where the blood-brain barrier prevents pathogens from entry , but wheDue to their location and therapeutic resistance , brain nTo address this issue, we applied a viral metagenomics approach to focus sequencing on nuclease-resistant (encapsidated) viral nucleic acids after filtration of tissue homogenates through a 400nm filter , 17 . The Alphatorquevirus genus includes 29 species , Betatorquevirus includes 12 species , and Gammatorquevirus includes 15 species  [The virome and PCR analyses identified two novel anelloviruses in a specimen from a patient with a skin melanoma that had metastasized to the brain collected in 1993 (Sample #5) Figures  and 3. Giquitous , and veriquitous –27. TherDV 1–15) . Human aDV 1–15) , brain tDV 1–15) , and nerDV 1–15) . The detDV 1–15) . Since tDV 1–15) , 32.Betatorquevirus genus. They were designated Torque teno mini virus Emory1  and Torque teno mini virus Emory2 , and their genomes were deposited in GenBank. The genome lengths for TTMV Emory1 and Emory2 are 2,830 and 2,938 nucleotides (nt), with a GC content of 37% and 36%, respectively. Both viral genomes contain the typical anelloviruses genome organization [Betatorquevirus.Phylogenetic analysis of the ORF1 gene, the most conserved open reading frame (ORF) among anelloviruses, showed that the two viruses clustered with other Torque teno mini viruses Figure , supportnization   and fluorescence in situ hybridization (FISH) on frozen tumor sections post-fixed with formalin with probes specific to Emory1 and Emory2. However, results for both methods were negative (data not shown). As a control, DNA of both viruses was confirmed present by PCR in the pre-fixation frozen specimens , melanomas (∼7%), renal cell carcinomas (∼6.5%), and breast carcinomas (∼5%) . In thisOur study did not identify polyomaviruses or cytomegalovirus sequences in the filtered and nuclease treated tumor homogenates, even though these viruses were previously reported in primary human brain tumors , 12. ThiAnelloviridae family lack homologs of genes that promote cell transformation in other DNA viruses such as polyomaviruses or papillomaviruses, which respectively contain oncogenic proteins large T antigen and E6 and E7 that inactivate the retinoblastoma (Rb) and p53 tumor suppressors. Anelloviruses are not known to integrate into host cell genomes [Our study does not provide any evidence that the newly discovered viruses had a role in the pathogenesis of the metastatic melanoma in which they were identified. Although anelloviruses nearly universally infect humans in their lifetime, no human anellovirus has yet been shown to be oncogenic . Genomes genomes , thereby genomes , 39. If Tumors undergo extensive angiogenesis, increasing the number of blood vessels within the neoplastic tissue . The disIn conclusion, our study identified two new species of betatorqueviruses and is the first demonstration of TTMVs in human brain tumor specimens. Furthermore, it provides proof-of-principle that metagenomic analysis can be used to identify viral genomes that are not integrated in human brain tumor genomes, likely reflecting their presence as infectious viral particles. Finally, it suggests that active virus infections are rare in human brain tumors, complementing prior studies that focused only on transcriptome or viral DNA integrated in tumor cells.We investigated 20 frozen human brain tumor samples retrospectively collected under institutional IRB approval, including primary tumors of Schwannoma, Medulloblastoma, Astrocytoma, Glioblastoma, Gliosarcoma, and Subependymoma, as well as metastases to brain from Carcinoma, Melanoma, and Adenocarcinoma Table .Unbiased viral metagenomics sequencing of nuclease-resistant nucleic acids was performed directly according to previously described protocols that detect both DNA and RNA viruses , 17. Bride novo into contigs using Ensemble Assembler [Sequence data was analyzed using a customized NGS pipeline as described previously . First, ssembler . The conGenome analysis was performed according to previously described procedures , includiSpecific PCR primers for each new TTMV were designed directly from the genomes obtained from the NGS data. Tumor DNA was extracted using QIAamp Viral RNA Mini kit (Qiagen). PCR was performed using primer sets specific for each virus: Emory1_AF, 5′-CGCCGAAAACCTTACAAAAA-3′; Emory1_AR, 5′-TTGGTGGTTGTGTGCTGAAT-3′; Emory2_AF, 5′-CCACCACAACAATTCCAAAA-3′; Emory2_AR, 5′-CAGTCTCCGCTCATTGGTTT-3′. PCR was carried out using the Ex Taq DNA Polymerase (Clontech) for 45 cycles using a touch down cycling condition as described before : 95°C foIn situ hybridization (ISH) and fluorescence in situ hybridization (FISH) were performed on frozen tumor sections post-fixated with formalin according to an established protocol [® 532, and mounted with ProLong Gold with DAPI (Molecular Probes).protocol . Digoxig


