To evaluate the 12-month total direct costs  of delivering subcutaneous depot medroxyprogesterone acetate (DMPA-SC) under three strategies — facility-based administration, community-based administration and self-injection — compared to the costs of delivering intramuscular DMPA (DMPA-IM) via facility- and community-based administration.We conducted four cross-sectional microcosting studies in three countries from December 2015 to January 2017. We estimated direct medical costs  using primary data collected from 95 health facilities on the resources used for injectable contraceptive service delivery. For self-injection, we included both costs of the actual research intervention and adjusted programmatic costs reflecting a lower-cost training aid. Direct nonmedical costs  came from client interviews conducted during injectable continuation studies. All costs were estimated for one couple year of protection. One-way sensitivity analyses identified the largest cost drivers.Total costs were lowest for community-based distribution of DMPA-SC (US$7.69) and DMPA-IM ($7.71) in Uganda. Total costs for self-injection before adjustment of the training aid were $9.73 (Uganda) and $10.28 . After adjustment, costs decreased to $7.83 (Uganda) and $8.38  and were lower than the costs of facility-based administration of DMPA-IM . Costs were highest for facility-based administration of DMPA-SC ($12.14) and DMPA-IM ($11.60) in Burkina Faso. Across all studies, direct nonmedical costs were lowest for self-injecting women.Community-based distribution and self-injection may be promising channels for reducing injectable contraception delivery costs. We observed no major differences in costs when administering DMPA-SC and DMPA-IM under the same strategy.Designing interventions to bring contraceptive service delivery closer to women may reduce barriers to contraceptive access. Community-based distribution of injectable contraception reduces direct costs of service delivery. Compared to facility-based health worker administration, self-injection brings economic benefits for women and health systems, especially with a lower-cost client training aid. The DMPA-SC product available to Family Planning 2020 countries is Pfizer’s Sayana® Press, which delivers the contraceptive drug through the BD Uniject™ injection system, allowing for easier administration by lay health workers with minimal training and for women to self-inject. Recent studies demonstrated the operational feasibility of these administration modalities and acceptability to women and health workers Evidence on the costs of contraceptive service delivery in LMIC is generally scarce. Only a few studies have estimated the costs of delivering injectable contraceptives — mainly DMPA-IM Therefore, we sought to investigate whether the costs to administer DMPA-SC differed from the costs to administer DMPA-IM and whether these costs differed by delivery strategy. We conducted these costing studies in parallel with studies evaluating the method continuation rates of DMPA-SC under facility-based administration in Burkina Faso, community-based distribution via Village Health Teams in Uganda, and self-injection in Senegal and Uganda, all compared to DMPA-IM The main objective of this study was to assess the costs of delivering DMPA-SC and DMPA-IM using different strategies in three sub-Saharan African countries: Burkina Faso, Uganda and Senegal. We reported costs from a health system perspective and also accounted for women’s travel and time costs to travel to, wait for and receive services. Specifically, we sought to understand the total direct costs of delivering DMPA-SC and DMPA-IM, including commodity costs; costs of provider time, medical supplies and drugs for the treatment of side effects; and travel and time costs to women. We did not seek to directly compare results across countries. However, we can draw some lessons by looking at the costs by delivery strategy, especially in Uganda where we assessed three delivery approaches  in two studies conducted in a very similar setting .We then used the cost estimates as input in a follow-up cost-effectiveness study 2We received in-country approval for conducting the costing studies from the Comité d’Ethique pour la Recherche en Santé in Burkina Faso, Mulago Research Ethics Committee of Uganda and Comité National d’Ethique pour la Recherche en Santé of Senegal. We obtained consent to participate in this study from each health worker interviewed.2.1Burkina Faso introduced DMPA-SC through the facility-based delivery strategy in place for DMPA-IM 2.2We conducted four microcosting studies across the three countries. Microcosting is a cost estimation method that involves collecting detailed data on the resources used (input quantities) and the value of those resources (input prices) in the delivery of a health service The direct medical costs for service delivery included the costs of contraceptive commodities, health worker time to deliver family planning services , medical supplies and drugs for the treatment of side effects. For self-injection, we also included the resources used for training women to self-inject: health worker time to train the client and necessary supplies .In addition, we estimated the direct nonmedical costs (women’s travel costs and time) by interviewing women enrolled in the continuation studies . To thisWe estimated the economic costs of contraceptive service delivery to account for donated health commodities as well as for the time of volunteers involved in contraceptive service delivery . We estimated annual costs per couple years of protection (CYP) — equivalent to receiving four injections of these 3-month injectable contraceptives 2.3We estimated health workers’ time costs by multiplying the self-reported time spent with a client for each family planning service by the average salary per minute of the health workers. We included facility-based health worker time costs, valued at the average government salary pay by grade. We valued volunteers’ time using the salary of a nursing assistant. For the Uganda Village Health Teams study, in addition to health workers’ time, we also included time and travel costs for Village Health Teams to reach the communities where they met with women and the travel costs to reach their facility for replenishment of supplies or data reporting.To estimate costs of medical supplies and tests, we asked health workers to list all medical supplies and tests used in the delivery of each contraceptive method and the percentage of women on whom these were used. We calculated the costs by multiplying the quantity of supplies and pregnancy tests used per client with the corresponding unit price Costs of treatment of side effects included the costs of health worker time spent on counseling and/or treating a client experiencing side effects, and the drugs they typically prescribed for treating these side effects. We used similar approaches as described above to calculate the costs for health workers’ time. To calculate drug costs, we multiplied each unit price by the total quantity prescribed for each side effect and by the probability of experiencing the side effect over the first 12 months of use based on data from the continuation studies . We calcTo estimate the costs of medical equipment related to safe disposal of injectable contraceptives, we divided the cost of a safety box by the number of DMPA-SC and DMPA-IM units that could fit in one safety box We estimated costs of training women to self-inject DMPA-SC under a research study design. These costs included the time spent by study health workers to train clients to self-inject and the costs of training material calculated by multiplying the quantity of each supply by the respective unit cost. We estimated costs for retraining women by multiplying the training costs by the percentage of women who were retrained.We adjusted costs of training supplies to better reflect current programmatic implementation in Uganda. Namely, we replaced the self-injection training booklet given to women under the research study with a lower-cost, one-page (front-and-back) training instruction sheet with the same information.We derived travel and time costs for women to receive injectable contraceptives using client-level data collected during method continuation studies We calculated roundtrip transport costs per visit to the facility or Village Health Team using the self-reported data. We calculated annual costs for DMPA-SC or DMPA-IM users by multiplying the costs per visit by four when women went to a facility/Village Health Team for all injections. Instead, for self-injecting women, we included the costs of the first visit and the (minor) costs for subsequent visits in case women sought help at the health facility for side effects.2.4We performed data analysis in Microsoft Excel . We converted costs in local currency to 2016 US$ using annual average exchange rates by country; US$1 corresponded to 3419 Ugandan shillings and 595 West African Francs  3We estimated supplies for self-injection training at $3.60 for Uganda and $3.58 for Senegal before adjustment, with the training booklet ($2.00) accounting for the largest share of the costs. The adjusted training aid costs reflect the replacement of the booklet with the one-page instruction sheet ($0.07), and the estimated self-injection training costs for Uganda and Senegal were $1.70 and $1.68, respectively .Total direct medical costs were lowest for community-based distribution by Village Health Teams in Uganda  because of lower Village Health Teams time costs. Community-based distribution also saved costs for tests and supplies typically used during the first facility visit. In Uganda, the costs were similar for DMPA-IM and DMPA-SC delivered under the same strategy (community-based). We attributed the $0.54 higher cost for four DMPA-SC injections in Burkina Faso relative to DMPA-IM to differences in reported side effects and the associated costs of treatment Women’s time and travel costs estimates ranged from $0.97 to $4.66 across countries and delivery strategies . The lowWhen we included both direct medical costs and direct nonmedical costs, the adjusted total direct costs of self-injection in Uganda and Senegal were lower than the total direct costs of DMPA-IM delivered by facility-based health workers .Fig. 1On4Research studies on DMPA-SC delivery alongside DMPA-IM provided a unique opportunity to analyze the costs of this new contraceptive technology across different delivery strategies. In both Uganda and Burkina Faso, the costs for DMPA-SC were similar to the costs for DMPA-IM when delivered under the same strategy; hence, there is no clear cost advantage of one product over the other. In Uganda, community-based distribution of DMPA injectable contraception resulted in lower costs for both service delivery  and for women  when compared to facility-based DMPA-IM delivery. Bringing contraceptive services closer to women by leveraging and allowing community health workers to provide short-term contraceptives may help increase access to and uptake of contraceptives — especially in rural areas, where women may face higher financial barriers that limit their ability to travel to a facility to receive services The option to self-inject may further reduce financial and logistical barriers for women. Women’s travel and time costs accounted for 32% to 46% of total costs when health workers administered the injections compared to 9% to 20% of total costs when self-administering. As the one-way sensitivity analysis for these Senegal and Uganda self-injection studies showed, there was a much wider range in women’s travel costs over the four injections for women who were receiving DMPA-IM injections from providers than for women who were self-injecting; the relative cost savings for women who self-inject will also vary widely depending on the distance between their home and the facility and, hence, their travel costs.The upfront costs to train women to self-inject can be high, as observed under the research study. As the sensitivity analysis showed, the training aid was an important driver of costs. Simple modifications to the training aid used can reduce client training costs, likely without affecting the quality and effectiveness of the training. As self-injection is offered and scaled up in more settings, these cost drivers can be reduced to make self-injection training more affordable and sustainable.Our cost estimates are comparable with previous study results, which found facility-based delivery of DMPA-IM to cost between $8.50 and $20 per CYP This study had several limitations. First, the study sites that we used for the costing analysis were not representative of their districts or of the country in which the study was set, and the sample sizes were small. The need to select facilities from those involved in the pilot introduction of DMPA-SC or the research study of self-injection drove this limitation. Second, self-reported estimates of health worker and client resources may be inaccurate due to recall bias. In addition, this analysis looked at the annual costs of DMPA (four injections); however, women’s 12-month use of injectable contraception across methods and delivery modalities may differ. Finally, we did not include some delivery or programmatic costs in this study, such as costs for training and supervising health workers to deliver injectables, facility operational and management costs, and supply chain costs.One-way sensitivity analysis for the costs for facility-based delivery of DMPA-SC and DMPA-IM in Burkina Faso.Fig. A1One-way sensitivity analysis for the costs for self-injection of DMPA-SC and facility-based delivery of DMPA-IM in Senegal.Fig. A2The following are the supplementary data related to this article.


