The Stress-Prevention@Work implementation strategy has been demonstrated to be successful in reducing stress in employees. Now, we assess the economic return-on-investment to see if it would make for a favourable business case for employers.Data were collected from 303 health-care workers assigned to either a waitlisted control condition (142 employees in 15 teams) or to Stress-Prevention@Work (161 employees in 15 teams). Main outcome was productivity losses measured using the Trimbos and iMTA Cost questionnaire in Psychiatry. Measurements were taken at baseline, 6, and 12 months post-baseline.The per-employee costs of the strategy were €50. Net monetary benefits were the benefits  minus the costs  and were the main outcome of this investment appraisal. Per-employee net benefits amounted to €2981 on average, which was an almost 60-fold payout of the initial investment of €50. There was a 96.7% likelihood for the modest investment of €50 to be offset by cost savings within 1 year. Moreover, a net benefit of at least €1000 still has a likelihood of 88.2%.In general, there was a high likelihood that Stress-Prevention@Work offers an appealing business case from the perspective of employers, but the employer should factor in the additional per-employee costs of the stress-reducing interventions. Still, if these additional costs were as high as €2981, then costs and benefits would break even.This study was registered in the Netherlands National Trial Register, trial code: NTR5527. Work-related stress is common in the health workforce  and then at 6 and 12 months post-baseline (t1 and t2). The control group was given access to the intervention after 12 months. The study design is therefore best described as a matched cohort study in two parallel groups with clustering at the team level. More details are provided in Hoek et al.  were recruited for participation and split into 15 teams (with 252 nurses) for the experimental condition where Stress-Prevention@Work was offered and matched with another 15 teams (with 221 nurses) as waitlisted controls. An independent researcher matched teams on working conditions and size and allocated the teams to the intervention or control group. This researcher did not have information about the perceived stress levels in the teams. Since the intervention focused on the organisation and not on individual workers, individual randomisation was not feasible. The main outcome of the study was productivity losses, assessed using the Trimbos and iMTA Cost questionnaire in Psychiatry (TiC-P), which is a self-completed questionnaire identifying days absent from work (absenteeism) and days working while sick (presenteeism). As we were specifically interested in productivity losses, only these items of the TiC-P were used . As the paper compromised an investment appraisal, we looked at both costs and  benefits, which are described below. Measurements were conducted in May 2016  raising awareness that proper management of work-related stress is important; (2) screening for determinants of work stress; (3) setting intervention goals and selecting appropriate preventive interventions; (4) implementing the selected stress management intervention in the workplace ; and (5) evaluating the strategy’s impact on work-related stress. Types of interventions ranged from guidelines for communicating about work stress to more extensive, tailor-made interventions, possibly involving intermediaries/consultants. The interventions were either at organisational or employee level. For example, organisational interventions included a guideline to start a dialogue between employees and their manager(s) about the presence of work-related stress within the organisation or team. Individual interventions could consist of online self-help modules to reduce work-related psychosocial risk factors  during office hours on working through all the steps of the Stress-Prevention@Work implementation strategy. This is equivalent to €17.40 per employee, when valuing 1 h of work of an employee at €34.75 (for the year 2014) in line with the Dutch guideline for costing in health-economic evaluation /16 = €6.50 per employee.Finally, the Human Resource Management department at the health service invests 150 h of work annually to promote the Stress-Prevention@Work strategy across all 4500 employees. This represents costs of (150 h × €34.75)/4500 = €1.60 per employee.This investment appraisal looked at the costs associated with the Stress-Prevention@Work implementation strategy and compared these costs with the economic benefits derived from greater work productivity through lesser absenteeism and presenteeism. On the costs side, the following items are included:The sum total of costs of Stress-Prevention@Work is therefore 6.25 + 15.63 + 17.40 + 6.50 + 1.60 = €47.38 per employee per annum. This is rounded to €50 per employee. It is assumed that these costs are incurred by the employer. Moreover, it is assumed that more extensive interventions take place outside office hours.t0, t1 and t2) and multiplied by €278.The economic benefits from Stress-Prevention@Work are generated by greater work productivity via lesser absenteeism and lesser presenteeism. As before, we value 1 h of work at €34.75. Thus, 1 day of sick leave costs 8 h × €34.75 = €278. The number of days of sick leave was estimated using the TiC-P at each of the three measurements points  were assessed over the last 4 weeks. The area under the curve (AUC) method was used to compute the cumulative costs (CC) over the full follow-up period of 12 months, while interpolating the costs in the months between the measurement points. This was done with the following equation: Here, it should be noted that there are three measurements at Finally, the difference between the experimental and control condition in the cumulative costs represents the economic benefits when one condition has lower cumulative costs than the other condition. It was hypothesised that the experimental condition would be associated with lower cumulative costs and hence with monetary benefits.The analyses were conducted in several steps.First, we checked for baseline imbalances using descriptive statistics that are presented in Table t1 and t2 were imputed for which we used regression imputation with predictors of the outcome for precision and with predictors of missingness to account for possible selective dropout. This way, a regression model was estimated to predict the observed values of a variable based on other variables, which was then used to impute values in cases where the value of that variable was missing.Second, we adhered to the intention-to-treat principle as per the CONSORT and CHEERS guidelines  in a regression model with the group variable  as predictor. The C is the intervention cost of €50 per employee. We also computed the return-on-investment, ROI, as ROI = NB/C. These metrics, especially NB and ROI, are key to an investment appraisal and serve to decide if investing in the Stress-Prevention@Work implementation strategy is worthwhile as seen from the employer’s business perspective. All analyses were carried out in Stata 14.1 and Microsoft Excel 2010.Finally, with the NBs in hand, we completed the investment appraisal by computing the cost–benefit ratio as C/NB, where While our main analysis is based on robust techniques, we also carried out several sensitivity analyses.t1 and t2. In the main analysis, the intention-to-treat analysis was performed using regression imputation (RI) of missing observations at follow-up. In the sensitivity analysis, the intention-to-treat analysis was repeated using linear mixed modelling (LMM), to see if the costs followed the same trajectory over time as estimated under the main analysis.First, the study was subject to substantial dropout and this may have affected the cost estimates at Second, the main analysis was conducted without adjusting for baseline costs of presenteeism and absenteeism, but there was a small  difference between both conditions. Hence, the main analysis was repeated including costs of presenteeism and absenteeism at baseline as a covariate to adjust for this slight baseline imbalance.Third, given the relatively high dropout, a sensitivity analysis was conducted in which missing observations were imputed using multiple imputations (5 times) by chained equations with predictive mean matching in which ‘‘real’’ observed values from similar cases are imputed instead of imputing regression estimates. This technique is often used to account for non-normality of data which is often the case for costs.Last, cumulative costs in the base case were calculated on a relatively conservative basis, where the costs at baseline were assumed to be stable for the first 8 weeks (in favour of waitlisted control condition). Hence, in a sensitivity analysis, cumulative costs were calculated using the following formula: In total, 473 employees (in 30 teams) were approached. Of these, 221 (in 15 teams) were assigned to the waitlisted control group and another 252 (in 15 teams) to the matched intervention group. At baseline, 143 participants in the control group responded to the baseline questionnaire and 161 in the intervention group. These participants constituted the intention-to-treat sample. However, we had no information about the team membership of one individual in the control condition and that information was required for conducting the design-based regression analysis to account for clustering of employees in teams. Hence, the intention-to-treat sample was brought down to 142 participants. In the intervention group, there were 161 participants. Table t1 and t2 were imputed using regression imputation as outlined before. Predictors in these regression imputation models were costs of absenteeism and presenteeism at baseline. Other predictors were job satisfaction, female gender and age.The flow of participants through the study is depicted in Fig. Table t0, t1 and t2 and employing the AUC method, we computed