Hes1 dimer inhibitor, agalloside (2), which can accelerate the differentiation of neural stem cells was isolated by using Hes1-immobilized beads. 2), which can accelerate the differentiation of neural stem cells is described. Six natural products, including one new natural product, which bind to Hes1 were rapidly isolated by a developed “target protein oriented natural products isolation” (TPO-NAPI) method using Hes1-immobilized beads. Of the six compounds, 2 inhibited Hes1 dimer formation at both the protein- and cellular level. Neural stem cells treated with 2 differentiated to neurons with longer neurites than cells treated with varproic acid or retinoic acid. Moreover, 2 exhibited specificity for neurons. This promotion of differentiation was supported by an increase in the mRNA expression of the proneural genes, Mash1 and Ngn2, which were inhibited by Hes1.The Hes1 dimer inhibitor, agalloside ( Neural stem cells (NSCs) can differentiate into neural cells such as neurons, astrocytes and oligodendrocytes. NSCs have been discovered in the adult mouse braini.e., their proliferation and differentiation.Basic-helix-loop-helix (bHLH) transcription factors control the fate of neural stem cells, Recent technical innovations in small molecule screening using huge small molecule libraries have led to the successful use of reverse chemical genetics using immobilized target proteins.2) accelerated NSC differentiation by disrupting dimer formation of Hes1, a repressor-type bHLH transcriptional factor. To the best of our knowledge, this is the first example of the acceleration of NSC differentiation by a Hes1 dimer inhibitor.Here we report target protein oriented natural products isolation (TPO-NAPI) using a protein-immobilized beads-HPLC method for NSCs differentiation activators. Six natural products, including one new compound, were isolated. One of these compounds, agalloside (S-transferase (GST) fused Hes1-immobilized beads (GST-Hes1-beads) were mixed with our library of natural product extracts. The extracts provided many HPLC peaks, corresponding to the various natural product components . Although there have been no reports of small molecules binding to Hes1, we previously identified Hes1 dimer inhibitors from our natural product library using the Hes1 dimer plate assay.1)1 in ESIA schematic of an approach for Hes1-binding natural products is shown in mponents . After imponents . The retAquilaria agallocha (leaves) collected in Thailand are shown in A. agallocha; many UV-absorbing peaks are evident. After mixing with GST-Hes1 beads, followed by washing and release of the bound compounds by addition of EtOH and heating, the supernatant provided several peaks. Comparison of the HPLC results with those from GST-beads (control) led to the isolation and identification of 7,4′-di-O-methylapigenin 5-O-β-d-xylosyl-β-d-glucoside (2),O-β-d-xylosyl-β-d-glucoside (3),4)5)Garcinia mangostana  collected in Thailand was rapidly isolated by just one HPLC separation step. A macrolactam, BE-14106 (6),Actinoalloteichus cyanogriseus IFM11549 from a soil sample collected at the Sakazuki forest in Chiba, Japan. A new natural product, a hydroxypiperidine with three conjugated double bonds, was named inohanamine (7) and was also isolated using the HPLC peak guide approach. Compound 7 was isolated from an AcOEt extract of Streptomyces sp. IFM 11584 collected at Inohana Park in Chiba, Japan. The structure of 7 was determined by 1H- and 13C-NMR, HRMS, HMBC, COSY and NOE , which are the core flavanone structures of 2 and 4, respectively , 2 was investigated further. The electrostatic charge distributions of compounds with similar structure (2–4) were examined using DFT calculations (see ESI2) has an obvious electron deficient area which might be one of the reasons its activity against Hes1.With these isolated Hes1-binding natural compounds in-hand, their ability to inhibit Hes1 dimer formation was examined using the Hes1 dimer plate assay.ectively . The flaectively , did notectively . Nonspecectively .49 The rectively . Compounhibition . This res see ESI. Only ag2) (5 and 10 μM) clearly decreased Flag-Hes1 dose dependently, indicating that 2 inhibits Hes1 dimer formation in cells.To examine Hes1 dimer inhibition in cells, HA- and Flag-tagged Hes1 expression vectors  were prepared and transfected into C3H10T1/2 cells , accordi2 to accelerate NSCs was evaluated (2) (5 and 10 μM) for four days. A confocal microscope was used to obtain images of differentiated neural cells after immunostaining class III β-tubulin (Tuj1) in neurons, and glial fibrillary acidic protein (GFAP) in astrocytes and nuclei (TO-PRO-3). The number of neurons and the length of the neurites were calculated for over 3000 cells and 800 neurites in each sample. Agalloside (2) exhibits potent neurite outgrowth promoting activity . Comparison of the number of neurons and astrocytes vs. the total number of cells showed increased neuronal differentiation with agalloside (2) (2 were 36.5% (5 μM) and 39.3% (10 μM), which represent a 36.2 and 46.6% increase compared to those of control. The differentiated astrocytes with 2 were 28.0% (5 μM) and 33.5% (10 μM), which show 0.0 and 19.6% increase compared to those of control. Agalloside (2) showed neuron specificity in differentiation.Next, the ability of valuated . Multipoactivity . The medside (2) . The dif2 on mRNA expression of pro-neural genes (activator-type bHLH factors), the expression levels of Mash1, Ngn2 and NeuroD2 were examined (Mash1 expression by directly binding to the Mash1 promoter.2) up-regulates Mash1 after 24 h (2) cancelled the N-box dependent repression activity of Hes1 dimers2) disrupts Hes1 dimer formation in NSCs, leading to upregulation of Mash1. This is the first report of the upregulation of a pro-neural gene by a Hes1 dimer inhibitor. Although it remains unknown whether