Workplace bullying is regarded as one of the most devastating stressors at work for those targeted, and the bullying-mental health relationship is well-documented in the literature, even under lower levels of exposure. However, less is known about when and for whom these negative behaviors have more effect. Perceived control over outcomes in life  has normally been related to good health and well-being, while relying on chance and/or powerful others  have been related to stress and poor health. In situations with reduced individual control like bullying, however, these mechanisms may act differently. Hence, the aim of the present study was to investigate whether internal and external locus of control, respectively, moderates the bullying-mental health relationship. Data were gathered in 2014–2015 from 1474 Russian employees (44% response rate), and analyzed using Mplus and SEM modeling. Included measurement scales were the Negative Acts Questionnaire-Revised, the General Health Questionnaire-12, and Levenson’s Locus of Control scale. Although the prevalence of high intensity bullying was low, the results showed the expected positive relationship between exposure to bullying behaviors and psychological strain. Furthermore, this relationship was moderated by locus of control. In line with our expectations, internal locus of control did not have the generally assumed positive effect on strain when exposed to bullying behaviors. On the other hand, external locus of control seems relatively beneficial when facing bullying behaviors. The results of this study thus support that exposure to bullying and its associated behaviors are unique stressors where personal characteristics seem to play a different role than normally expected when facing other kinds of stressors. Over the past 20 years, research has provided converging results from many countries suggesting that exposure to some level of mistreatment and harassment at work is a severe stressor that occurs across all sectors in working life, finding its targets among all age groups, all organizational levels, as well as affecting men and women alike . Such exDespite the overwhelming evidence indicating stressor–strain relationship between bullying and health, not all targets react in the same way or to the same degree when facing this predicament . First oLocus of control refers to the tendency to perceive outcomes in life as a result of one’s own actions and thus being within one’s own control , as opposed to being determined by external factors, such as chance or powerful others  . People So, theoretically, workplace bullying may result in negative outcomes for employees, especially when this mistreatment threatens people’s resources  , or if tEven though theoretically it is reasonable to postulate that locus of control may act as an important moderator in the bullying-mental health relationship, this prediction has largely been ignored empirically . One excH1. Exposure to bullying behaviors is positively related to psychological strain.H2. Internal locus of control acts as a catalyst in the relationship between exposure to bullying behaviors and psychological strain, in the sense that this relationship is stronger among targets holding an internal locus of control orientation.H3. External locus of control acts as a buffer in the relationship between exposure to bullying behaviors and psychological strain, in the sense that this relationship is weaker among targets with an external locus of control orientation.A questionnaire survey fronted with information sheet was distributed electronically to Russian employees in 2014–2015 by internal Human Resource personnel within several organizations countrywide. All levels of the organizations were aimed for and regular biweekly reminders were sent to those invited to participate in the study. Participation was voluntary and actual participation seen as a statement of consent. The invitation to participate in the study was sent to 3365 employees. Altogether, 1474 responded (44% response rate), of whom 1048 (71.1%) were women and 426 (28.9%) were men. The mean age was 36.02 years (SD = 9.58). Furthermore, 83.7% had a higher education (Bachelor’s degree or higher), 14.8% had a technical degree, and 1.5% had finished middle or secondary school. In terms of organizational type, 85.9% worked in a private company, 13.7% worked in a local public organization, and 0.4% worked in a foreign public organization see .Never) to 5 (About daily). This scale showed a very strong internal consistency with a Cronbach’s alpha value of 0.96.Exposure to bullying behaviors was measured with the Negative Acts Questionnaire-Revised NAQ-R; . This scNot at all) to 4 . The Cronbach’s alpha value for this scale was 0.80, showing satisfactory internal consistency. GHQ-12 is evaluated to be a useful screening tool for the assessment of mental distress , and a two-factor structure was supported in a confirmatory factor analysis , these scales were used as independent measures of locus of control, as suggested by the scale author (Locus of control was measured with e author .Strongly disagree) to 6 (Strongly agree). The Cronbach’s alpha values for this subscale was 0.76, showing satisfactory internal consistency.Internal locus of control was measured with eight items  and measures to what extent one feels in control over outcomes in life. Responses were given on a 6-point scale from 1 (Strongly disagree) to 6 (Strongly agree), and the Cronbach’s alpha value for this subscale was 0.82, again showing satisfactory internal consistency.External locus of control  was also measured with eight items , and assesses to what extent a person believes that outcomes in his/her own life are dependent upon powerful others. Responses were given on a 6-point scale from 1  23.0. was used for analyzing demographics and scale reliability (α). Mplus 7.4.  was usedThe construct validity of the included scales was investigated by means of a CFA in Mplus. The hypothesized measurement model  was tested and compared with two alternative models. An inspection of the fit indices  indicate2  = 3493.39, p = 0.000; CFI = 0.96, TLI = 0.96, RMSEA = 0.04]. Then an interaction model was tested (see p = 0.000), internal locus of control , and the product term  were related to psychological strain. The full model explained 7% of the variance in psychological strain. The relationship between bullying and strain was strongest for those with high scores on internal LoC  as compared with those having low scores . Hence, both H1 and H2 were supported.In order to test the relationship between locus of control, exposure to workplace bullying and psychological strain, SEM in Mplus was used. Firstly, the direct effect model was tested with bullying and internal locus of control as predictors of psychological strain see . The fitsted see , 3, with2  = 2584.20, p = 0.000; CFI = 0.98, TLI = 0.97, RMSEA = 0.04). In the interaction model, with standardized variables, both bullying , external locus of control , and the product term  were related to psychological strain (see p = 0.000) as compared with those having high scores . The full model explained 8% of the variance in psychological strain.The third hypothesis, proposing that external locus of control acts as a buffer in the relationship between workplace bullying and psychological strain was also supported see . The dirrain see , 4. The To sum up, the results in this study indicate that those with high external locus of control are less affected than those with low external locus of control, when exposed to bullying behaviors. Meanwhile, those with high internal locus of control are somewhat more affected than those with low internal locus of control, when exposed to higher levels of bullying behaviors at work. As such, our two main hypotheses are supported.In the literature on workplace bullying, studies investigating when and for whom bullying results in negative outcomes are strongly called for . Hence, The results in this study indicate that, when exposed to higher levels of bullying, the bullying-psychological strain relationship is strongest among those targets with high internal locus of control and among those with low external locus of control. This may be contrary to what one initially expects from the theory, given that generally internal locus of control is related to positive well-being outcomes, while external locus of control is related to negative outcomes . One posAnother explanation for the findings in this study, may be due to the fact that people high in internal- and low in external locus of control tend to employ other coping styles when facing stressors, as those with high internal locus of control use problem-focused behaviors more often than those with high external locus of control . NormallThe present results have some important theoretical implications. First of all, the results challenge main stream theories which suggest that certain personality characteristics make people more vulnerable and other more resistant when facing stressors such as exposure to bullying. This does not seem to be the case, indicating either that such dispositions may act differently depending on the nature of the stressor or indicating that bullying is a stressor different from other typical social stressors at work. Hence, theoretical models should account for the fact that personal characteristics normally seen as negative/positive for people’s coping, health and well-being, may act differently than expected under exposure to bullying see also . TheoretThe results in this study emphasize the importance of including moderators when studying the bullying-mental health relationship, in that the examination of direct effects may underestimate the impact of the predictor variable, at least its impact on some particular groups of targets. Also, even though certain personal characteristics are risk factors for poor health and well-being in general, they may in fact act in the opposite way when exposed to bullying behaviors. In particular, relying on individual resources that normally protect people from work stressors, in our case locus of control, seem not to reduce the risk of impaired health under exposure to bullying behaviors at work. For therapists, family physicians and counselors working with targets with health problems after bullying, this is important knowledge. Furthermore, organizational based anti-bullying policies and programs are often advised in studies like the present one, with proper policies and procedures to handle bullying complaints. Based on the present study, such programs are not to be implemented in order to protect some generally vulnerable workers, but should be developed as much for the protection of the otherwise healthy and stress resistant employees.The use of cross-sectional data hinders the possibility to draw causal explanations for the findings in this study, and studies with longitudinal designs are recommended in order to conclude further. Although the use of self-report data is assumed to increases the risk of common method variance , the finWorkplace bullying, even in less frequent forms, is related to reduced health and well-being among those targeted, possibly depriving its targets from experiencing control over outcomes in life. In line with such assumptions, the present study shows that this relationship is dependent upon the nature of the targets’ locus of control. Having an external locus of control seems to be beneficial when in this predicaments, as the relationship between bullying and strain were lower for these targets as compared to those low in external control. Targets with an internal locus of control, however, seem to fare worse when exposed to bullying. Those holding an internal locus of control orientation had a stronger relationship between exposure to bullying and strain, as compared to those low in internal locus of control. Hence, it seems that people high in internal- and people low in external locus of control are the most negative harmed when exposed for higher levels of workplace bullying. A possible explanation may be that when expected resources fail to work an increase in exposure to bullying behaviors results in greater harm than if the expectation of personal control is already absent. Having an external locus of control may also involve blaming others for one’s misfortune more than oneself, which may result in lesser feelings of shame and guilt. Hence, these targets report fewer symptoms of psychological strain when exposed to bullying behaviors.The study was approved by the ethics committee of the Department of Organizational Psychology at Birkbeck, University of London. Participation was voluntary and actual participation seen as a statement of consent.All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.SE holds training courses in the management of workplace bullying and advices employers on the prevention and management of workplace bullying. The remaining 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.