the annual cumulative costs. Including the intervention costs of €50 per employee in the experimental group, these cumulative costs averaged at €9893 per employee per year in the control group and a lower €6912 in the intervention group.Using the total cost estimates of the last 4 weeks at z = 1.77, p = 0.078, two-tailed).The average net benefit is the between-group mean difference of the annual cumulative costs, hence €9893 − €6912 = €2981 (hence in favour of intervention group). The net benefit was statistically evaluated using a bootstrapped and design-based regression model to account for the non-normality of costs and clustering of employees in teams. Under this model the net benefit was estimated to be €2981 (95% CI: − €329 to €6291) per employee per year, which only approached statistical significance  by cost savings within 1 year. Likewise, there is a 92.9% likelihood that the net benefit will be at least €500 (or more), which is a tenfold payout of the initial investment of €50. A net benefit of at least €1000 has still a likelihood of 88.2%. Finally, the mean (approximating the median) expected net benefit of €2981 is associated with a 51% probability. These probabilities (in %) are depicted in Fig. Finally, an NB of €2981 for an initial investment of €50 is equivalent to a cost–benefit ratio of 50/2981 = 0.017 and represents a return-on-investment of 2981/50 = 59.6, i.e., close to 60-fold payout per euro invested.The main analysis relied on regression imputation (RI) of missing observations at follow-up. In the sensitivity analysis, the intention-to-treat analysis was repeated using linear mixed modelling (LMM), to see if the costs followed the same trajectory over time as estimated under the main analysis. The comparison of both analyses (RI and LMM) is depicted in Fig. As noted before, there was one baseline difference  that nonetheless attracted attention as a potential confounder: the somewhat higher costs stemming from presenteeism in the experimental condition. In this case, it could be argued that higher baseline costs in the experimental condition would strengthen the null hypothesis that the intervention would not be less costly than the control condition.z = 3.31, p = 0.001).Next, the main analysis was repeated, but now with the costs of presenteeism at baseline as a covariate to adjust for possible confounding. In the main analysis the net benefit was estimated at €2981 (95% CI: − €329 to €6291) and after adjustment this increased to €3106 (95% CI: €1344–€4975), which resulted in a statistically significant finding .Using predictive mean matching (PMM) as imputation technique resulted in a net benefit of €3117 (95% CI: − €530 to €6764), which was not statistically significant .The alternative formula to calculate cumulative costs resulted in a net benefit of €3013, which was not statistically significant  per one euro invested. The outcomes are surrounded by some uncertainty. Nonetheless, there is a likelihood of 96.7% that the initial investment will at least break even after 1 year and there is an 88.2% likelihood that the net benefits will amount to €1000 per employee in a year. Hence, the overall picture is in favour of the Stress-Prevention@Work implementation strategy, which is in line with recent views that  health promotion can benefit workers and employers  at baseline. A sensitivity analysis adjusting for this possible confounder showed that the net benefits were underestimated in the main analysis and increased from €2981 to €3106, indicating that the main analysis was more conservative.t0 and t1 and then dropped at t2, which contrasts with the control group where costs increased between t0 and t1 and then dropped off at t2. Lastly, an analysis was performed in which missing data were imputed using multiple imputation with PMM. Given that these replications provided similar results, this attested to the robustness of our main analysis. When comparing responders with non-responders (dropouts), non-responders demonstrated higher baseline costs of absenteeism (€389 vs €321), but lower baseline costs of presenteeism (€52 vs €74). Other characteristics  were similar in both groups.Second, loss to follow-up was substantial and could have biased the estimates at follow-up. However, we used intention-to-treat analysis by imputing missing observations. This was done with regression imputation with both predictors of outcome  and by predictors of loss to follow-up (to counter the effects of selective dropout). In one sensitivity analysis, linear mixed modelling was used instead and replicated the pattern of how costs stayed much the same in the experimental group between Third, absenteeism and presenteeism were based on self-report over the last month and this may have caused some recall bias. It should be noted that it is hard to see how the costs of presenteeism could have been measured without relying on self-report. Also, it was a conscious choice to keep the recall period short (last 4 weeks) and not, say, the last 3 months to minimise recall bias. Ideally, we would have liked to cross-validate the self-reported data of employees with company-registered sickness absence data. This was, however, not possible due to privacy regulations.Fourth, stress reduction may also have impacted on staff turnover and the employees’ ability to continue working until the age of retirement. Now, the intervention’s impact on resignation and early retirement remains unknown, as such assessments would have required a much longer follow-up and larger sample size.Fifth, the effect of the  intervention(s) may be dependent on how these intervention activities are undertaken . For example, we have assumed that more extensive interventions take place outside office hours; however, this assumption might be harmful to the level of compliance achieved in implementing the intervention and demotivate participants as the employer invites them to participate in an intervention to reduce work stress and then tells them to do it in their spare time. Hence, it is recommended that employer and employee both keep this in mind and maintain an open conversation. Further studies should examine the extent to which this issue plays an important role in the compliance and effectiveness of more extensive additional interventions.Sixth, the waitlisted control design may have caused a more beneficial effect of Stress-Prevention@Work compared to the waitlisted control condition, as the use of a waitlisted control condition has been associated with an overestimation of effect sizes in psychotherapy  and the benefits (stemming from lesser productivity losses). This suggests that the added per-employee cost of one or another stress intervention can be substantial well before the employer begins to see that the costs begin to exceed the benefits. It should be noted, however, that in the Netherlands, mental health care is most often covered by the health-care insurance .There is a business case to be made of encouraging the uptake of a stress-reducing intervention for health-care workers by Stress-Prevention@Work. This implementation strategy costs approximately €50 per employee, but is associated with a high likelihood that these initial investments are more than recouped by the employer because of lesser absenteeism and presenteeism. It should also be kept in mind that the cost of any subsequent stress-reducing intervention has to be subtracted from the expected net benefits of €2981. It is therefore recommended that the Stress-Prevention@Work portal not only offers information that helps to choose from the wide range of available stress-reducing interventions, but in addition should offer information about the per-employee costs of those interventions. That would help to better inform decision-making processes and would in addition help to reduce uncertainty about the health-economic viability and sustainability of stress-prevention at work.


Aspergillus thermomutatus chrysovirus 1 (AthCV1), isolated from Aspergillus thermomutatus, on A. fumigatus, A. nidulans and A. niger. Protoplasts of virus-free isolates of A. fumigatus, A. nidulans and A. niger were transfected with purified AthCV1 particles and the phenotype, growth and sporulation of the isogenic AthCV1-free and AthCV1-infected lines assessed at 20 °C and 37 °C and gene expression data collected at 37 °C. AthCV1-free and AthCV1-infected A. fumigatus produced only conidia at both temperatures but more than ten-fold reduced compared to the AthCV1-infected line. Conidiation was also significantly reduced in infected lines of A. nidulans and A. niger at 37 °C. AthCV1-infected lines of A. thermomutatus and A. nidulans produced large numbers of ascospores at both temperatures, whereas the AthCV1-free line of the former did not produce ascospores. AthCV1-infected lines of all species developed sectoring phenotypes with sclerotia produced in aconidial sectors of A. niger at 37 °C. AthCV1 was detected in 18% of sclerotia produced by AthCV1-infected A. niger and 31% of ascospores from AthCV1-infected A. nidulans. Transcriptome analysis of the naturally AthCV1-infected A. thermomutatus and the three AthCV1-transfected Aspergillus species showed altered gene expression as a result of AthCV1-infection. The results demonstrate that AthCV1 can infect a range of Aspergillus species resulting in reduced sporulation, a potentially useful attribute for a biological control agent.This study determined the effects of  Mycoviruses have been reported in a wide variety of fungi . In natuWhile the vast majority of mycoviruses appear to have little or no obvious impact on their host, there are reports of significant changes in the phenotype and growth characteristics of some fungi due to viral infection. These alterations include reduction in growth and virulence of plant pathogenic fungi ,9,10,11,Botryosphaeria dothidea chrysovirus 1 [Magnaporthe oryzae chrysovirus 1-A [Magnaporthe oryzae chrysovirus 1-B infection [Botryosphaeria dothidea