the Hes1 dimer regulates directly or indirectly, Hes1 also regulates the expression of Ngn2.Ngn2 is required for neural differentiation. Increased Ngn2 expression was clearly detected after treatment with 2 for 24 h (2) might inhibit the suppression of Ngn2 expression by Hes1. Moreover, the neuronal specificity of 2 for the differentiation of NSCs is supported by reports that Mash1 and Ngn2 suppress astrocytic gene expression.NeuroD2 is a late response activator-type bHLH factor, and Mash1 acts upstream of NeuroD.2 expressed NeuroD2 more highly than the control after 48 h  and Ngn2, which activate the transcription of neurogenesis genes such as NeuroD2  in Hes1, partial proteins of Hes1 were synthesized (2) was mixed and incubated with each protein immobilized beads . After washing the beads, binding compound (2) was released by addition EtOH. Using HPLC, the binding amounts were compared as their UV absorption (2) binding. These partial Hes1 proteins have an HLH domain. On the other hand, the amount of agalloside (2) decreased after mixing with the beads of Part B (104-281aa) and Part E (151-281aa), which do not have an HLH domain. 2) binding region in Hes1 can be predicted as the HLH domain. We also predicted the interaction between the HLH domain and agalloside (2) by performing in silico docking analysis. The sugar region of agalloside (2) might interact and disrupt the hydrophilic interaction between Arg46, Glu76 and Lys77, which would be important to make the helix and loop units binding. Moreover, the flavonoid core seems to bind the hydrophobic pocket, which consists with Ile50, Leu54 and Leu57 of the helix , which was isolated by the TPO-NAPI method using Hes1 immobilized beads. We demonstrated the isolation of six natural products, including one new compound, by TPO-NAPI. This method is useful for isolating natural products which bind to target proteins. In principle, the binding activity of ligands to proteins gives compounds the chance for a desired bioactivity. We thus believe that this strategy using TPO-NAPI to identify modulators bHLH factors will provide good candidates for development as regenerative drugs.In conclusion, we report here the first example of a naturally occurring Hes1 dimer inhibitor, agalloside (ca. 3.6 nmol) in PBS was added to pre-washed glutathione Sepharose 4B beads  and they were mixed at 4 °C for 1 h. The GST-Hes1 beads were washed five times by NET buffer , then the beads were suspended in NET buffer (250 μL). A MeOH or EtOAc extract of natural resources  was added to above GST-Hes1 freshly prepared beads (bed volume 100 μL) and the mixture was gently mixed for 2 h at 4 °C. The beads were then washed by a rotated-mixer at 4 °C for 10 min three times with NET-N buffer . 70% EtOH (150 μL) was then added to the washed beads and the suspension was heated at 100 °C for 3 min. The beads were gathered by centrifugation  and the supernatant was centrifuged at 15 000 rpm for 15 min. The one-third of the supernatant was analyzed by HPLC. The control GST-beads were also prepared in the same procedure of GST-Hes1 beads. GST  in PBS was added to pre-washed glutathione Sepharose 4B beads . If there is the obvious difference in the peak intensity between the results of GST-Hes1-beads and GST-beads (control), such extracts were obtained as “hit” extracts which had the Hes1 binding natural products.To prepare GST-Hes1 beads, GST-Hes1  for 2 h at 4 °C. After the removal of protein solution, to block remaining activated units on the well, the wells were incubated for 2 h at 4 °C with 100 μL of 10 mM ethanolamine , then washed twice with 200 μL of PBST (PBS containing 0.05% Tween 20). The Hes1 bound microplate wells were incubated with 50 μL of Cy3-labeled-Hes1 in NET-N buffer  for 24 h at 4 °C. After removal of protein solution, each well was washed twice with 200 μL of PBST, then each compound solution  was added. After incubation for 1 h at RT in the dark, each well was washed twice with 200 μL of PBST, then dried under reduced pressure 1 h in the dark. The fluorescence intensity was measured in a microplate reader . The Cy3 dye was excited at 530 nm and emitted at 590 nm. Usually, the assays were carried out in three individual wells, and the mean value and SD were calculated.Nunc Immobilizer™ Amino 96 well plate, white  was used for immobilizing of Hes1. The wells were incubated with 100 μL of Hes1 (10 μg mLSupplementary informationClick here for additional data file.


Novel research training approaches are needed in global health, particularly in sub-Saharan African universities, to support strengthening of health systems and services. Blended learning (BL), combining face-to-face teaching with computer-based technologies, is also an accessible and flexible education method for teaching global health and related topics. When organised as inter-institutional collaboration, BL also has potential for sharing teaching resources. However, there is insufficient data on the costs of BL in higher education.Our goal was to evaluate the total provider costs of BL in teaching health research methods in a three-university collaboration.A retrospective evaluation was performed on a BL course on randomised controlled trials, which was led by Stellenbosch University (SU) in South Africa and joined by Swedish and Ugandan universities. For all three universities, the costs of