Pleurotus ostreatus. The present study demonstrated that infection with mycovirus PoV-ASI2792 (PoV) caused phenotypic and physiological changes in fungal cells and mycelia. The microscopically determined growth rate of the virus-infected strain was lower than that of the virus-cured strain, due to the conglomerate phenomenon during the mycelial growth process. An exploration of the viral effects of PoV on fruiting bodies yield showed significantly lower than that on virus-cured P. ostreatus. A colorimetric assay of polyphenol oxidase activity in the strains showed very weak activity in the virus-infected strain. To estimate the activity levels of enzymes related to the growth and fruiting body formation, the relative expression levels of genes encoding various extracellular enzymes such as Carbohydrate-Active Enzymes (CAZymes) were measured by quantitative RT-PCR. The expression levels of the assayed genes were significantly lower in virus-infected than in virus-cured P. ostreatus. Together, these results indicate that PoV infection affects the spawn growth and fruiting body formation of P. ostreatus via decreased expression and activity of some extracellular enzymes including lignocellulolytic enzymes.Isogenic virus-cured and virus-infected fungal strains were previously obtained and compared to investigate mycoviral diseases and, specifically, the influence of viral infection on the vegetative growth of  The first reported mycovirus  was found in 1962 in the cultivated mushroom Agaricus bisporus. The mushroom virus causes abnormal mycelial growth and malformed fruiting body production5.Mycoviruses infect and replicate in all major fungal taxa, including both pathogenic and edible fungi. The fungal viruses are commonly latent, and hosts do not exhibit symptoms upon infection. Nevertheless, some mycoviruses cause severe morphological and physiological changes in fungal hosts, such as alterations of growth rate, colony morphology, spore production, pigmentationPleurotus ostreatus, mycoviral infections can cause symptoms such as irregular mycelial growth rate, decreased fruiting body yield, and malformed fruiting bodies, and thus occasionally affect cultivation of the mushroom. As mentioned above, several mushroom diseases have been reported to be associated with mycoviruses, such as oyster mushroom spherical virus (OMSV)6, oyster mushroom isomeric virus (OMIV)7, P. ostreatus spherical virus (POSV)8, oyster mushroom dsRNA virus (OMDV)9, P. ostreatus virus 1 (PoV1)10, and P. ostreatus ASI2792 virus 3.In the edible mushroom P. ostreatus have been isolated and characterized. However, studies of mycovirus–host interactions have been insufficient to reveal the mechanisms involved in infection of P. ostreatus because it is difficult to secure definite isogenic virus-free or -cured strains to compare to virus-infected strains3. Because different genotypes of the same fungal species can produce inconsistent data with regard to identical mycovirus strains, various approaches have been used to obtain isogenic virus-cured strains, such as cycloheximide or ribavirin treatment, hyphal tip cutting, single-sporing, protoplast regeneration, or thermal stress treatment14. Even when the virus is successfully cured from a virus-infected fungal cell, impairments caused by the treatment make the cured fungal cell inappropriate for investigations of fungal–mycoviral interactions. In the method for securing an artificial virus-infected strain, infectious cDNA clones of a fungal virus for artificial infection were transformed into the virus-cured fungal strain upon integration of the infectious clones into the fungal genome based on Koch’s postulates15. However, this method is also challenging because of the difficulty in the case of multi-segmented mycovirus15 and the alteration of gene expression with non-homologous chromosomal integration, such as ectopic recombination16. Therefore, in our previous investigation of PoV in P. ostreatus, virus-curing was performed by the mycelial fragmentation method followed by single colony isolation17. A virus-cured strain (free version) of a naturally infected isolate can be obtained by a technique based on selecting progeny or fungal hyphae without the virus15. Virus-cured isogenic lines, which have identical genetic backgrounds, can then be established to explore direct mycovirus–fungal host interctions. As reported, we successfully obtained a PoV-cured strain as well as the PoV-infected strain from a malformed fruiting body, and verified the presence of the virus and its effect on the irregular colony growth of P. ostreatus3.Mycoviruses infecting various hosts including Pleurotus ostreatus, a white-rot basidiomycete, degrades its substrates  with various carbohydrate-active enzymes (CAZymes) such as lignocellulolytic enzymes associated with lignin degradation18. CAZymes secreted by fungi are classified into several families, including two high-priority classes, i.e., glycoside hydrolase (GH) and auxiliary activities (AA)18. These enzymes assist in the acquisition of nutrients to enhance mycelial growth and fruiting body production in Pleurotus spp. The expression levels of genes encoding extracellular enzymes associated with the degradation of lignin, cellulose, hemicellulose, and pectin exhibit clear distinctions depending on the culture conditions, such as available nutrients (substrate composition); temperature; levels of moisture, light, carbon, and oxygen; and presence of infectious diseases, in cultivated mushrooms20. Because it is difficult to prevent disease in mycovirus-infected strains of cultivated mushrooms, other fungal species have been used to investigate the gene expression and activity levels of extracellular enzymes, such as lignocellulolytic enzymes, in mycovirus-infected strains4. However, there is little literature about the effect of mycoviruses on the gene expression levels and activities of extracellular enzymes, even though mycoviruses can cause drastically decreased fruiting body production in cultivated mushrooms, including Pleurotus spp.P. ostreatus. Expression and activity levels of extracellular enzymes such as lignocellulolytic enzymes were analyzed throughout PoV infection of P. ostreatus to investigate the relationship between the mycovirus and the discernible phenotypic changes in growth and fruiting bodies. To our knowledge, this is the first report describing the effects of a mycovirus infection on the gene expression of representative CAZymes (CAZy GH and CAZy AA class enzymes) and non-CAZymes during the different developmental stages of P. ostreatus.In the present study, we explored the novel biological functions of PoV, which causes phenotypic aberrations or alterations in fungal growth and fruiting bodies, through the comparison of isogenic virus-cured and -infected strains of P. ostreatus ASI2792, we obtained abnormal mycelial cells, confirmed the presence of the PoV mycovirus (PoV-ASI2792)3, and examined the deleterious effects of mycoviral infection on the growth of fungal colonies using isogenic virus-cured and virus-infected fungal strains. In the present study, we attempted to better understand the relationship between PoV infection and the vegetative characteristics of P. ostreatus based on a pair-wise comparison of isogenic strains. Two virus-cured lines, P. ostreatus AS2792-VF10 (POVF10) and P. ostreatus AS2792-VF12 (POVF12), were randomly selected on the basis of normal phenotypes using previous data3. Whenever a comparative analysis of isogenic strains is performed, the status of PoV infection should be confirmed based on colony morphology and viral genome assay. For this comparative study of one PoV-infected line (POV) and the two virus-cured lines (POVF10 and POVF12), the presence or absence of the PoV mycovirus was verified for each strain via gel electrophoresis and RT-PCR using P. ostreatus mycelium. The genome segment and a partial cDNA from the PoV mycovirus were only observed in the mycelia from the virus-infected strain , which exhibited delayed mycelial growth caused by abnormal hyphal branching, were compared to those of two virus-cured isogenic strains, POVF10 and POVF12. The highest yield of all tested mushroom strains was obtained from the first flush, and yields gradually reduced upon subsequent flushes. Fruiting bodies of the POV strain were only obtained from three flushes, whereas fruiting bodies of the POVF10 and POVF12 strains were obtained from more than four flushes , whereas PoV RNA accumulation in the POV strain was significant throughout two developmental stages of P. ostreatus  and -infected (POV) strains were analyzed using qRT-PCR. Total RNA from three developmental stages—vegetative mycelium (M), mycelium spawn (B), and mature fruiting body (F)—was used to compare the mRNA expression of 12 genes encoding 10 different extracellular enzymes  in the virus-infected and -cured strains Figs.  and S4.Fmnp2 and mnp3) and laccases (pox1 and pox3), which belong to the group of auxiliary activities (AA) redox enzymes involved in lignin degradation in the CAZy database, exhibited severely down-regulated expression during the vegetative mycelium stage in the POV strain. Among the four AA genes, the most significant difference in expression was observed for mnp2, which had 337-fold higher expression in the mycelium of the POVF12 strain than in the POV strain  and a lipase gene were inconsistent between the two virus-cured strains at the mycelium spawn stage. A gene encoding polygalacturonase was not affected by PoV in the spawn stage of P. ostreatus , CAZy GH enzymes , and non-CAZymes (protease and lipase) in the vegetative mycelium of P. ostreatus. Furthermore, these extracellular enzymes were substantially down-regulated in the mycelium spawn due to PoV infection. Two laccase genes from the CAZy AA family and four genes encoding cellulase, β-glucosidase, chitinase, and amylase from the CAZy GH group exhibited down-regulated expression in the fruiting body upon PoV infection. Our results indicate that PoV affects growth and/or fruiting body formation in P. ostreatus by decreasing or suppressing the expression of some CAZymes and non-CAZymes. Further studies are necessary to investigate these hypotheses. Edible mushrooms have been used as a resource for enzymes for pharmaceuticals, chemical production, biofuels, food, and consumer products because they are safe and highly nutritious. In particular, effective lignocellulolytic enzymes, which strongly degrade complex lignocellulosic substrates into soluble substances, are produced from cultivated mushrooms34. The results