chrysovirus 1 [Aspergillus fumigatus chrysovirus [A. niger chrysovirus [Like many mycoviruses, most chrysoviruses are associated with latent infections of their fungal hosts . Howeverovirus 1 ; weakeneirus 1-A ; the absnfection ; hypovirovirus 1 ; aconidiysovirus ; and acoysovirus .Fusarium graminearum viruses [Fusarium graminearum strain PH-1 via protoplast fusion. An RNA-Seq-based transcriptome analysis of each of the virus-infected PH-1 cultures showed that all four mycoviruses affected the transcriptome profiles and each mycovirus regulated the expression of a totally different set of host genes. However, transcriptome profiles of the naturally infected Fusarium graminearum strains do not appear to have been included in that assessment. Also, Sun et al. [Cryphonectria parasitica together with the Cryphonectria hypovirus 1-EP713.The effects of mycovirus infection on gene expression have been studied for some fungi. In a study of four different  viruses  a hypovin et al.  showed tA. thermomutatus (isolate Ath-1) infected with Aspergillus thermomutatus chrysovirus 1 (AthCV1). This paper presents the results of a subsequent study to investigate the ability of AthCV1 to infect other Aspergillus species  and the biological and molecular impacts of AthCV1 on these species.We previously reported  sector fA. thermomutatus infected with AthCV1 was used as a source of virus particles for protoplast transfection of A. fumigatus , A. niger  and A. nidulans . Clinical isolates of Afu-13 and Ang-9 were provided by Dr David Holland, Clinical Director Infection Services, Middlemore Hospital, Auckland, New Zealand and And-1 was provided by Wendy McKinney, Mycology Reference Laboratory, Auckland Hospital. Cultures were maintained on Potato Dextrose Agar  in the dark at 37 °C and 20 °C.An isolate of A. thermomutatus was grown in Yeast Extract Peptone Dextrose broth  and incubated on a shaking incubator at 180 rpm in the dark for 2 days. Approximately 10 g of fungal mycelium was harvested on a filter paper using vacuum filtration, ground to a fine powder in liquid nitrogen and transferred to a 50 mL Falcon tube containing 20 mL of sodium phosphate buffer (SPB)  and 10 mL chloroform. The mixture was incubated for 30 min on ice on an orbital shaker, at 230 rpm and then centrifuged at 10,000× g for 30 min at 4 °C. The upper aqueous phase was centrifuged at 120,000× g for 2 h at 4 °C and the resultant pellet was re-suspended in 1 mL SPB (0.02 M pH 7.0) for 4 h at 4 °C. The suspension was clarified by centrifugation at 10,000× g for 20 min at 4 °C, the supernatant centrifuged at 120,000× g for 2 h at 4 °C and the pellet was re-suspended in 0.5 mL SPB  overnight at 4 °C. Following centrifugation at 10,000× g for 20 min a 50-µL drop of the supernatant was negatively stained with 2% uranyl acetate (pH 4.0) and observed for virus particles using a Phillips CM12 TEM. The virus was detected by virus-specific reverse transcription polymerase chain reaction (RT-PCR), using virus specific primers that amplified a 639 bp product from the coat protein (genome segment 2), as described by Ejmal et al. [AthCV1 was purified as follows: Virus-infected l et al. .4·7H2O, 0.2M C6H5Na3O7·2H2O, pH 5.5). The mycelium was then coarsely chopped and transferred to a flask containing 17 mL protoplast buffer. Three mL of filter sterilized Novozyme buffer  containing 200 mg of Lysing Enzymes from Trichoderma harzianum  was added to the mycelial suspension, which was incubated for 4 h at 28°C in a shaker at 85 rpm. Protoplasts were passed through a 75-µm strainer and collected in a 50-mL tube containing 30 mL KC buffer (0.6 M KCl and 50 mM CaCl2) and centrifuged at 4000× g for 10 min. The protoplast pellet was then washed twice with 10 mL sorbitol-Tris-calcium chloride (STC) buffer  and centrifuged at 4000× g for 10 min before being was re-suspended in 0.5 mL STC and kept on ice. For transfection, 130 µL PEG 4000 (60% in sterile water) was mixed with 70 µL potassium chloride-Tris-Calcium Chloride (KTC) buffer  and added to a tube containing 200 µL of purified virus particle suspension and 5 µL 0.05 mM spermidine . A 200-µL aliquot of the protoplast suspension was then added to the suspension and mixed by twirling the tube for 10 s before it was incubated on ice for 30 min. Following incubation, a mix of 200 µL of polyethylene glycol (PEG) 4000 and 100 µL KTC was added to the previous suspension and gently twirled again. Following incubation at room temperature for 20 min, 40-µL aliquots of the mixture were added to 5 mL warm agar medium  containing 0.7% agar), gently mixed and spread on SMM plates, which were parafilm sealed and incubated at 37 °C. Individual colonies were picked off, grown on fresh PDA plates and sub-cultured three times, at weekly intervals, then checked for the presence of the virus by RT-PCR as described by Ejmal et al. [Protoplast preparation and virus transfection were undertaken as described by Ejmal et al. . Brieflyg for 10 min. The spores were re-suspended in 10 mL distilled water before being counted in a Neubauer chamber, as described by Aneja [Sporulation rate comparisons were conducted at 20 °C to represent environmental temperature and 37 °C to represent human body temperature. Five virus-free and five virus-infected lines were grown from single spores, inoculated at the edge of PDA plates (9 cm diameter) and incubated in the dark until the mycelium reached the opposite side of the plate. To harvest conidiospores, the plates were washed with 40 mL aqueous 0.05% Tween 80, which was then filtered through cheesecloth and centrifuged at 8000× by Aneja .For linear growth comparison, five replicates of isogenic virus-free and virus-infected single spore isolates were individually inoculated at the edge of PDA plates and grown at 37 °C and at 20 °C. The growth was measured every 24 h until the mycelium reached the far edge of the plate. At the completion of each experiment cultures were tested for the presence of the virus using RT-PCR as described by Ejmal et al. .t-test, using SPSS version 21 .To compare biomass production five isogenic virus-free and virus-infected single spore isolates, of each species, were grown at 37 °C and at 20 °C in the dark. Plugs of the resultant mycelium were individually transferred to conical flasks containing 200 mL YPD Broth and incubated with shaking at 180 rpm, in the dark. The resultant mycelium was vacuum filtered and 100 mg of each sample retained for virus screening by one-step RT-PCR by Ejmal et al. . The remA. nidulans ascospores was tested as follows: First ascospores were processed to eliminate contamination from mycelium and conidia, based on the methods of O’Gorman et al. [A. niger, the sclerotia were isolated from cultures on PDA plates incubated at 37 °C for 2 weeks in the dark, according to the method of Utkhede and Rahe [AthCV1 transmission through n et al.  and Giran et al.  and indin et al. . To testand Rahe , with miand Rahe .A. thermomutatus, A. fumigatus, A. niger and A. nidulans were grown on sterile cellophane film overlaid on PDA media. The cultures were incubated at 37 °C for 5 days in the dark, as described by Zhang et al. [Both AthCV1-infected and uninfected cultures of g et al. , to provg et al.  was usedA. fumigatus, A. niger and A. nidulans, where a reference genome was available (fungi.ensembl.org/index.html), the Tuxedo protocol [galaxy-qld.genome.edu.au) and Tophat2, version 0.6 [Aspergillus genome databases (www.aspergillusgenome.org), Ensemble Fungi database (fungi.ensembl.org/index.html) and the UniProt Knowledgebase (www.uniprot.org).For protocol  was usedsion 0.6  used to sion 0.6 . Cuffmersion 0.6  was thension 0.6  used to A. thermomutatus, where no reference genome was available, the Trinity de novo transcriptome assembly software (version 0.0.2) [For n 0.0.2) ,39 was uA. fumigatus (Afu-13), A. niger (Ang-9) and A. nidulans (And-1), confirmed that all three species were successfully transfected with AthCV1 purified particles , A. niger (Ang-9) and A. nidulans (And-1) at 37 °C and 20 °C, together with the results for A. thermomutatus previously published [A. fumigatus, AthCV1 infection significantly reduced (p ≤ 0.05) asexual sporulation at both temperatures, with the effects being more extreme at 37 °C than at 20 °C. As would be expected there was no sexual reproduction observed in either the AthCV1-free or AthCV1-infected treatments at either temperature as A. fumigatus is a heterothallic species. In the homothallic species A. nidulans conidial production was significantly reduced (p ≤ 0.05) in the AthCV1-infected line at 37 °C but not at 20 °C, while there was a significant increase (p ≤ 0.05) in the number of ascospores produced by the AthCV1-infected line at both 37 °C and 20 °C. In A. niger asexual sporulation was significantly decreased (p ≤ 0.05) in the AthCV1-infected line at 37 °C while there was no significant difference between AthCV1-free and infected lines at 20 °C. A. niger also formed sclerotia in the AthCV1-Infected line at 37 °C but not at 20 °C. There was no sexual reproduction in A. niger cultures at either temperature.The results for the effects of AthCV1 on sporulation of ublished  are presAspergillus species at 37 °C and 20 °C, together with the results for A. thermomutatus previously published [A. fumigatus on PDA plates was significantly greater (p ≤ 0.05) in AthCV1-infected cultures at 20 °C but not at 37 °C. In contrast, there was a significant decrease in mycelial dry weight associated with AthCV1 infection at 37 °C but not at 20 °C. In addition, while the AthCV1-free culture produced a uniform grey-colored mycelium, the mycelium of the AthCV1-infected line formed small white sectors that subsequently merged together. This was more prominent at 37 °C than at 20 °C, eventually covering most of the plate  results indicated that for AthCV1-infected cultures some genes were highly upregulated while others showed downregulation or were undetectable , compared with the isogenic virus-free