the BL course were evaluated using activity-based costing with an ingredients approach. For SU, the costs of the same course delivered with a classroom learning (CL) approach were also estimated. The learning outcomes of both approaches were explored using course grades as an intermediate outcome measure.In this contextually bound pilot evaluation, BL had substantially higher costs than the traditional CL approach in South Africa, even when average per-site or per-student costs were considered. Staff costs were the major cost driver in both approaches, but total staff costs were three times higher for the BL course at SU. This implies that inter-institutional BL can be more time consuming, for example, due to use of new technologies. Explorative findings indicated that there was little difference in students’ learning outcomes.The total provider costs of the inter-institutional BL course were higher than the CL course at SU. Long-term economic evaluations of BL with societal perspective are warranted before conclusions on full costs and consequences of BL in teaching global health topics can be made. Global health needs strong health systems . To thisThe African Capacity Development on Health Systems and Services Research (ARCADE HSSR) project focused on building research capacity in African countries . The proReviews on BL , 13, 14 BL is increasingly popular , 23, butOne of the first courses delivered using BL approaches in ARCADE HSSR was a course on randomised controlled trials (RCTs). The course was taught using CL at SU from 2009 as part of the master of science in epidemiology programme . The shiThis retrospective economic evaluation was conducted to estimate the total provider costs of BL in teaching a course on RCTs as a three-university collaboration in the ARCADE HSSR framework. A cost description was performed for all three institutions – SU, KI, and MU. In addition, at SU, a CL course was included in the evaluation as an historical comparator. The learning outcomes of BL were also explored and compared to those of CL at SU, using course grades as an intermediate outcome measure. As both the BL and CL versions of the course had the same learning content and objectives, course grades were a good source of outcome data available for the retrospective evaluation. The students’ final grades on both the BL and CL courses were formed based on assignments (50%: three assignments in the CL course and four in the BL course) and a final exam (50%). The grading scale on the course was 0–100 and the pass mark was 50% (50), which is the standard on postgraduate-level courses.In terms of running the courses, the final year of running each course was evaluated . Both the CL and the BL courses had the same learning objectives, and the same themes were covered in teaching . Descriptive statistics on both courses are presented in www.moodle.org). SU used a classroom with videoconferencing equipment for teaching the course, and KI used a meeting room with similar functionality. Students from MU participated in the teaching sessions in a classroom using their own laptops, because of low student numbers and non-availability of videoconferencing equipment for such a small number of students. The course also required a bridge for videoconferencing to connect all sites and allow for interaction. The sites used Microsoft Lync software to connect through the bridge. The faculty used Camtasia software in 2013 to make videos of the teaching content, which were made available for students to watch online.The BL course included 16 hours less of scheduled CL time compared to the CL course at SU . The BL All costs were examined from the providers’ perspective, using activity-based costing. Using activity-based costing, all activities needed to produce the course were first specified, after which cost ingredients were categorised for each activity. An ingredients approach was used to collect data on resources used in order to identify all necessary inputs regardless of the funding source. This approach consisted of identifying and valuing all resources required to set up and run the BL and CL courses and calculating the total costs for both approaches. Resource use was tracked from the start of the project and course planning until the end of 2013. All inputs were tracked by and linked to the site that incurred the cost, even if the purchase or work input benefitted all sites to some extent. This illustrated the needs of institutions with different roles, KI having the overall managerial role of ARCADE HSSR, SU representing the course organiser, and MU having a smaller participant role in this BL activity.We conducted a document review to achieve an overview of activities related to BL in the ARCADE HSSR project, by examining the project's grant agreement . The actAn overview of the collection and valuation of inputs is presented in For participants who were identified by key informants for their time contribution to the project or courses, time input to meetings was not calculated separately to avoid double-counting, and only travel costs retrievable from the project expenses  were included as meeting-specific costs. Only hours used to participate in the meeting were included for those meeting participants that were not otherwise included in staff time. This time was valued based on professional positions. These