of this study suggest that these industrial enzymes should be purified from virus-free commercial mushroom lines.CAZy GH enzymes hydrolyze the glycosidic bond between two or more carbohydrates or between a carbohydrate and a non-carbohydrate moiety in basidiomycetesThe factors associated with viral diseases should be further investigated even though substantial data have already been accumulated on the symptoms of infection. Comprehension of the importance of virus-free spawn and the pathogenic mechanisms of viral infection are required for appropriate and efficient production of cultivated mushrooms. Our data can also be applied to investigations of the unexplored relationships between mycoviral infection and phenotypic changes in other mushroom species that are major agricultural products for food, animal feed, enzymes, medicinal compounds, and organopollutant degraders.P. ostreatus—were used for this study3. The fungal cultures were maintained on potato dextrose agar (PDA) plates at 25 °C in the dark for 7 days. Sawdust agar plates  were used for the measurement of the radial growth of the colonies under the same culture conditions. Small mycelial cultures were grown on cellophane overlays under the same culture conditions3 and collected for the extraction of dsRNA and total RNA. The harvested mycelia were stored at −70 °C for further analyses. PDA plates containing each cell wall-disturbing agent, sodium dodecyl sulfate (SDS) (0.1 and 0.3%) and Congo red (0.005 and 0.01%), were used to assay cell wall integrity of fungal strains at 25 °C in the dark for 5 days. All measurements of radial growth rate on culture plates were performed in at least three biological and three technical replicates. Each inoculum of P. ostreatus strains for sawdust-bottle cultivation was grown in potato dextrose broth (PDB) at 25 °C in the dark for 7 days with continuous agitation at 200 rpm.Previously described strains—the PoV-ASI2792 (PoV)-cured and PoV-infected isogenic lines of the edible mushroom P. ostreatus hyphae. Culture plates containing the fungal hyphae were directly observed at a magnification of 200 × . The ProRes CapturePro software  was used to observe changes in the branching pattern.Fungal mycelia were cultured for 2 days on PDA and viewed under differential interference contrast microscopy  to analyze the branching pattern of the P. ostreatus, sawdust and beet pulp were used as the principal substrates. Sawdust, beet pulp, rice bran, and cotton-seed meal were mixed at a 50:30:10:10 ratio (W/W/W/W) and the mixture was adjusted to a water content of 65% in a polypropylene bottle, as described previously35, with slight modifications. Each bottle was inoculated with mycelia grown in PDB and incubated in the dark at 25 °C for 30 days. After the surface of substrate was completely covered with mycelia (M), the sawdust bottles (B) were transferred to horizontal racks in a cropping room at 22–25 °C and 80–85% relative humidity under semidarkness. The mushroom spawn was a pure culture of mycelia growing on a solid substrate. The spawn bottles were then opened, and the mats were rehydrated 2–3 times a day to keep the mycelia moist and induce fruiting body (F) formation36. Mycelial spawn and fruiting bodies were stored at −70 °C for further study.For fruiting body cultivation of 38. The flush efficiency (FE) was also measured; it was calculated as the ratio of the number of flushed bottles to the total number of bottles39.The weights of the harvested fruiting bodies were recorded. The total weight of all fruiting bodies from four flushes was measured and used to calculate total yield and biological efficiency (BE). BE was defined as the ratio of fresh fruiting body weight to substrate dry weight40. Fungal strains were grown for 5 days on modified Bavendamm’s medium  in darkness, and assessed by gauging the appearance of a brown oxidation zone around the colonies. The radial growth of the colonies and the diameter of the dark brown zone on Bavendamm’s medium were measured using a calipers . The analyses of polyphenol oxidase activity were performed in three biological and four technical replicates.Polyphenol oxidase activity was measured using the Bavendamm assayP. ostreatus were extracted as previously described41. The dsRNA was prepared from the corresponding total nucleic acid preparations. A miniprep method for detection of dsRNA41 was applied with slight modifications. The dried mycelia were ground to a fine powder, and the dsRNA was isolated from 200 mg of mycelial powder via CC41 cellulose column chromatography. The dsRNA was dissolved in 40 μl of RNase-free water. Total RNA from fungal samples was purified using TRI Reagent Solution . The quality and quantity of dsRNA and total RNA were evaluated using electrophoresis on 0.8% agarose gels and/or a UV SPECTROstar Nano system .The dsRNA and total RNA from mycelia, fruiting bodies, and/or sawdust spawn of via the comparative threshold cycle method using the LightCycler 96 qualitative detection module (Roche), as described previously42. In addition, relative gene expression levels of target genes in the mycelia, fruiting bodies, and sawdust spawn were performed with the same qRT-PCR method for the viral gene. Detailed primer information is listed in Supplementary Table P. ostreatus was used as the reference gene30.We synthesized cDNA from approximately 0.1–1 μg of dsRNA or total RNA from the mycelia, mature fruiting bodies, and/or sawdust spawn using the GoScript Reverse Transcription system . RT-PCR analysis was carried out for the PoV gene encoding the RNA-dependent RNA polymerase (RdRp) protein using the primer pair pov-F and pov-R. The level of viral gene expression in the mycelia and fruiting bodies was performed using the results of qRT-PCR with the PoV-specific primer pair. qRT-PCR was performed in a reaction mixture containing 2 μl of first-strand cDNA and 100 nM each of povq-F and povq-R primers in a total volume of 20 μl. SYBR green-based qRT-PCR was performed using FastStart Essential DNA Green Master mix  on a LightCycler 96 system (Roche). Statistical analysis of viral gene expression were calculated Mycelial colony diameter, fruiting body yield, and area and color intensity of the brown oxidation region were analyzed via one-way analysis of variance using SPSS software , with p < 0.05 considered to indicate significant differences between constructs. When a significant F-value was obtained, Duncan’s multiple range test was used to test the significance of the effect (p < 0.05) between virus-cured and virus-infected strains. Relative gene expression levels obtained using qRT-PCR were analyzed using the same statistical test.Supplementary Information.


The affiliation for author Gerardo Botti is incorrect. The correct affiliation is: Scientific Director, Istituto Nazionale Tumori, Fondazione “G. Pascale”, IRCCS Italia, Naples, Italy.


Borrelia burgdorferi. Until recently, ticks were considered as the vectors of a few viruses that can infect humans and animals, such as Powassan, Tick-Borne Encephalitis and Crimean–Congo hemorrhagic fever viruses. Interestingly, however, several new studies undertaken to reveal the etiology of unknown human febrile illnesses, or to describe the virome of ticks collected in different countries, have uncovered a plethora of novel viruses in ticks. Here, we compared the virome compositions of ticks from different countries and our analysis indicates that the global tick virome is dominated by RNA viruses. Comparative phylogenetic analyses of tick viruses from these different countries reveals distinct geographical clustering of the new tick viruses. Some of these new tick RNA viruses (notably severe fever with thrombocytopenia syndrome virus and Heartland virus) were found to be associated with serious human diseases. Their relevance to public health remains unknown. It is plausible that most of these newly identified tick viruses are of endogenous origin or are restricted in their transmission potential, but the efforts to identify new tick viruses should continue. Indeed, future research aimed at defining the origin, the ecology and the spillover potential of this novel viral biodiversity will be critical to understand the relevance to public health.Ticks are vectors of several pathogens that can be transmitted to humans and their geographic ranges are expanding. The exposure of ticks to new hosts in a rapidly changing environment is likely to further increase the prevalence and diversity of tick-borne diseases. Although ticks are known to transmit bacteria and viruses, most studies of tick-borne disease have focused upon Lyme disease, which is caused by infection with  Arachnida, order Parsitoformes, and suborder Metastigmeta that is further divided into three families, Ixodidae (hard ticks), Argasidae (soft ticks) and Nuttalliellidae [Ixodidae is the largest family and it includes four genera and over 700 species of tick which are distributed around the globe [Borrelia burgdorferi [Ticks are obligate blood-feeding ectoparasites that belong to the class iellidae . Ixodidahe globe . The Worgdorferi  was firsgdorferi . Thirty gdorferi . Phylogegdorferi . This sugdorferi . These oHyalomma marginatum marginatum), but recent outbreaks (in Turkey) have been linked to migratory birds dispersing both ticks and pathogens to new areas [Ixodes spp.) of vectors have shown the ability to transmit the virus. Therefore, the potential exists for a pathogen such as CCHF to, like B. burgdorferi, expand in geographic distribution rapidly if conditions permit. Overall, the threat posed by ticks and their associated pathogens is great and additional research is needed to better understand issues such as the role of reservoirs, the dynamic ranges of vectors, and the community ecology of the enzootic reservoir and vector cycles. A combination of these factors is what leads to the spillover of pathogens into humans.While Lyme Disease illustrates the ability of tick-borne pathogens to rapidly expand in a dynamic environment, another important tick pathogen, Crimean Congo Hemorrhagic Fever (CCHF), shows the ability of these pathogens to persist endemically across a vast geographic area (30 countries), despite the notable virulence  . First dTicks are now well recognized as vectors of several highly pathogenic viruses. However, until a decade ago, only a few tick viruses were known and, among these, flaviviruses were the most well characterized for their geographical prevalence and disease association. Powassan virus is a tick-borne flavivirus present in North America and it is phylogenetically