line. These included genes related to fungal sporulation . The average RNA size was rulation . The numAspergillus species. Some genes showed expression change in only one species, such as the transcription factor gene steA (AN2290), for which changes were observed only in A. niger. For other genes, the variation was just in the degree of expression, as for the orthologue of the heat shock protein gene, hsp30/hsp42 (AN5781). In other instances, the difference was in the direction of regulation, such as the internal alternative NADH dehydrogenase ndiA (AN10660), which was completely repressed in A. nidulans but upregulated by 25-fold in A. fumigatus and by 18-fold in A. niger. The orthologue of this gene in N. crassa has an NADPH dehydrogenase activity and a role in spore germination with mitochondrial inner membrane localization (S. cerevisiae) overexpression of the orthologue (ndi-1) of this gene (ndiA) can result in apoptosis-like cell death, which is associated with an increase in the production of reactive oxygen species (ROS) in mitochondria [nosA/AN1848, which in the AthCV1-infected lines was reduced 326-fold in A. niger, 20-fold in A. nidulans and 2-fold in A. fumigatus but was unchanged in A. thermomutatus.As shown in lization . In yeasMycoviruses have received much attention in relation to their potential as biological control agents for harmful fungi. Two necessary properties of biocontrol agents are (i) the ability to reduce the growth and virulence of the target fungus and (ii) rapid and efficient transmission of the virus from the inoculum to the target fungal population. Hyder et al.  found thA. fumigatus protoplasts with Aspergillus fumigatus tetramycovirus (AfuTmV-1) [Aspergillus fumigatus chrysovirus (AfuCV) [Botryosphaeria dothidea with Botryosphaeria dothidea chrysovirus 1 (BdCV1) [Rhizoctonia solani with Rhizoctonia solani partitivirus 2 [Cryphonectria parasitica with a reovirus from C. parasitica [Rosellinia necatrix with Rosellinia necatrix partitivirus (RnPV1) [Rosellinia necatrix mycoreovirus 3 (RnMYRV-3) [A. fumigatus, A. niger and A. nidulans with AthCV1 particles from naturally infected A. thermomutatus, to produce isogenic virus-free and virus-infected lines, in order to evaluate the effects of the virus on these fungal hosts.Vegetative incompatibility is a major barrier to studying the effects of viruses on a range of fungal hosts and their use in the field, as it can be a serious barrier to horizontal virus transmission between different fungal species and even different strains of the same fungal species. However, in vitro, this obstacle may be overcome by the use of protoplast fusion ,46,47,48fuTmV-1)  and Aspe (AfuCV) , as well (BdCV1) , Rhizoctivirus 2 , Cryphonrasitica , Roselli (RnPV1)  and RosenMYRV-3) . In the A. thermomutatus, to significantly reduced growth for A. niger at 37 °C and A. nidulans at 20 °C and a significant increase in the growth for A. fumigatus at 20 °C. An increase in linear growth due to virus infection was previously reported by Nuss [C. parasitica (strain Euro7) cultures infected with Cryphonectria hypovirus 1 (CHV-1/Euro7) showed faster growth than the isogenic virus-free isolate. However, linear growth on agar plates does not necessarily correlate with total biomass production, as seen in our experiments for A. fumigatus, A. nidulans and A. niger  and the chrysovirus, Helminthosporium victoriae virus 145s (HvV145S) [A. fumigatus formed non-sporulating sectors when infected with A. fumigatus partitivirus 1 (AfuPV-1) or with AfuCV. However, the basis of fungal sectoring can have a number of underlying causes other than virus infection.In the current investigation, sectoring (areas with different appearance) were observed in the virus-infected cultures of all four g, BdCV1 , HelmintHvV145S) . In a stHvV145S) , A. fumiAspergillus species did not respond to AthCV1 infection in the same way. The response was often temperature dependent; A. fumigatus reacted consistently at both temperatures with significantly reduced conidial production , ascospore production was significantly increased at both 37 °C (90%) and 20 °C (170%) compared to uninfected. A. thermomutatus  produced ascospores only when infected with AthCV1  through ascospores in Botrytis cinerea [S. cerevisiae [Fusarium graminearum [Since vertical transmission and dissemination of virus infection is largely dependent on spores, the rate of infection in spores is an important factor. For many fungi, virus transmission through conidia is often high (<100%) ,58,59, w cinerea , dsRNA vrevisiae  and two A. thermomutatus, compared to no ascospores in the virus-free line and a significant increase in ascospore production in A. nidulans, together with the decrease in conidial production, could be the result of physiological stress due to AthCV1 infection, as stress is often a trigger for a change from asexual to sexual reproduction in fungi [Aspergillus sections Flavi [Circumdati [A. japonicas [A. niger at 37 °C. Frisvad et al. [A. niger although most of the A. niger strains used in their study failed to produce sclerotia despite the multiple attempts to enhance the process. It is possible that mycovirus infection affected their two strains that produced sclerotia, however the strains used in their study were screened for the presence of mycoviruses. In summary, the findings in this study indicate that AthCV1 infection enhances sexual sporulation in A. nidulans and A. thermomutatus and possibly also in A. niger where sclerotia were produced at 37 °C.The production of large numbers of ascospores in AthCV-1 infected in fungi . Therefons Flavi  and Circrcumdati  and A. japonicas , were prd et al.  were theAspergillus species demonstrate that the effect of AthCV1 is both species dependent and temperature dependent.In the current study variability in the nature of the sectoring phenotype and differences in sporulation, mycelial growth diameter and mycelial dry weight associated with AthCV1 infection in the four different Aspergillus species, knowledge of which metabolic pathways are affected might possibly be exploited for better control of the fungus. Consequently, preliminary gene expression data, based on pooled samples of three replicate cultures for each treatment, were obtained to identify changes in the expression of known reproduction-related genes as a starting point for future, more extensive studies. In response to AthCV1 infection changes in expression were detected in a total of 223 different genes, across the four Aspergillus species examined. Genes specifically related to sporulation and/or reproduction numbered 54, those showing greater than five-fold changes (either + or −) are presented in Given that AthCV1 has significant effects on asexual and sexual spore reproduction in some A. nidulans, Paoletti et al. [MAT2 represses MAT1 expression and vice versa. The current findings reflect this with the two homothallic species A. thermomutatus and A. nidulans where MAT1/NFIA_071100 and MAT1/AN2755 were downregulated in the AthCV1-infected treatment by 8-fold and 16-fold respectively whereas, MAT2 was upregulated by 4-fold in the AthCV1-infected A. nidulans and expressed in the AthCV1-infected A. thermomutatus but not in the virus-free line.An important family of genes for sexual reproduction in fungi are the Mating Type (MAT) genes which enable homothallic fungi to switch their mating type. In i et al.  have shoA. nidulans, other highly upregulated genes that contribute to the increase in sexual sporulation include: gprk/AN7795 [www.aspergillusgenome.org) (13-fold), fhbA/AN7169 [mst1/AN5674  [nudF/AN6197 (positive regulation of conidiation and ascospore production) [cryA/AN0387 (37-fold decrease) that codes for a blue light- and UVA-sensing cryptochrome that represses sexual development by regulating other regulators such as VeA, NsdD and RosA [hogA/AN1017 (6-fold decrease) which is a putative mitogen-activated protein kinase (MAPK), is highly up-regulated under osmotic stress conditions required for sexual development and sporulation [A. nidulans conidiation was hugely reduced and this seems consistent with the increased number of ascospores, as it is known that ascospore production can inhibit conidiation and vice versa [conF/AN8640 (21-fold reduction) which contributes in conidia germination and in the protection of conidia against desiccation [noxR/AN6046 (13-fold reduction) which is important for conidiophore development and conidia production [aspB/AN6688 (12-fold reduction), which plays a role in growth emergence and conidiation [In AthCV1-infected k/AN7795  (39-foldlopment)  and nudFand RosA  and hogArulation . In AthCce versa . Changesiccation , noxR/ANidiation ,76.A. thermomutatus there was a 7-fold increase in expression of csnB/NFIA_070550, which is essential for cleistothecia and ascospore formation [laeA/NFIA_010750, an orthologue of the global regulator of secondary metabolism (laeA/AN0807), was downregulated by 1913-fold. This gene coordinates asexual development in response to light, is involved in the regulation of secondary metabolism and is required for the formation of Hulle cells [fhbA/NFIA_030070), which is involved in the positive regulation of sterigmatocystin production and in the negative regulation of sexual sporulation [In AthCV1-infected ormation ,78. The  laeA/AN07, was dole cells ,82,83. Trulation , was dowA. niger that were affected and possibly contributed to the huge decrease of conidiation in the AthCV1-infected treatment at 37 °C included: the putative C2H2 transcription factor (flbC/An02g05420) which showed a 31-fold downregulation and has a predicted role in conidiation and is expressed in germinating conidia [adv-1 in N. crassa which is required for normal sexual and asexual development [ppoA/An04g05880 (20-fold decrease in expression), the miss expression of the A. nidulans orthologue ppoA/AN1967 leads to a decreased level of conidiation (http://www.aspergillusgenome.org); and An05g00480 (5-fold reduction) which