participants contributed to the meeting content, but as none of the key informants had mentioned them as key for the project or courses, we considered the inclusion of full costs excessive.The cost of videoconferencing equipment was covered by approximating the cost of the spaces that had the necessary equipment, and this cost was included in the running cost of BL. SU had purchased the bridge for the videoconferencing system, which was included in the start-up costs of BL in ARCADE HSSR. As Microsoft Lync was not specifically purchased for the project, the initial once-off licence fee was not costed, but the yearly fee for three devices was included as an approximation of a recurring cost of using the program in running the course. Several other communication cooperation systems were tested in ARCADE HSSR  at the project level, thus the cost for 1 year's use of each of these programmes is included in the ARCADE HSSR start-up costs of the BL course. The BL start-up costs also included an once-off licence fee for the Camtasia software. Other purchases in ARCADE HSSR that participants considered necessary for starting up BL included two computers, two microphones, and a webcam, which were all purchased at KI. The annualisation and depreciation of these costs are discussed at the end of this section.The valuation of the inputs was done by expenditure reviews of project financial records. For those BL resources not included in project records and resources needed in the CL version of the course, national market prices were used to approximate costs. A common overhead is added to instances of outside purchasing of services from the university in the main study setting of South Africa. This figure, 10%, was used as the overhead cost on the final sum, to account for resources that serve several departments or programmes .Costs were collected in local currencies where this information was available (KI and part of SU costs). Costs from MU and part of the SU costs were collected from EU reporting, where all costs were presented in euros. All currencies were first converted to US dollars using the average exchange rate of the year when the cost had taken place . Costs wAll equipment costs were classified as capital costs, as the useful life of all the items was more than 1 year, and start-up costs were considered capital expenditure. Annualisation was undertaken by using 2013 interest rates for each country's 10-year government bonds as the discount rate –31 and 5Work related to BL in general, done as part of the ARCADE HSSR project, was vital for achieving the knowledge, acquiring the equipment, and formulating the materials needed to deliver the RCT course as BL. Thus, interviewees were also asked about inputs needed for planning of the ARCADE HSSR project as a whole and on general BL activities within the project, as well as what share of these inputs was relevant to the RCT course. The averages of these estimations were used to allocate part of the general project inputs to the RCT BL course. Key informant estimations were also used to allocate staff hours related to BL activities in the ARCADE HSSR project to start-up and operational needs. As start-up inputs will benefit BL activities at the institutions and the courses for many years, these costs were annualised, and the annual cost for 1 year was included in the final costs. As the BL course had been running for 3 years, one-third of the ARCADE HSSR project costs allocated to running the RCT course were included in the costs of running the 2013 RCT BL course.Three input categories were formed to present the collected data under both start-up and running costs. Planning and management included inputs related to the course and project planning and management; central activities, such as general ARCADE HSSR meetings; and coordination and administrative work. The ICT capacity category included all inputs needed for ICT capacity building and maintenance. The course development and delivery consisted of inputs needed for the course module review and development, as well as course delivery. Space costs included only teaching space costs, as meeting space costs were included in the meetings category.A one-way sensitivity analysis was conducted to assess the role of possible uncertainties in the data. Staff costs were identified as the key driver of total cost and also as most subject to recall bias; thus the total cost of time inputs was varied by 20% up and down. As space costs vary considerably depending on location and limited data were available for evaluation of space costs with videoconferencing equipment, space costs were varied by 20% up and down. Meeting costs were very specific to the project under evaluation (e.g. the number of meetings needed and number of people travelling); thus they were varied on a larger scale, 50% up and down. Furthermore, as significant variation existed in estimations of what proportion of costs related to BL activities in ARCADE HSSR project should be allocated to the RCT BL course, this parameter was varied to represent the lowest and highest estimations.Thirteen master's students participated in the RCT course that was delivered as CL at SU in 2010 . AltogetExplorative findings on course grades suggest that there was little difference between the groups in learning outcomes. The mean final grade of students