related to the tick-borne encephalitis virus group that was reported from ticks in Europe . AlthougThis metaviromics approach has led to the identification of a plethora of new tick viruses ,18. ThesMost of the well-characterized tick-borne viruses were identified between 1950 and 1975. Long after the first realization that ticks were acting as the vector of pathogenic viruses such as Nairobi sheep disease virus and Louping ill virus in 1931, the first large-scale project to characterize tick viruses was undertaken by the Rockefeller Foundation [Flaviviridae and is serologically related to Tick-Borne Encephalitis Virus complex (TBEV) that also includes Karshi, Kyasanyur forest disease, Langat, Louping ill, Alkhurma, Omsk hemorrhagic fever and Royal Farm viruses. As reflected in the name, POWV was first identified in 1958 in a child with encephalitis from Powassan city in Ontario, Canada [Haemaphysalis longicornis ticks in Russia in 1972. In 1997, a close genetic relative of POWV was identified in Ixodes damimni and was named Deer Tick Virus (DTV) [I. scapularis, POWV is common in I. cookei and I. marxi and medium-sized mammals such as Marmota monax (Woodchucks), while DTV is common in I. scapularis and small mammals, such as White-Footed Mice (Peromyscus leucopus) [Powassan virus (POWV) belongs to the family , Canada . Later, us (DTV) . Phylogeeucopus) . I. scapularis and P. leucopus, it is expected that DTV-associated cases will become more common compared to POWV [Infection with POWV in humans is mostly reported in the United States and the estimated fatality rate is 10% . The vir to POWV . Orthonairovirus in the family Nairoviridae and is an enveloped virus with a tripartite RNA genome of negative polarity. The main vector of CCFHV is Hyalomma marginatum, a tick that is widespread in the regions of Africa, Europe, Asia and the Middle East. The virus was first found in Crimea in 1944 and later in the Congo in 1969 [CCHFV belongs to the genus  in 1969 . Infecti in 1969 .Phylogenetic analysis of CCHFV isolates from different countries reveals the existence of at least seven distinct genetic clades, indicating that a tremendous amount of diversity exists . InteresPhlebovirus of the family Bunyaviridae. The initial outbreak consisted of 171 patients from six provinces of China, indicating a widespread infection. After the initial identification of SFTSV from human samples, epidemiological surveillance indicated the presence of this virus in Haemaphysalis longicornis ticks. These ticks predominantly feed on farm animals including goats, sheep, cattle, and horses, but they can also be found on companion animals including dogs and cats. Phylogenetic analyses indicate a close genetic relatedness among the SFTV isolates of humans and animals, indicating a shared origin [SFTSV was first isolated in 2009 from patients in China who presented with fever, thrombocytopenia, leukocytopenia, and multi-organ dysfunction . More red origin . Notablyd origin . Coltivirus in the family Reoviridae. The principle vector of CTFV is the Rocky Mountain Wood Tick, Dermacentor andersoni. Thus far, human cases of CTFV infection have been reported from Wyoming, Montana, Utah, Oregon, Idaho and Colorado [CTFV is an RNA virus with a segmented genome of positive polarity and it belongs to the genus Colorado . A recenColorado . Amblyomma americanum), suggesting a tick-borne virus infection in the index cases. During the period 2012–2013, a total of six cases of heartland virus were identified and all patients were men older than 50 years. Viral surveillance studies indicate the persistent infection of heartland virus in Amblyomma americanum ticks as evident by the presence of the virus in ticks collected over several years [Heartland virus is a newly identified phlebovirus that was first isolated from two northwestern Missouri farmers who were hospitalized with fever, leukopenia, and thrombocytopenia in 2009 . Genomesal years . Additioal years .While the traditional approach of virus identification using cell culture or via animal models was very successful, several tick viruses remained unidentified as shown by many recent metaviromics studies ,39,40,41Flaviviruses were the most common, followed by viruses belonging to the families Nodaviridae, Tetraviridae, Picornaviridae, Caulimoviridae, Virgaviridae, Narnaviridae, Luteoviridae and Sobemoviridae. Worldwide, the most common constituents of tick viromes were single-strand negative-sense RNA viruses belonging to the orders Bunyavirales and Monogenavirales [Chuviridae and Rhabdoviridae , Norway nairovirus and Beiji nairovirus (China). Genomes of these viruses are comprised of three segments of single-strand negative-sense RNA that code for the nucleocapsid proteins (segment S), envelope proteins (Segment M) and the polymerase (segment L), but, to date, only two of the three segments have been identified, namely the S and L segments. Notably, the M (envelope) segment of bunyaviruses is also known to be the most divergent part of the viral genome and, thus, despite being present in the analyzed tick samples, the M segment could have eluded identification due to high genetic diversity . Among the newly identified tick nairoviruses, South Bay virus (SBV) is the most well characterized. The virus was first identified in I. scapularis ticks collected from the United States and was determined to be present in 20% of individual ticks [These viruses belong to the family al ticks . The seqal ticks . OverallPhenuiviridae of the order Bunyavirales. Tick phleboviruses include the well-characterized SFTSV and Heartland Virus, and several recently identified viruses such as Black-legged tick phlebovirus type 1, 2 and 3 (United States), Norway Phlebovirus 1, American dog tick phlebovirus, Beiji Phlebovirus (China) and Timbillica virus   . Phlebovfections ,18,39. Sectively ,40. CompChuvirus of the order Mononegavirales. These viruses have linear or circular genomes that consist of a single-strand negative-sense RNA that codes for all viral proteins. Tick mononegavirus was first identified in I. scapularis [I. scapularis and A. americanum ticks from several states within the United States [Ixodes spp. and were genetically clustered , H. longicornis (50 of 91), and H. campanulata (18 of 24) had the highest positive rates, followed by H. flava (5 of 45). The remaining tick species—including R. sanguineus (1 of 3), I. sinensis (1 of 1), and I. granulatus (1 of 3)—all contained JMTV-positive samples. Evidence of JMTV infection was also reported from a wide range of rodents and cattle. Although from an unpublished report, a JMTV-related virus (Alongshan virus GenBank no. AXE7876.1) was reported to be found in humans with febrile illness. Another relative of JMTV, Mogiana tick virus was recently found in R. microplus ticks from Brazil [The most interesting of the new tick viruses is Jingmen tick virus (JMTV), which is also the first well-characterized segmented flavivirus . The JMTus ticks . Interesm Brazil . Finallygicornis  of 91, am Brazil ,49,50,51Undoubtedly, our knowledge of the viruses present in ticks has been substantially increased in the last few years, but a deep dive into data generated by metagenomics indicates that most of the studies designed to characterize the tick virome used pools of ticks as study samples and, thus, it is plausible that rare or less abundant tick viruses have eluded identification. This hypothesis is further supported by the observation that, of several well-known tick viruses, only a few have been found using metagenomics. Two ways to improve the analysis of the tick virome include using the metagenomics of large numbers of individual ticks and/or the enrichment of samples for viruses that are more likely to infect mammalian cells. Tick homogenates can be inoculated in cell lines or animal models to enrich viruses with the potential to infect vertebrate hosts, followed by metaviromic-based identification.Another important issue is our limited knowledge of the origin and ecology of these newly discovered tick viruses. The tick virome is comprised of innate and/or acquired viruses. Innate viruses can be endogenous viruses, those integrated in the genome, and viruses that are exclusively present in ticks and maintained through vertical transmission. So far, a thorough analysis of tick eggs and larvae has not been reported and this could shed some light on the nature of innate virome of ticks. Similarly, only a few acquired tick viruses have been characterized for their transmission and ecology. The studies of these viruses, such as Powassan or CCHFV, indicate that these viruses are acquired by ticks from mammalian reservoir hosts during the blood meals associated with the tick lifecycle. These viruses then remain within the tick and vertebrate host cycle until they incidentally spill over and infect humans. Interestingly, the complete host range of these viruses is still not fully understood since most of these viruses cause a transient viremia in the vertebrate host that may or may not be detected using PCR-based assays. We believe the development of serological assays for tick viruses will substantially improve our knowledge of virus ecology and host tropism. Serological assays can also reveal the real infection prevalence of known or new tick viruses in vertebrate hosts, since the presence of antibodies can indicate both prior and ongoing viral infection.Lastly, it is important to surveil domestic, feral and wild animals for both the presence of tick viruses and evidence of viral infections. It is plausible that the ongoing transmission of tick viruses in synanthropic animals is providing tick viruses an interface to evolve and establish infection in humans. Infection with Powassan virus in a range of wild rodents and other mammals is well documented and this supports the maintenance and evolution of tick viruses in synanthropic hosts . The ide