is expressed in germinating conidia and has a predicted role in positive regulation of conidiophore development and conidium formation (www.aspergillusgenome.org). In addition, An14g02540, the orthologue of the sclerotium regulator in A. oryzae (sclR/AO090011000215), that encodes a transcription factor with a role in hyphal morphology and the promotion of sclerotial production [A. nidulans heat shock protein 30 (hsp30/AN2530) [A. fumigatus, Afu3g14540 (hsp30), has been found to be expressed in a high abundance in conidia and is known to increase in response to the antifungal amphotericin B and to hydrogen peroxide [Genes of  conidia ,85,86; Aelopment ; ppoA/Anoduction . Also, A sclR/AO0011000215peroxide .A. fumigatus revealed the greatest upregulation (6427-fold increase) for Afu1g04410, which has no known function and the greatest downregulation (18094-fold reduction) for the DNA N-glycosylase, putative (Afu7g05320), the orthologue of which in S. cerevisiae has a predicted role in oxidized purine nucleobase lesion DNA N-glycosylase activity and molecular function in DNA damage repair (www.uniprot.org). Other A. fumigatus genes that showed altered transcription levels due to AthCV1 infection were: ppoA/Afu4g10770 (7-fold reduction), an orthologue of A. nidulans ppoA/AN1967, which is involved in the response to oxidative stress, negative regulation of sexual sporulation and positive regulation asexual sporulation [A. fumigatus [N. crassa (ndi-1) and has a predicted role in oxidation-reduction process and spore germination [A. fumigatus included: Scytalone dehydratase arp1/Afu2g17580 (9-fold increase), which is involved in conidial pigment biosynthesis and for which mutants display increased C3 complement binding [ayg1 which is involved in conidial pigment biosynthesis with a role in polyketide shortening and melanin biosynthesis in Aspergillus fumigatus [pksP/Afu2g17600 (12-fold increase) which is involved in biosynthesis of the conidial pigment and asexual spore wall assembly [arp2/Afu2g17560 (16-fold increase) which is involved in conidial pigment biosynthesis and expression of conidia-enriched protein [Galleria mellonella [Transcriptome analysis of the heterothallic rulation  and Afu1umigatus ; and Afumination . In addi binding ; Afu2g17assembly ; arp2/Af protein ,94. The llonella .Aspergillus species included in this study exhibited changes in phenotype  a specific virus strain can cause different effects on different Heterobasidion strains and (ii) the impact of a single virus strain on a certain Heterobasidion isolate can differ according to the changes in environmental conditions. The diversity in response of the four Aspergillus species to AthCV1 supports the proposition that even if a particular mycovirus is found to have a hypovirulent effect on a certain fungal isolate, it does not necessarily follow that it will have the same effect on other isolates of the same or closely related species. Consequently, any evaluation of the potential of mycoviruses as biological control agents should be conducted on a range of fungal isolates in a range of environmental conditions. In the current study, differences in the expression of the internal alternative NADH dehydrogenase NdiA (AN10660) in different Aspergillus species is probably a good example of this phenomenon as it was upregulated by 25-fold in A. fumigatus and by 18-fold in A. niger but entirely repressed in A. nidulans.In response to AthCV1 infection all four of the henotype  and genehenotype . For somr et al.  who studA. fumigatus and A. niger may reduce the ability of the fungus to spread and compete with uninfected fungal isolates in the field and thereby limit the impact of AthCV1 on Aspergillus, the high number of ascospores produced by the AthCV1-positive A. nidulans and A. thermomutatus and the enhancement of sclerotia production in A. niger may improve the survival of these fungi, as these structures typically resist adverse environmental changes better than conidiospores. Moreover, the presence of virus could directly or indirectly, increase diversity of Aspergillus by stimulating sexual reproduction and possibly increase the percentage of vegetatively compatible fungal strains allowing virus spread between them.In-vitro transfection can be used to experimentally extend the host range of a mycovirus in order to investigate the potential effects on new fungal hosts. This ability to overcome the barriers caused by hyphal incompatibility and genetic diversity may, if it results in stable transfected lines, extend the application of mycoviruses, such as AthCV1, for fungal disease control. However, it is important to understand the wider implications of specific phenotypic changes , observed under controlled experimental conditions, to the epidemiology of the fungus in the environment. For example, while the low conidiation rate of AthCV1-infected The use of mycoviruses as effective biological control agents requires consideration of multiple factors . The first objective is to find a virus that is capable of inducing serious impact on its fungal host. Once such a virus is identified, intensive research on how and when to use the virus is required, for example, direct application to the site of infection in the case of chestnut blight control . A major


DR1-4) as a sensitive probe for quinoidal structures in curved π-conjugated molecules. The quinoidal contribution in S-DR1-4 was found to increase with increasing the curvature of the curved structure, which was quantitatively analyzed using NBO analysis and the natural orbital occupation numbers computed by the CASSCF method. The curve effect on the singlet-triplet energy gap was examined by the CASPT2 method. The singlet-triplet energy gaps for the highly π-conjugated diradicals were determined for the first time using the CASPT2 method. Substantial quinoidal contribution was found in the curved structures of the delocalized singlet diradicals S-DR1-4, in contrast to its absence in the corresponding triplet states T-DR1-4.Curved (non-planar) aromatic compounds have attracted significant research attention in the fields of basic chemistry and materials science. The contribution of the quinoidal structure in the curved π-conjugated structures has been proposed to be the key for materials functions. In this study, the curve effect on the quinoidal contribution was investigated in Kekulé-type singlet diradicals (S- Curved (non-planar) aromatic compounds like fullerenes, buckybowls, and carbon nanotubes have attracted considerable attention from researchers in the fields of basic chemistry as well as materials science ,9,10,11.DR1-4 [q) in curved π-conjugated molecules  (DR1 were optimized to obtain C2 symmetry at the UB3LYP/6-31G(d) level of theory with Gaussian 09 (revision D.01) software . The broken-symmetry (BS) method [DR1 (See ψA (HOMO in the restricted Hartree-Fock (RHF) method) increased from 1.66  to 1.80  with increasing angle of bend (θ) in the diradical structures. The bent angles (θ) were obtained after the structural optimization of DR1 in C2 symmetry at the fixed angles of C1–C2–C6 = C10–C9–C5 = 180, 160, 140, and 135°, respectively. On the other hand, the occupation number in orbital ψB (LUMO in the RHF method) decreased from 0.35  to 0.21  with increasing θ. q) was given by q (%) = (ψnA/2.0) × 100, ψnA being the number of electrons in the HOMO orbital. The q values increased from 83 to 90 with increasing θ. The θ-dependent changes in the occupation number and q value indicates that the bonding interaction between the two phenyl-rings, leading to the formation of the quinoidal structure, increased with increasing θ. In fact, the C5–C6 distance was found to decrease from 143.0 pm (θ = 0°) to 139.6 pm (θ = 29°). The increase in quinoidal contribution was also proved by the decrease in C1–C2 bond distance and the increase in the Wiberg bond order (BO) [DR1. First, the curve effect on the diradical character was investigated on the 4,4′-dimethyl-1,1′-biphenyl-4,4′-diyl diradical  method  was used[DR1 See . The natder (BO)  (entries1A) S- and triplet (13B) T-DR1 were computed using the complete active-space second-order multiconfigurational perturbation theory (CASPT2) [DR1 were destabilized with increasing θ, as reflected by the ΔErel,S/ΔErel,T values in Erel,S and ΔErel,T, were calculated with respect to the singlet and triplet absolute energies, respectively, in the planar structure (θ = 0°). The singlet–triplet energy difference, ΔEST = ES − ET, increased substantially from 11.6  to 19.3 kcal mol–1 . As judged by the curve effect on the electronic energies of S-DR1 and T-DR1, i.e., ΔErel,S/ΔErel,T, the triplet state was destabilized more significantly than the singlet one with increasing θ. For example, at θ = 29° ΔErel,S and ΔErel,T were calculated to be +21.0 and +28.7 kcal mol–1, respectively (entry 4). The curve effect on ΔEST is rationalized by the quinoidal contribution to the curved structure of the singlet state S-DR1, which stabilized the singlet state, but not the triplet state. The electronic energies of singlet ground state (1(CASPT2) ,62, inclDR1 was also rationalized by the curve effect on the dihedral angle (θd = C4–C5–C6–C7) of the biphenyl moiety  (entries 5–8), 2,7-dimethylphenanthrene-2,7-diyl diradical (DR3) (entries 9–12), and 2,6-dimethylanthracene-2,6-diyl diradical (DR4) (entries 13–16) to analyze the quinoidal contribution to the curved singlet states. As found for DR1, the quinoidal contribution (q) to the singlet state of DR2-4 increased with increasing θ (entries 5–16). For DR2, the q value increased from 88.5 to 92.5 with increasing θ. The q values for other diradicals DR3 and DR4 were also found to increase with increasing their curved character: from 79 to 93.5 for S-DR3 and from 82.5 to 91.0 for S-DR4. Consistent with the increase in quinoidal contribution, the C1–C2 bond in the singlet state became shorter than that in the triplet state, indicating that the quinoidal contribution to the singlet state increases with increasing θ. Furthermore, the Wiberg BOs of C1–C2 increased with increasing θ in DR2-4: from 1.68 to 1.76 for S-DR2, from 1.47 to 1.75 for S-DR3, and from 1.54 to 1.73 for S-DR4. As for DR1, the significant curve effect on the singlet–triplet energy gap (ΔEST) was also computed for DR2-4. The energy gap increased with increasing θ, because the destabilization with increasing θ in the triplet states T-DR2-4 were larger than those in the corresponding singlet states. Similar computations were conducted for the π-extended Kekulé-type diradicals, viz., 2,6-dimethylnaphthalene-2,6-diyl diradical  of the aromatic ring. In other words, the quinoidal contribution increases with increasing θ of the aromatic ring. The increases in the quinoidal contribution in the curved diradicals are consistent with the curve effect on the quinoidal character of hoop-shaped molecules, which has been intensively investigated in the last decade. The curved structure can increase the π-conjugation length with decreasing the HOMO-LUMO gap, which should be smaller than that in the planar molecules having the same number of π-electrons. The molecular design is expected to be appropriate for future soft-materials.In this study, the quinoidal contributions in curved aromatic structures were quantitatively analyzed by computing the curve effect on the diradical character of