taking the BL and CL courses was 67.3 (SD 7.6) and 64.3 (SD 5.8), respectively.The economic costs of the RCT courses are presented as start-up and running costs and are summarised under three main activities . The totStaff costs were the major cost category within most activities and in total costs . For theIn the BL course's start-up costs, meetings were the most significant source of costs, representing 50% of the total start-up costs. The source of these costs was five international meetings that were relevant for starting up the BL activities within the project. Three of these were general ARCADE HSSR project meetings, where only part of the content was related to BL and therefore only part of the total meeting costs were allocated to BL, according to interviewee estimates (50% for one meeting and 25% for two meetings). One international meeting was solely focused on BL, and one focused directly on transforming the RCT course to BL.Of the start-up costs of the BL course, 13% were equipment and licence costs, whereas such costs were less than 1% of running costs. Teaching space costs represented 17 and 31% of running costs of the BL and CL courses, respectively. The average per-site cost of the RCT BL course was USD 22,700, for example decreasing the SU costs by 23% when compared to the site-specific cost.At SU, the costs of starting up and running the RCT course as BL were 115% higher than starting up and running the course as CL . Of the Varying the estimates of the allocation of ARCADE HSSR costs to the RCT BL course had the highest impact on total costs in the sensitivity analysis . AllocatOur research indicated that delivering the course as BL incurred more than double the costs that were estimated for the CL format of the course at the leading university, SU, which also had the highest number of participating students. However, costs remained higher even after evening out the costs of BL between participating universities and after considering the per-student costs. Staff costs seemed to drive the total cost for both approaches, similarly to other studies . No notaEven though the BL course included fewer classroom hours, the results show that staff hours for course development and delivery were significantly higher for the BL course than the CL course. This finding was also highlighted by lecturers involved in developing and delivering BL courses in the ARCADE projects . This reIn the present evaluation we were able to both compare costs and explore one outcome of BL and CL for only one institution. Furthermore, our comparator (CL) at one institution was an historical control. There is a need for rigorous evaluation studies, such as RCTs, with a comparable time frame and outcomes that have a broader spectrum of the hypothesised benefits of BL – expanding the coverage of teaching health systems research and enhancing student learning. Before these types of rigorous evaluations are available, no firm conclusions should be made on cost-effectiveness of BL compared to CL.The findings of this evaluation are very context-specific, presenting an example of the costs of applying BL as an inter-institutional collaboration between three sites. As this evaluation included only the providers’ perspective, no conclusions can be made on the full societal costs of BL, as for example students’ costs were not included. This evaluation included the first BL course offered in the ARCADE HSSR project, and even if the third year of running the course was looked at in terms of the running costs, the findings represent the transition phase from CL to BL. Interviewees often highlighted that setting up and running the more recent BL courses implemented within ARCADE HSSR has been easier. Nonetheless, the present evaluation provides a full cost description of offering a BL course, including all inputs needed to start-up and run the courses, and not just actual purchases by a specific project, and it can thus be used as preliminary data on provider cost structures related to the shift from CL to BL in teaching health research methods to masters-level students as an inter-institutional collaboration.This pilot-scale evaluation offers new insight into the total provider costs of BL as organised in an inter-institutional collaboration with a multicountry setting. Intuitively, such efforts could have a great advantage in global health education, in bringing lecturers and students together from different settings , thus poThis contextually bound pilot economic evaluation focused on costing an inter-institutional BL course on RCTs, a key skill in health systems and global health research. The evaluation demonstrated that BL had substantially higher costs than the traditional CL approach in South Africa. Despite these costs, BL, especially when implemented with the intention of sharing limited teaching resources between institutions and increasing accessibility of education, has the potential to support global health research capacity building in resource-constrained settings. Further work investigating the long-term costs of BL, including societal perspectives and analysis of consequences, as well as strategies for cost reduction are warranted to determine the true cost-effectiveness of BL compared to traditional teaching approaches. Further exploration of capacity building approaches in LMICs are also needed.