We observed Ebola virus antibodies in 1 insectivorous bat genus and 6 frugivorous bat species. Certain bat species widespread across Africa had serologic evidence of Zaire and Sudan Ebola viruses. No viral RNA was detected in the subset of samples tested (n = 665). Ongoing surveillance of bats and other potential animal reservoirs are required to predict and prepare for future outbreaks.To clarify the role of bats in the ecology of Ebola viruses, we assessed the prevalence of Ebola virus antibodies in a large-scale sample of bats collected during 2015–2017 from countries in Africa that have had previous Ebola outbreaks  or are at high risk for outbreaks (Cameroon). We analyzed 4,022 blood samples of bats from  We collected and preserved samples the same way we did for free-ranging bats.During November 2015–August 2017, we collected samples from free-ranging frugivorous and insectivorous bats in Guinea, Cameroon, and the DRC. We captured bats at night using ground mist nets or harp traps in roosting and foraging sites. We set up ground mist nets (12 × 3.2 m) of 30-mm and 60-mm mesh sizes at different heights (1–7 m) to maximize capture of different species. We opened nets or harp traps just before sunset and checked for bats every 1–2 hours. Captured bats were released the same night immediately after sampling. Using bat whole blood taken by venipuncture of the propatagial or brachial vein, we dropped blood samples directly onto Whatman 903 filter paper . We air-dried and preserved samples individually in plastic bags containing silica desiccant and stored them in hermetic boxes; 2–3 weeks later, we transferred dried blood spots to −20°C until needed for analysis. Data recorded in the field included information on capture site , capture method, morphology , sex, age class , and species . We collected negative control samples (n = 145) from a captive-born insectivorous bat species (103 6 beads) in 96-well flat-bottom filter plates  on a plate shaker at 300 rpm for 16 h at 4°C in the dark. After washing, we added 0.1 μg/mL of goat anti–bat biotin–labeled IgG  per well and incubated for 30 min at 300 rpm. After another round of washing, we added 50 µL of 4 µg/mL streptavidin-R-phycoerythrin  per well and incubated for 10 min at 300 rpm. Reactions were read with BioPlex-200 . We expressed results as median fluorescence intensity (MFI) per 100 beads. We included 3 samples on every plate to validate interassay repeatability.We tested dried blood spots with a Luminex-based serologic assay adapted for bats . The asshttps://www.r-project.org/). We considered a blood sample reactive if the MFI of the reaction was above the cutoff. We defined Ebola virus antibody positivity as reactivity to glycoprotein and nucleoprotein of the same lineage, as was done in our previous study  or m2000sp methods , which are known for a high performance recovering nucleic acids from dried blood spots (https://blast.ncbi.nlm.nih.gov/Blast.cgi) to identify the most similar bat species. For samples with no or low similarity (<97%) hits with species in GenBank, we performed phylogenetic analyses with newly obtained sequences and reference sequences for different bat species using maximum-likelihood methods implemented with PhyML (http://www.atgc-montpellier.fr/phyml/) to determine genus.We confirmed bat species identification recorded in the field on a subset of samples by using molecular tests. We amplified an ≈800-bp fragment of mitochondrial cytochrome b using primers cytb-L14724 (forward) and cytb-H15506 (reverse) , identification was possible only at the genus level; for some Molossidae bats, we could not distinguish between Mops and Chaerephon genera because of the lack of sequences in GenBank (Pteropodidae) of 12 species and 2,199 (54.7%) insectivorous bats (7 families) of >27 species. The insectivorous bat families sampled, in order of decreasing frequency, were Hipposideridae (31.9%), Molossidae (13.4%), Miniopteridae (5.8%), Rhinolophidae (2.1%), Vespertilionidae (0.8%), Nycteridae (0.5%), and Emballonuridae (0.12%). Overall, 54.7% of bats were female and 43.8% were male; for 1.5% (n = 60) of bats, sex was unknown. Most (77.9%) bats were adults, and 9.6% were juveniles; for 12.5% (n = 502) of bats, age could not be determined or was not recorded.We analyzed blood samples from 4,022 wild bats from 21 different regions in Cameroon (n = 10), Guinea (n = 8), and the DRC (n = 3) ; Table 1 GenBank . For 87 >1 antigen was 734 (18.2%) by the mean + 4×SD method, 274 (6.8%) for the change-point method, 175 (4.4%) for the binomial method, and 457 (11.4%) for the exponential method. Blood samples frequently reacted with glycoprotein antigens; samples reacted most with Zaire and Sudan Ebola virus antigens and least with Reston  were positive for Zaire or Sudan Ebola virus antibodies  bat species.We estimated specific reactivity to Zaire and Sudan Ebola viruses by bat species. We did not include Bundibugyo and Reston because recombinant nucleoproteins were not available. Among insectivorous bats, only blood samples from tibodies . Among fM. condylurus bat samples from Gabon and Mops sp. bat samples from Cameroon. Data were available for 5,405 frugivorous bats of 17 species from 12 genera from West , West Central , and East (Zambia) Africa  have been tested. Overall, blood samples from 8 frugivorous bat species have been found reactive with Zaire Ebola virus antigens. Blood samples from E. helvum, H. monstrosus, and R. aegyptiacus bats from several countries across Africa have been reported to be seropositive. Reactivity has been observed with samples from E. gambianus bats in Ghana (10.8%) and Guinea. Reactivity was observed with large sample sets from E. franqueti bats derived from Gabon and the Congo and a small sample set from Ghana but not Guinea, Cameroon, or the DRC. M. pusillus and M. torquata bats tested positive for Zaire Ebola virus antibodies in studies in which large sample sets were collected. Among L. angolensis bat samples, only those from Cameroon have tested positive for antibodies.For comparison, we compiled data regarding Zaire Ebola virus serology in bats of known species from previous studies  and this study  , 6. Data) Africa . No ZairH. monstrosus , M. torquata , and E. franqueti [116 from Cameroon]), all were negative for Zaire Ebola virus RNA. Of the 371 samples from bat species E. helvum , L. angolensis , M. pusillus , R. aegyptiacus , E. gambianus (25 from Guinea), and Mops sp. (20 from Cameroon), all were negative for Zaire Ebola virus RNA.We screened 665 samples from the DRC (n = 193), Cameroon (n = 399), and Guinea (n = 73) by RT-PCR for the presence of Zaire Ebola virus RNA. Of the 294 samples originating from bats previously documented to carry Zaire Ebola virus RNA  and 8 frugivorous bat species  exhibited Zaire Ebola virus antibodies  in only 1 study; virus replication was seen in all species, and fecal shedding was seen in E. wahlbergi bats  was confirmed to be seropositive. Zaire and Sudan Ebola viruses circulate in different species across Africa, with potential co-circulation of both viruses in some species. Although we have data on >8,000 bats from >40 species, this sample size is small, given the high numbers of bats that constitute colonies. This study illustrates the complexity of tracking the animal reservoir of Ebola viruses, not only because sampling of wild bats without performing euthanasia is difficult and time-consuming but also because of the absence of a reference standard for serologic tests. To clarify the significance of Ebola virus antibodies, documenting the extent to which viral RNA and shedding can be detected in species with antibodies is crucial for predicting and controlling the risk for new outbreaks. Efforts must continue not only to sample bats but also other animals to elucidate where the virus circulates in wildlife.In conclusion, we demonstrated higher rates of Ebola virus antibodies in frugivorous than in insectivorous bats. The total number of frugivorous species shown to be Zaire Ebola virus seropositive has increased to 8, and 1 insectivorous bat species (Description of serum sample dilution testing, mean fluorescence intensity cutoff values, and Ebola virus antibody cross reactivity.


Fragaria, Rubus and Ribes spp., and conserved accessions of the plant genetic resources of Rubus spp. maintained at research stations in Finland, for the presence of viruses using small interfering RNA (siRNA) -based diagnostics (VirusDetect). The advance of the method is that unrelated viruses can be detected simultaneously without resumptions of the viruses present. While no virus was detected in pre-basic mother plants of Fragaria and Ribes species, rubus yellow net virus (RYNV) was detected in pre-basic mother plants of Rubus. Raspberry bushy dwarf virus (RBDV), black raspberry necrosis virus (BRNV), raspberry vein chlorosis virus (RVCV) and RYNV were detected in the Rubus genetic resource collection. The L polymerase encoding sequence characterized from seven RVCV isolates showed considerable genetic variation. The data provide the first molecular biological evidence for the presence of RYNV in Finland. RYNV was not revealed in virus indexing by indicator plants, which suggests that it may be endogenously present in some raspberry cultivars. In addition, a putative new RYNV-like badnavirus was detected in Rubus spp. Blackcurrant reversion virus (BRV) and gooseberry vein banding associated virus (GVBaV) were detected in symptomatic Ribes plants grown in the field. Results were consistent with those obtained using PCR or reverse transcription PCR and suggest that the current virus indexing methods of pre-basic mother plants work as expected. Furthermore, many new viruses were identified in the collections of plant genetic resources not previously tested for viruses. In the future, siRNA-based diagnostics could be a useful supplement for the currently used virus detection methods in certified plant production and thus rationalize and simplify the current testing system.Pathogen-free stocks of vegetatively propagated plants are crucial in certified plant production. They require regular monitoring of the plant germplasm for pathogens, especially of the stocks maintained in the field. Here we tested pre-basic mother plants of  Virus-free and genetically true-to-type propagation materials are crucial for certified production of small fruits. Certified production of small fruits has been carried out in Finland since 1977 . Basic min vitro cultures, and cryopreserved [Fragaria x ananassa (Weston) Royer] grown on 3800 ha, raspberry on 429 ha, and currants (Ribes nigrum L. and R. rubrum L.) on 1740 ha [The national plant genetic resources program of agriculture, horticulture and forestry was established in Finland in 2003 with the aim to conserve genetic resources and promote their sustainable use. The horticultural plants are conserved in clonal field collections, reserved , 7. Becareserved . The mosAphis idaei van der Goot) and the European large raspberry aphid (Amphorophora idaei Börner) are the most common vectors that transmit viruses to red raspberry (Rubus idaeus L.) in Northern Europe [Cecidophyopsis mites transmit viruses to Ribes species [Chaetosiphon fragaefoli Cockerell) transmitting viruses to Fragaria