In June 2016, a disease characterised by intestinal haemorrhage with a mortality rate of approximately 5% was observed in a duck farm in Shandong province, China. Here, we report the isolation and characterisation of a reovirus from duck tissue samples by inoculating duck embryos and duck embryo fibroblasts (DEF). The isolate replicated in DEF and Vero cells and formed syncytia. Sequence analysis revealed that the viral genome was 23,434 nt in length with typical structure organization, consisting of 10 dsRNA segments ranging from 3998 nt (L1) to 1190 nt (S4) in size, and was genetically distinct from previous Chinese duck-origin reoviruses. Phylogenetic analyses showed that the isolate was most closely related to the recently reported duck reovirus D2533/6/1-10 isolated in Germany, forming a monophyletic branch different from known reference avian reoviruses. Experimental infection results indicated that the isolate replicated transiently in ducklings and was shed via faeces. Infection with the isolate caused epithelial cell damage and lymphocyte apoptotic death in the bursa of Fabricius, which may result in immunosuppression in infected ducklings. The role of the isolate in current duck haemorrhage enteritis remains to be determined, but its damage to the bursa warrants further investigation of the duck immune response. They are members of genus Orthoreovirus in the family Reoviridae. The viral double stranded RNA genome contains 10 segments divided into three size classes based on their electrophoretic motility on a sodium dodecyl sulfate-polyacrylamide gel: large , medium  and small (S1–S4)5. The genome is predicted to encode eight structural proteins  and several nonstructural proteins . ARVs have been associated with a variety of diseases in domestic fowl, including chickens 6, turkeys (infectious enteritis)7, ducks and geese 10. In wild birds, reoviruses have been detected in white stork, grey heron, rock pigeon and so on4. ARV isolates were traditionally classified using standard serological procedures, but more recently have been characterised by molecular methods based on genome sequences and deduced amino acid analyses. However, for pathogenic isolates, it is difficult to assign a new isolate to a pathogenic group or pathotype according to its genetic background due to the lack of clearly defined virulence determinant(s) of the avian reovirus.Avian reoviruses (ARVs) are widely distributed in a variety of avian species, including wild birds11, and the disease was seen only in Muscovy ducks (Carina moschata) and mule ducks, an infertile hybrid cross between male Muscovy and female Pekin ducks (Anas platyrhynchos). Genetic analysis showed that the virus was closely related to MDRV isolated in Europe12. Since 2006, outbreaks of disease characterised by spleen necrosis in Pekin ducklings were observed, and the aetiological agent was identified to be a reovirus distinct from previous MDRV isolates10. Currently, the reovirus has been tentatively classified as a “novel” duck reovirus (NDRV) to distinguish it from the “classical” MDRV14. Virus infections were subsequently diagnosed widely in mainland China, affecting almost all domestic ducks and geese17. In June 2016, an outbreak of disease characterised by intestinal haemorrhage was observed in a commercial duck farm in Shandong Province, with a mortality rate of around 5% before the age of 4 weeks. In this study, we conducted an aetiological investigation and isolated a reovirus genetically distinct from previous duck reoviruses identified in China. The pathogenicity of the isolate was evaluated in ducklings.In China, Muscovy duck reovirus (MDRV) infection was first recognised in domestic Muscovy ducks in 20015 TCID50/mL . Analysis of the NGS data revealed that Ych was a reovirus distinct from the known China-origin duck reovirus isolates. To confirm the isolate, we designed a primer pair specific for the Ych λC gene on the basis of the NGS sequence data, and reverse transcription-polymerase chain reaction (RT-PCR) was performed using the RNA template extracted from the original duck tissue sample. A gene fragment identical to that revealed by deep sequencing was obtained. We further analysed the electrophoretic mobilities of the viral double-strand RNA segments and revealed that the ten genomic segments displayed clearly distinguishable migration patterns from those of duck reovirus DRV-HC and ARV-S1133 Fig. . It was 18. Proteins encoded by representative avian reovirus homologous segments were identified in the genome  and 91.7–98.5% (aa), which was isolated from Pekin ducklings in Germany20.Nucleotide and amino acid (aa) sequences of all segments of Ych were compared with representative members of The open reading frames (ORFs)of the nucleotide sequences of ten segments were used to construct the phylogenetic trees Fig. . PhylogeThe serotype relationship of Ych with duck reovirus DRV-HC and ARV-S1133 was determined by virus neutralisation test Table . The thrCompared with uninfected controls, no abnormal clinical signs were noted in ducklings infected by oral/intranasal or subcutaneous inoculation during the experimental period, and neutralising antibody was not detected in sera collected from the infected ducks on 9, 12 and 16 dpi. However, virus-specific RNA could be sequentially detected from cloacal swabs of some infected ducklings from 1–4 dpi Table , suggestHistopathological examination of the tissues of infected ducklings revealed inter-follicle oedema of the bursa of Fabricius Fig. . The epi22, and high morbidity and mortality with multifocal hepatic and spleen necrosis at necropsy have been recorded for the majority of duck and goose reovirus infections25. In this work, a reovirus Ych was isolated from tissue samples of diseased Pekin ducks, characterised by intestinal haemorrhage. The isolate could induce syncytia formation in infected cell cultures but did not cause duck embryo death as previously described for pathogenic duck reoviruses. Genomic analyses showed that Ych shared the common genome structural characteristics and the predicted polycistronic S1 genome segment, but it was genetically distant from known reovirus isolates identified in China10. It is interesting that the greatest sequence similarity values of the ten genome segments were seen with the recently described reovirus strain D2533/6/1-10, which was isolated from a bursa sample of Pekin ducks in Germany20. Phylogenetic analysis of all genes revealed that the two isolates form a distinct branch from reference ARVs, suggesting that they might have a common evolutionary origin. Based on the relatively small amount of ARV sequence data available in GenBank, no attributable genomic reassortment or intra-segment recombination was found for the evolutionary origin of the newly emergent duck reovirus. Cross neutralisation results demonstrated that Ych belongs to a serotype distinct from the “novel” duck reovirus and chicken reovirus S1133.ARVs have been demonstrated to be involved in numerous diseases in commercial poultry and the pathogenicity of isolates differs considerably. Differences in pathogenicity have been described for a number of avian reoviruses26. It was noticed that the German strain D2533/6/1-10 was isolated from a sample of duck bursa although its pathogenicity was not further evaluated20. Our experimental infection results demonstrate that the virus replicated in the bursa of Fabricius. These findings are also in agreement with previous reports that the intestine and bursa of Fabricius were the initial sites of replication of some chicken and turkey reovirus isolates28. Microscopic examination revealed damage to the bursa epithelial cells and increased number of apoptotic lymphocytes, suggesting that Ych infection might induce a transient and possibly permanent immunosuppression in infected ducklings. This might explain to some extent that no specific neutralising antibody against the virus was detected from the sera of infected ducklings until the end of observation period. However, the fact that virus was not detected in the spleen, liver, kidney or lung samples of the experimentally infected ducks suggests that Ych was noninvasive for these internal organs, thus affecting also the humoral immune response. This scenario has been reported in chickens for reoviruses possessing low pathogenicity29.In this study, inoculating ducklings with Ych by oral, intranasal or subcutaneous routes, which are likely natural transmission routes for avian reoviruses, did not produce clinical disease and gross lesions, as were observed in the field cases. However, virus