Human influenza A viruses (IAVs) cause global pandemics and epidemics, which remain serious threats to public health because of the shortage of effective means of control. To combat the surge of viral outbreaks, new treatments are urgently needed. Developing new virus control modalities requires better understanding of virus-host interactions. Here, we describe how IAV infection triggers cellular apoptosis and how this process can be exploited towards the development of new therapeutics, which might be more effective than the currently available anti-influenza drugs. Influenza A and B viruses are common causes of seasonal epidemics. Infected individuals display mild symptoms like cough, sore throat, nasal discharge, fever, headache, and muscle pain . HoweverInfluenza A virus (IAV) in particular is a potential threat to global health. In contrast to influenza B virus which is only found in humans, IAV can cause pandemic outbreaks when a novel subtype emerges, typically from an animal origin . In the Anti-influenza drugs that target influenza neuraminidase (NA) have been used to prevent and treat influenza virus infections for many years. In particular, oseltamivir, zanamivir, and peramivir exert antiviral effects , but cerDeveloping new virus control modalities requires better understanding of virus-host interactions. Here, we attempt to summarize our knowledge in virus-host cell interactions with a particular focus on programmed death of infected cells (apoptosis). We propose a concept of using apoptosis-inducing drugs as a new class of potential anti-influenza agents. These small molecules can facilitate apoptosis of infected cells, without affecting non-infected cells and, therefore, limit IAV replication and spread. The concept can be expanded to other viral diseases.Orthomyxoviridae family . H. H62]. HWe propose a model for this effect in 50) for A-1155463 is lower than that for ABT-263. In addition, half-maximum cytotoxic concentration (CC50) value of A-1155463 is higher than that of A-1331852, whereas EC50 of both are lower than that for ABT-263 . Thus, The typical approach in antiviral drug discovery has been to identify virus inhibitors that target various stages of virus replication and to preserve infected cells from death  23,24,3,81,82,83However, Bcl2i must be used as a prophylactic rather than a therapeutic drug because of the following issues. Although the induction of apoptosis has been shown to be selective for infected cells in vitro, inhibition of Bcl2 proteins may have off-target effects in vivo ,75. Our Cellular antiviral responses including IFN response and apoptosis are employed in order to inhibit virus replication and spread. IAV has evolved to gain mechanisms to disconcert these responses to ensure its replication. Based on our knowledge on host-virus interaction, we can explore ways to develop pharmacological interventions to control IAV infections. In particular, our advance in understanding apoptosis has shown potential in developing apoptosis-inducing molecules as antiviral drugs against flu. A-1155463 could serve as a lead compound in this process. Prophylactic treatment with A-1155463 may prevent development of severe disease. Successful prevention of flu using Bcl2i could provide an alternative therapeutic option for IAV, against which current treatment is limited. Having wider treatment options could reduce the use of drugs targeting virus proteins, and thus slow down the rise of drug-resistant virus strain through evolutionary selection pressure. Timely use of Bcl2i may also reduce the use of antibiotics, which are utilized for treatment of secondary bacterial infections. This will limit the development of emerging antibiotic-resistant bacteria. Exploring a new class of antiviral drugs is crucial, and further investigations on the antiviral properties of Bcl2i could lead to development of new drugs to prevent other viral diseases, associated with HIV, ZIKV, HBV, and VZV (29–31).