species has not yet been detected in Finland but was recently detected in Sweden, a neighboring country [The maintenance of vegetatively propagated plants free of pathogens is challenging in the field, because plants such as small fruits tend to become infected with viruses . The rasn Europe –12, wher species . Strawbe country .The aim of this study was to test viruses in pre-basic mother plants and conserved accessions of plant genetic resources for the presence of viruses. The plants were tested using a newly developed method, deep sequencing of virus-derived small interfering RNAs (siRNAs) in plants, which relies on the ability of plants to recognize and inactivate double-stranded RNA by splicing it into fragments of certain sizes , 16–19. Fragaria, Rubus and Ribes spp. suitable for Finnish climate were maintained at the Natural Resources Institute Finland (Luke) in Laukaa  (Rubus breeding lines (not yet approved as pre-basic mother plants) and indicator plants of Fragaria and Rubus maintained in vitro were tested . In addition, similar representative samples were taken from the pre-basic mother plants of Fragaria and from the Rubus stock plants used as virus indicators and maintained in vitro.Pre-basic mother plants of economically important cultivars and other cultivars of 25°99’E) . The pla25°99’E) . Also, ae tested . Each prRubus was developed in the 1980s. Accessions in the collection originate from different parts of Finland and consist of material selected based on interesting breeding traits, such as winter hardiness. After several rounds of selection, the Rubus collection currently consists mainly of Rubus cultivars bred in Finland. They are maintained in pots in the field during the summer and in cold storage during the winter at Luke in Piikkiö by the Baltic Sea . Plants of the Rubus collection had not been previously tested for viruses. The plants were sampled at Luke-Piikkiö in June 2015 . Samples were collected from one to three plants and parallel samples were taken from each cultivar or plant stock. Several leaves, mostly those showing symptoms, were sampled from each plant. In addition, samples were collected from Ribes nigrum and Ribes rubrum that showed distinct symptoms of malformed leaves and conspicuous yellowing, respectively . Equal amounts of total RNA (100 ng) were combined from each leaf sample to obtain six pools, each of which contained 21–43 samples (Fragaria (GEN17), Fragaria and Rubus (GEN18), Rubus and Ribes (GEN19), and Ribes (GEN20)  . Samples (GEN20) . Pool HXThe RNA pools were sent to Fasteris SA  for sequencing of the small RNAs. RNA samples were subjected to acrylamide gel electrophoresis in Fasteris, and small RNAs < 45 nt long were purified from the gel. Single-stranded 3’ adapters and barcoded 5’ adapters were ligated to the small-RNA oligonucleotides, followed by reverse-transcription and amplification by PCR to generate DNA colony template libraries. The PCR products were purified and diluted to 10 nM prior to high-throughput DNA sequencing by Illumina Genome Analyzer (HiSeq 2500). The raw siRNA reads obtained by Illumina were deposited to the European Nucleotide Archive (ENA) with accession numbers ERP108051 (gene pools GEN19-GEN20) and PRJEB30660 (gene pools HXR1 and HXR2).http://cshprotocols.cshlp.org/content/2010/11/pdb.prot5515. For PCR analysis of RNA viruses, RNA was extracted from frozen leaves with the RNeasy Plant Mini Kit (Qiagen). Total RNA was treated with DNase I (Thermo Scientific) and cDNA was synthesized using the Revert Aid RT Kit (Thermo Scientific) and random hexamer primers. The quality of the cDNA was checked in control PCR reactions using the Phire Plant Direct PCR Kit (Thermo Scientific).For PCR analysis of DNA viruses, DNA was extracted from fresh or frozen leaves using DNeasy Plant Mini Kit , the cetyl trimethyl ammonium bromide (CTAB) method, or a combination of organic extraction and the CTAB method , http://For PCR analysis, cDNA was diluted 10-fold to minimize the concentration of possible compounds that may inhibit PCR reactions. PCR was carried out using puReTaq Ready-To-Go PCR Beads , or Phusion DNA polymerase or PCR Phire Hot Start II DNA polymerase , and virus-specific primers . PCR prohttp://bioinfo.bti.cornell.edu/tool/VirusDetect/.The reads of 21–24 nt from high-throughput DNA sequencing data were assembled into contigs using Velvet software . The conNucleotide and amino acid sequences were aligned using MultAlign . PhylogeFragaria and Ribes, respectively (Sequencing of the pooled RNA from the plant samples resulted in 10.0–15.9 million reads (21–24 nt) per pool. No virus was detected in sample pools GEN17 and GEN20, which contained samples from pre-basic mother plants of ectively .Fragaria, a few breeding lines of Rubus, and a few virus indicator plants of Fragaria and Rubus. GEN19 contained samples from pre-basic mother plants of Rubus and Ribes, a few breeding lines of Rubus, and virus indicator plants of Rubus (Raspberry bushy dwarf virus (RBDV) and raspberry yellow net virus (RYNV) were detected in pool GEN18, whereas only RYNV was detected in pool GEN19 . GEN18 cof Rubus .Mapping of the virus-derived siRNA reads to the RBDV RNA1 and RNA2 showed high coverage of 72.6% and 80.3%, respectively, and high depth of coverage of 27.9 and 19.6, respectively . High coRubus plant genetic resources  of all identified viruses, except BRNV, which had a low coverage of 11.6–24.2% . RYNV was detected by PCR in 10 samples from pool GEN19   that were found to contain viruses were also tested by PCR or RT-PCR to confirm the results. As expected, RYNV and RBDV were detected in pool GEN18 . RYNV waol GEN19 . The RYN. rubrum .The sequence variability of RYNV was further characterized because only a partial sequence of the open reading frame 3 (ORF3)  and twoRubus samples from pre-basic mother plants and from plants in the collection of genetic resources , the Rubus collection of plant genetic resources (isolates MM-3dg and MM-4), and also the RYNV isolate PK-16 from raspberry cultivar Pisan keltainen (Rubus idaeus f. chlorocarpus Krause) were identical (Rubus breeding line Z23 (Z23-97 and Z23-98) were identical and also were very similar to each other, with substitutions at only three nucleotide positions. Fourteen of the 22 RYNV isolates sequenced in this study differed at the nucleotide level  of ORF3 of RYNV was amplified and sequenced from 22 RYNV-positive 4 nt of Os Tables . RYNV-Cadentical . The twode level . Sequencde level .Rubus. They included MM-78, MM-79, and MM-80 (raspberry cultivar Maurin Makea); TH-87 and TH-88 ; and Jatsi-109 (raspberry cultivar Jatsi) in pre-basic mother plants of raspberry and also in raspberry isolates MM-3dg, MM-4 (cultivar Maurin Makea), and PK-16 (cultivar Pisan keltainen) in the Rubus collection of genetic resources , HK-23 and HK-24 (cultivar RU158 Hoolin kanta), Heija-28 (cultivar RU24 Heija), Heisa-33 and Heisa-34 (from cultivar RU18 Heisa), Ville-37 (from cultivar Ville), or IS-39 and IS-40 (from cultivar Indian Summer) from the Rubus collection of genetic resources  and nearly identical to RYNV-Ca (96.8–98.9% aa identity)  over theSequence variability of RYNV was further characterized by cloning and sequencing PCR products of RYNV isolates from raspberry cultivar Maurin Makea. Analysis of 10 PCR clones indicated variability at several nucleotide positions. Most of this variability occurred at the earlier-detected degenerate nucleotide positions rather than at unique positions . Five ofThe previously characterized isolates RYNV-Ca and RYNV-BS differ in their genomic organization . To char. In phylogenetic analysis, the three identical RYNV-like isolates formed a clearly distinct clade supported by a high (99%) bootstrap value . Hence, they may represent a new badnavirus species or, alternatively, be remnants of an endogenous virus sequence.In addition to isolates of RYNV, a few sequences resembling RYNV were detected in three samples from raspberry cultivars RU168 Krusenbergs (isolates Krus-11 and Krus-12) and HY 6230 (isolate HY-20) Tables . These t. In phylap value . Hence, Rubus collection of genetic resources in the field. For example, two or more RVCV variants were found in raspberry cultivars RU53 and RU54. Therefore, some of the PCR products were cloned and sequenced, and isolates designated as RVCV-8 (cultivar RU53), RVCV-9-3, RVCV-9-4 and RVCV-10  was most similar—99.2% at the nucleotide level  and 100%Rubus collection of plant genetic resources. These plants have not been tested for the presence of viruses before. In addition, two viruses (BRV and GVBaV) were identified in symptomatic Ribes plants grown in a field trial.In this study, we tested the presence of viruses in pre-basic mother plants and conserved accessions of plant genetic resourses using the newly developed virus detection method (deep sequencing of virus-derived siRNAs). Four viruses  were detected in the Rubus by siRNA-based diagnostics, and no virus was detected in pre-basic mother plants of Fragaria and Ribes species. The results confirm that thermotherapy combined with meristem tip culture and regular virus indexing generally produce virus-free plants [Rubus idaeus ‘Malling Landmark’, R. idaeus ‘Malling Delight’ and Rubus occidentalis ‘Cumberland’ as indicator plants. However, the results of this study indicate that graft inoculation to indicator plants may be insufficient for indexing method to detect RYNV. RYNV  is one of the viruses associated with raspberry mosaic disease complex, together with BRNV  and raspberry leaf mottle virus  [Rubus viruses  appeared common among plants from the Rubus collection of genetic resources grown in the open field in Finland. Of 40 analyzed plant samples, 17 were found to contain two or three viruses. RBDV and BRNV have been previously identified in both cultivated and wild raspberries in Finland [Rubus arcticus L.) in Finland [Only one virus (RYNV) was detected in pre-basic mother plants of e plants . RYNV anviridae) . Both RYviridae) . Sequencviridae) , 33, 34. Finland , 39, 40. Finland . RBDV is Finland . It has  Finland , 42–45.Rubus, which suggests that it may actually be an endogenous virus that is present in some raspberry cultivars, as suggested [Rubus collection of plant genetic resources, RYNV sequences were variable. All isolates, except the one from Maurin Makea, were unique for their sequence. Some samples also contained sequences that resembled RYNV but were, however, notably different from them. The nucleotide sequence identity of the sequenced ORF3 region of these RYNV-like isolates was <84% as compared with