shedding from the infected ducklings was detected by RT-PCR, suggesting that the virus multiplied transiently in the infected duck intestine. However, virus shedding via faeces implies that infected ducklings may transmit virus laterally to pen mates as seen in other reoviruses in chickens and turkeys30. Haemorrhagic enteritis as seen in field cases was not reproduced in this study. Our experimental infection results demonstrate that the bursa of Fabricius may serve as a site of Ych replication in infected ducklings, and whether the increase of apoptotic lymphocytes could result in lymphoid cell deletion is under investigation. It was possible that under field conditions, the virus infection induced immunosuppression, predisposing the ducklings to infection with other pathogens, which would worsen the disease and lead to death.Describing the aetiological role of an ARV isolate can be complicated because some clinical disease states are often difficult to reproduce experimentallyIn summary, diagnostic attempts at Pekin duck haemorrhagic enteritis led to the isolation of a new reovirus, Ych, genetically distinct and antigenically different from previous duck reovirus strains identified in China. Experimental infection of ducklings indicated that the virus was able to replicate in the bursa of Fabricius and induce epithelial cell damage and lymphoid cell apoptosis. The virus-induced damage to this immune organ implies that the newly emergent reovirus infection may serve as a priming or concurrent agent in the development of duck enteritis. However, the role of Ych in haemorrhagic enteritis needs to be further evaluated in combination with other agents or substances.Twenty-day-old Pekin ducks that died at a farm in Yucheng, Shandong Province were necropsied. Pathological examination revealed mild to moderate swollen liver and intestinal mucosa haemorrhagic plaques. Samples of liver, spleen and brain tissue were inoculated onto tryptic soy agar  plates containing 2% fetal bovine serum and incubated at 37 °C for 48 h for bacterial examination. Virus isolation was performed later. The intestinal mucus, liver and spleen samples were pooled and homogenised in sterile phosphate-buffered saline solution (pH 7.2) to give a 20% suspension (w/v). After centrifugation at 8,000 × g for 10 min, the supernatant was filtered through a 0.22 μm pore-size sterile filter and inoculated into five 9-day-old duck embryos (0.2 mL/embryo) by the chorioallantoic membrane route. Embryos were candled daily and were chilled at 4 °C to death on 5 days post-inoculation (dpi) to harvest allantoic fluid for subsequent passage. Since no consistent embryo death was observed during the serial embryo-passage, we inoculated the allantoic fluid of the fourth passage onto confluent monolayers of duck embryo fibroblasts. The syncytium formation of the infected cells was visualised by Giemsa staining. To test the growth of the isolate in Vero cells, the DEF-adapted virus suspension was inoculated onto the monolayer of Vero cells using the conventional method.To determine whether the isolated virus had a lipid envelope, the DEF-adapted virus suspension was clarified by low speed centrifugation after two freeze/thaw cycles and treated with chloroform  for 10 min at room temperature. The chloroform layer was then removed by centrifugation and the infectious virus titre of the supernatant was quantified in DEF culture after serial dilution. For nucleic acid type determination, virus replication ability was tested in DEF culture with or without addition of 5-bromo-2′-deoxyuridine  in the maintenance medium. Duck Tembusu virus and duck enteritis virus were used as RNA and DNA virus controls, respectively.® Ultra™ RNA Library Prep Kit for Illumina  and submitted for NGS with the Illumina HiSeq4000 .Based on the fact that the isolate was a non-enveloped RNA virus, we extracted viral RNA from the concentrated virus. Briefly, the virus-infected DEF cell suspension was centrifuged at 8,000 × g for 30 min at 4 °C to remove cell debris. The clarified supernatant was further centrifuged by ultracentrifugation at 100,000 × g for 2 h. After removing the supernatant, the pellet was resuspended in distilled water to 1/100 volume of the original supernatant and total RNA was extracted from the suspension with TRIzol . Using the extracted RNA as template, an RNA library was prepared with NEBNextOn the basis of the preliminary sequence data obtained from NGS, a set of gene-specific oligonucleotide primer pairs Table  were deshttps://www.ncbi.nlm.nih.gov/orffinder/). The putative protein functions of respective ORFs were then inferred by BLASTp in the non-redundant protein database. The nucleotide and amino acid sequence identity were analysed using the Clustal W method of MegAlign software . Maximum likelihood phylogenetic analyses based on 10 ORFs nucleotide sequences were conducted using the General Time Reversible model using MEGA 7.0.14 software (www.megasoftware.net), and estimates based on bootstrap resampling were carried out with 1000 replicates.The initial complete genome was assembled and manually edited using ContigExpress software . ORFs were predicted using online software ORF finder  at 180 V for 7 h and nucleic acid bands were stained using rapid sliver staining kit according to manufacture’s guideline .To compare the electrophoretic mobilities of the genomic segments of the isolate with representative ARV strains, the virus was propagated in DEFs and total RNA was extracted from the ultracentrifugation-concentrated virus suspension using TRIzol as described above. The extracted RNA was then treated with DNase  and S1 Nuclease  to remove DNA and single-stranded RNA. The duck reovirus isolate HC and ARV S1133 were propagated in DEF and chicken fibroblasts, respectively, as described50/0.1 mL (50% tissue culture infective dose). After incubation at 37 °C for 1 h, the serum-virus mixture was inoculated into five wells of DEF monolayers in a 96-well plate. Cytopathic effect (CPE) was recorded for 5 days. The neutralising antibody titre was the highest serum dilution that resulted in the cells being completely protected.Antisera against the isolated virus, duck reovirus HC and ARV S1133 were separately prepared in 3-week-old SPF chickens  by four immunisations at 2-week intervals with inactivated virus suspension. Sera were collected 12 days after the last immunisation. For the cross-reactivity assay, heat-inactivated homologous or heterologous antiserum was 2-fold serially diluted and mixed with an equal volume of the virus suspension containing 200 TCIDad libitum access to feed and water. In experiment 1, twenty 2-day-old ducklings were divided into three groups and infected with the cell culture-prepared virus suspension as indicated in Table 4 TCID50 of the isolate. Group 2 were subcutaneously infected with the same dose, and group 3 were raised separately as mock-infected controls. Clinical signs were recorded for 16 days to evaluate the pathogenicity of the isolate for ducklings, and cloacal swabs were collected as indicated for detection of virus shedding. On 9, 12 and 16 dpi, serum samples were collected for antibody detection using the virus neutralisation test as described above. To evaluate the in vivo infectivity of the isolate, ten ducklings at 12 days old were infected by oral and subcutaneous inoculation with the same dose virus as described above. Five ducks were sacrificed on both 3 and 5 dpi, respectively, and liver, spleen, caecal tonsil, thymus and bursa of Fabricius samples were collected for virus detection by RT-PCR and re-isolation. Five uninfected control ducklings were examined in the same way at each sampling point.One-day-old Pekin ducklings without maternal antibody against duck reovirus were purchased from Nankou Hatchery . Ducklings were raised in negative pressured isolators with The viral RNA from tissue samples and cloacal swabs were extracted by viral RNA kit  and converted to cDNA using a Reverse Transcription System  with specific primers following the manufacturer’s instructions. Specific primers based on the λC gene were designed and synthesised . The length of RT-PCR product was 557 bp.In Situ Cell Detection kit  following the manufacturer’s instructions.Histological sections were routinely prepared from the bursa after samples were fixed in 10% neutral buffered formalin solution and paraffin embedded. The sections were stained with haematoxylin and eosin (HE). For apoptosis detection, TUNEL assay (dUTP nick end labelling) was conducted using an Animal infection experiments were approved by the China Agricultural University Animal Ethics Committee, in accordance with the guidelines of the Review of Welfare and Ethics of Laboratory Animals approved by the Beijing Municipality Administration Office of Laboratory Animals. Experiments involving reovirus infections were conducted in the Biosafety Level 2 facilities in College of Veterinary Medicine, China Agricultural Unversity.Supplementary material