previously characterized RYNV isolates. Thus, they may form a new virus species in the genus Badnavirus. Alternatively, they may represent sequences of endogenous badnaviruses that have integrated into the plant genome.RYNV was not revealed in virus indexing by indicator plants in pre-basic mother plants of uggested , 47. In Idaeovirus). RBDV in combination with RYNV or other aphid-transmitted viruses, such as RLMV and raspberry latent virus (RpLV), cause severe crumbly fruit symptoms in raspberries [The new RYNV-like virus and RYNV detected in this study were the most common partners in mixed virus infections, followed by RBDV . The cultivars are progeny from crossings by free pollination between wild and cultivated Rubus species. The original mother plants show no viral symptoms themselves, and virus-indexing using indicator plants has not revealed any virus. There is increasing evidence that endogenous forms of RYNV exist in some raspberry genomes [The Finnish RYNV isolates from raspberry cultivars Maurin Makea, Takalan Herkku, and Pisan Keltainen in the largest subcluster contained several variable degenerate nucleotides but, nevertheless, were identical at the amino acid level. Two of these isolates are from the pre-basic mother plants of  genomes , 47. The genomes –53.Rhabdoviridae, in which the viruses have a negative-sense RNA genome of 12000–14500 nt, and is common in Europe [Aphis idaei) and may easily spread between plants. Symptoms of RVCV include chlorosis of minor veins and reductions in plant vigor and raspberry yields, especially in mixed infections [RVCV belongs to family n Europe . Based on Europe . Our stufections . There ifections . TherefoRibes plants in a field trial in Piikkiö were shown to contain BRV (genus Nepovirus) in R. nigrum and GVBaV (genus Badnavirus) in R. rubrum. BRV is a bi-partite RNA virus that is the causative agent of the reversion disease of blackcurrants. Eriophyid gall mite of blackcurrant (Cecidophyopsis ribis Westwood) transmits BRV and also causes substantial damage, including distortion of leaves, galling, and sterility of buds [Ribes plants and is present in Europe and North America [Symptomatic  of buds . BRV was of buds , 56. GVB America .This study also confirms that siRNA-based diagnostics can be used to detect different viruses in plant samples without previous knowledge about the infecting virus. Recent studies show that VirusDetect reaches a similarly high sensitivity relative to RT-PCR in detection of plant viruses . HoweverIn summary, siRNA-based diagnostics revealed only one virus (RYNV) that is putatively endogenous in pre-basic mother plants suggesting that the current virus indexing methods are working as expected. On the other hand, several viruses were detected in accessions of plant genetic resources previously not tested for viruses. This is not unexpected as these plants are maintained in the field. In general, siRNA-based diagnostics proved useful for testing plants for viruses and could be a valuable supplement or replacement of some of the existing methods used for virus detection in pre-basic mother plants for certified plant production, and in the analysis of collections of plant genetic resources.S1 FigA, G), Y , M , W .Only the nucleotides that differ from those of RYNV-Ca are shown. Identical nucleotides are indicated by dots. The aligned region corresponds to the nucleotides 6282–6840 of the complete nucleotides sequence of RYNV-Ca. Wobble bases: R ((DOCX)Click here for additional data file.S2 FigEach nucleotide sequence indicates sequence from an independent PCR clone of MM-3dg. The uppermost sequence is the sequence obtained by direct sequencing of the PCR product. All sequences are different to each other except the sequences of MM-3-2 and MM-3-3, and MM-3-1 and MM-3-7 are identical to each other.(DOCX)Click here for additional data file.S3 FigEach amino acid sequence indicates sequence from an independent PCR clone of MM-3dg. The uppermost sequence is obtained by direct sequencing of the PCR product. X denotes the position containing degenerate nucleotides. The amino acid sequences of the clones 1, 2, 3, 6, 7 and 10 of MM-3dg are identical to each other, whereas the others differ from each other. There were totally five different sequences between the ten sequenced clones.(DOCX)Click here for additional data file.S4 FigOnly the nucleotides that differ from those of RYNV-Ca are shown. Identical nucleotides are indicated by dots. The aligned region corresponds to the nucleotides 6081–7920 and nucleotides 1–496 of the complete nucleotide sequence of RYNV-Ca. The open reading frames encoded by the sequences are indicated. 5’-terminal part of ORF1 in green, 3’-terminal part of ORF3 in bold and underlined, ORF4 in blue, ORF5 in red and ORF7 encoded in antisense strand in cursive and underlined.(DOCX)Click here for additional data file.S5 FigA, Multiple alignment of nucleotide sequences. Only nucleotides that differ from those of RYNV-Ca are shown. Identical nucleotides are indicated by dots. The aligned region corresponds to the nucleotides 6282–6840 of the complete nucleotides sequence of RYNV-Ca. B, Multiple alignment of amino acid sequences. Only amino acids that differ from those of RYNV-Ca are shown. Identical amino acids are indicated by dots.(DOCX)Click here for additional data file.S6 FigG, T), R , Y , M , S , W , N .Only the nucleotides that differ from those of FN812699 are shown. Identical nucleotides are indicated by dots. The aligned region corresponds to nucleotides 265–1091 of FN812699. Wobble bases: K ((DOCX)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.


Viral diseases, such as human immune deficiency virus (HIV), influenza, hepatitis, and herpes, are the leading causes of human death in the world. The shortage of effective vaccines or therapeutics for the prevention and treatment of the numerous viral infections, and the great increase in the number of new drug-resistant viruses, indicate that there is a great need for the development of novel and potent antiviral drugs. Natural products are one of the most valuable sources for drug discovery. Most natural triterpenoids, such as oleanolic acid (OA), possess notable antiviral activity. Therefore, it is important to validate how plant isolates, such as OA and its analogues, can improve and produce potent drugs for the treatment of viral disease. This article reports a review of the analogues of oleanolic acid and their selected pathogenic antiviral activities, which include HIV, the influenza virus, hepatitis B and C viruses, and herpes viruses. Viral diseases remain a major problem for humankind. It has been reported in some reviews that there is an increase in the number of viral diseases responsible for death and morbidity around the world ,2. Accor1), ursane (2), friedelane (3), hopane (4), lupine (5), and gammacerane (6)   10]. It. It1), u7) from the crude extract of Glycyrrha glabra roots, and it was found to be active against the herpes simplex virus . OA. OAA of 25, which was 3-fold more active than OA. Esterification of 25 with anhydrides resulted in compounds 26–28, which were 5-fold more active than OA with 28 showing remarkable activity [Due to the increase in the occurrence of drug-resistant virus strains, the improvement of effective treatments for the HIV infection is dependent on the identification of novel biomedical agents with low toxicity. Synthesis of oleanolic acid, as well as other closely-related triterpenes, such as betulinic acid and dihydrobetulinic acid, has led to anti-HIV agents ,54,55. Zactivity   virus. C-5 acetylamide and C-9 hydroxy of sialic acid were useful for their binding with hemagglutinin during viral entry into host cells [The influenza virus is a lethal respiratory virus capable of triggering significant damage to the population. Vaccines and antiviral agents are important for controlling the outbreak of influenza. The research into plant-based drugs against the influenza virus is promising, as some plants have been proven to have anti-influenza properties, some of these include: ientalis , Allium stulosum , Sambucuus nigra , and Psi guajava . OA and  guajava . These m guajava . Yang et guajava . Han et st cells . In a sist cells  , and were indicated as two antiviral compounds that seriously suppressed the duplication of the Hepatitis C Virus (HCV) genotype 1b replicon, and the HCV genotype 2a JFH1 virus. Furthermore, these compounds exhibited anti-HCV properties, partly by suppressing HCV NS5B RdRp properties, as noncompetitive inhibitors. Therefore, their results confirmed that natural products of OA and ursolic acid are potential HCV antivirals [Previous reports estimated that approximately 170 million people across the world are infected with the hepatitis C virus (HCV), which causes deaths globally . Althougepatitis . OA and tivirals .38–41) inhibited the secretion of HBsAg, and also decreased the secretion of HBeAg [39 showed major inhibition of HBV DNA duplication, which was significant when compared to the reference drug. Due to its great performance in vivo and in vitro, compound 39 is a potential novel anti-Hepatitis B virus drug candidate, with a different mechanism of action, which needs further investigation. Yan et al. (2006) reported interesting work on the synthesis of OA structures . These rof HBeAg . CompounAchyranthes aspera roots. The MeOH extract exhibited a weak anti-herpes virus effect (EC50 64.4 g/mL for Herpes simplex virus 1 (HSV-1) and 72.8 g/mL for Herpes simplex virus 2 (HSV-2)), whereas OA showed potent anti-herpes virus activity against both HSV-1 (EC50 6.8 g/mL) and HSV-2 (EC50 7.8 g/mL) [42) were found to exhibit moderate anti-HSV-1 activity [Herpes simplex virus (HSV) has two serotypes (HSV-1 and HSV-2), which target the oral and genital mucous membranes of humans, create latent infections in the sensory neurons, and may reactivate to cause recurring infections at the primary site . The eff.8 g/mL) . Keda etactivity .Triterpenoids are an important group of compounds widely found in nature. Previous reports on triterpenoids showed that OA and its analogues have countless beneficial effects, such as antiviral, anti-inflammatory, antitumor promotion, anticancer, and so forth. OA is relatively non-toxic and has been sold in China as a therapeutic for the treatment of human hepatitis. There are few reports on the antiviral activities of OA and its analogues. The treatment of viral diseases continues to present problems in current medicine, with various studies showing a great increase in the occurrence of drug resistant viruses . The mod