Bioaerosol sampling devices are necessary for the characterization of infectious bioaerosols emitted by naturally‐infected hosts with acute respiratory virus infections. Assessment of these devices under multiple experimental conditions will provide insight for device use.The primary objective of this study was to assess and compare bioaerosol sampling devices using a) an in vitro, environmentally‐controlled artificial bioaerosol system at a range of different RH conditions and b) an in vivo bioaerosol system of influenza virus‐infected ferrets under controlled environmental conditions. Secondarily, we also sought to examine the impact of NSAIDs on bioaerosol emission in influenza virus‐infected ferrets to address its potential as a determinant of bioaerosol emission.We examined the performance of low and moderate volume bioaerosol samplers for the collection of viral RNA and infectious influenza virus in vitroand in vivo using artificial bioaerosols and the ferret model of influenza virus infection. The following samplers were tested: the polytetrafluoroethylene filter (PTFE filter), the 2‐stage National Institute of Occupational Safety and Health cyclone sampler (NIOSH cyclone sampler) and the 6‐stage viable Andersen impactor (Andersen impactor).10copies of RNA/L air, 3.84 log10copies of RNA/L air and 6.09 log10copies of RNA/L air respectively at peak recovery. Infectious virus was recovered from the PTFE filter and NIOSH cyclone samplers on the peak day of viral RNA recovery.The PTFE filter and NIOSH cyclone sampler collected similar amounts of viral RNA and infectious virus from artificially‐generated aerosols under a range of relative humidities (RH). Using the ferret model, the PTFE filter, NIOSH cyclone sampler and the Andersen impactor collected up to 3.66 logThe PTFE filter and NIOSH cyclone sampler are useful for influenza virus RNA and infectious virus collection and may be considered for clinical and environmental settings. Early experiments indicated that influenza virus–infected ferrets treated with antipyretic compounds experienced increased nasal shedding, potentially leading to increased risk of transmission.Assessing the burden of influenza virus in the air is technically and operationally challenging in real‐world settings such as healthcare institutions.Filter‐based bioaerosol sampling devices, cyclone samplers, and cascade impactors are the most commonly used and accessible instruments for recovery and detection of viruses, though none were explicitly designed for this purpose.The primary objective of this study was to assess and compare bioaerosol sampling devices Table  using  in compliance with guidelines set by the Canadian Council on Animal Care and with approval of the SRI animal care committee.2.2Madin‐Darby canine kidney cells  were maintained in Eagle's minimum essential media (Wisent), 10% fetal bovine serum , and penicillin (100 UI/mL)/streptomycin . Influenza A/Ontario/2016 (H1N1)‐like and influenza A/Ontario/2016 (H3N2)‐like viruses were obtained from clinical samples at Sunnybrook Health Sciences Centre  and subsequently passaged and plaque‐purified.2.37 plaque‐forming units (PFU)/ml) and H3N2 (1.28 × 106 PFU/mL) influenza viruses were diluted in phosphate‐buffered saline  and nebulized separately  into a custom‐built, pressurized, artificial aerosolization chamber  was added to nasal wash sample supernatants after centrifugation at 800 g for 5 minutes at 4°C. The PTFE filter was vortexed with 2 ml VTM for 1 minute, and the NIOSH cyclone sample tubes and filter were vortexed with 2, 0.5, or 1 mL of VTM for 1 minute for stages 1, 2, and the NIOSH filter, respectively. All samples were stored at −80°C after processing. Viral titers from nasal wash and bioaerosol samples (from bioaerosol sampling devices) were quantified by plaque assay in MDCK cells and reported as PFU. Viral RNA from air samples was extracted using the MagMAX™ Viral RNA Isolation Kit (Thermo Fisher Scientific) and stored at −80°C. Reverse transcriptase‐quantitative polymerase chain reaction (RT‐qPCR) of all air samples was conducted using primers targeting IAV matrix gene2.6All data were initially tested for normality using the D'Agostino‐Pearson omnibus normality test. Normally distributed data were tested using either an unpaired t test or one‐way ANOVA to determine statistical significance. Following ANOVA testing, Tukey's multiple comparisons test was utilized to determine groups that were statistically different. Non‐normally distributed data were assessed using either the Mann‐Whitney test or the Kruskal‐Wallis test to determine statistical significance (GraphPad Prism 6 software). The ratio of viral RNA to infectious virus was determined by dividing the viral RNA collected (copies) by infectious virus collected (PFU).33.1To evaluate the ability of the PTFE filter and NIOSH cyclone sampler to collect influenza virus RNA and infectious virus, we aerosolized H1N1 and H3N2 influenza viruses into a custom‐built artificial aerosolization chamber , medium (47%‐53%), or high (78%‐83%) RH conditions. There was no significant difference between bioaerosol sampler collection at different RH conditions for RNA or infectious virus , but no fevers were noted. Nasal wash viral loads were highest on day 1 p.i., reaching 6.20 ± 0.22 log10 PFU/mL for untreated ferrets and 6.85 ± 0.26 log10 PFU/mL for treated ferrets, and declined until termination ‐treated influenza virus–inoculated ferrets Figure A. Treaten Figure C with no10 copies/L air for untreated and 3.66 log10 copies/L air for treated animals, while the NIOSH cyclone sampler collected 2.56 log10 copies/L air for untreated and 3.84 log10 copies/L air for treated ferrets on day 3 p.i. , mid (2.1‐4.7 μm), and lower 0.65‐2.1 μm) airway distributions in the respiratory tract‐2.1 μm aWe also inoculated ferrets from untreated and meloxicam‐treated groups and individually sampled each animal by nasal washing and using the PTFE filter Figure . Approxi4The merits of bioaerosol sampling for respiratory viruses are coming to the forefront as researchers have used this approach to detect virus from air emitted by individuals with laboratory‐confirmed influenza,4 copies/PFU for the PTFE filter and 7.55 × 103 copies/PFU for the NIOSH cyclone sampler. The ratios of collected viral RNA to infectious virus for both the PTFE filter and NIOSH cyclone sampler are slightly higher than the ratio for the nebulizer (3.66 × 103 copies/PFU), indicating a potential loss of infectivity during the aerosolization and collection process or due to other factors that may disrupt the viral envelope or otherwise affect infectivity. Despite this potential loss, both bioaerosol samplers recorded similar ratios, indicating their comparable ability to collect infectious virus. Both samplers were also able to collect as much as 104 PFU of virus. Previous work has suggested that dry sampling or sampling onto a dry surface such as a filter may limit the ability of bioaerosol samplers to collect infectious virus,The ratio of viral RNA to infectious virus is important for understanding the burden of influenza virus in the air and an indication of the influence of aerosolization, transit, and collection on the infectivity of the virus.IAV transmission is potentially enhanced at low temperature and low RH, while the reciprocal effect is observed when temperature and humidity increase.The use of ferrets to test IAV bioaerosol collection using three bioaerosol sampling devices is an excellent method for understanding the performance of these devices, though not all samplers could be tested concomitantly due to operational and special confines of the housing system required to maintain chamber equilibrium and temperature and RH constant. This environment is a controlled setting intended to reflect collection from emitting hosts. We found that the PTFE filter and NIOSH cyclone sampler were capable of collecting infectious virus from both groups of influenza virus–infected ferrets on day 3 p.i. The Andersen impactor collected impressive amounts of viral RNA, but no infectious virus was detected. This instrument collects at a higher flow rate (28.3 L/min) and has the ability to size fractionate particles, which are enticing attributes when choosing a bioaerosol sampler. However, the difficulties with maneuvering the apparatus with liquid media while inside the environmental chamber make it vulnerable to pump damage. The Andersen impactor is therefore best suited for controlled, experimental settings. Contrary to the Andersen impactor, the PTFE filter and NIOSH cyclone samplers were easy to manipulate inside the environmental chamber . These devices can be fixed to walls for static sampling or worn by personnel for personal breathing zone sampling. These attributes combined with the ability to collect viral RNA, and infectious virus makes these samplers more feasible for assessing the burden of influenza virus in the air in various settings, and more conducive for use in multicenter studies.It has been suggested that anti‐inflammatory NSAIDs such as meloxicamThere were several limitations in this study, including the choice of bioaerosol sampling devices; glass liquid impingers were not included since their use in a clinical environment would be prohibited. The use of liquid media instead of solid agar in the plates of the Andersen impactor may also be problematic since the volume of liquid or agar present in each plate is critical for proper size fractionation of particles and the potential loss of liquid during the collection process may lead to a misrepresentation of particle size distribution. Kormuth et al recently demonstrated the limited influence of RH on the infectivity of physiologically relevant fine aerosols and droplets,5In summary, the Andersen impactor has a high flow rate, the ability to size fractionate particles, and is commercially available. The cumbersome nature and cost of the Andersen impactor impacts the feasibility for widespread clinical and field use, but this instrument may be ideal for collection of IAV RNA in experimental and other well‐controlled settings. The NIOSH cyclone sampler is portable, lightweight, is able to size fractionate bioaerosols, collects IAV RNA and infectious virus, and works well under multiple RH conditions. It is not commercially available and requires time for processing and decontamination but can be used for personal sampling and whenever particle size fractionation is desired in clinical and environmental settings. The PTFE filter is portable, lightweight, disposable, and easily manipulated, and can collect IAV RNA and infectious virus with minimal processing. The PTFE filter is extremely versatile; it can be used in clinical and environmental settings, for both personal sampling and static sampling. The PTFE filter, NIOSH cyclone sampler, and Andersen impactor are all viable options for bioaerosol sampling of IAV, depending on the application, and may be considered for field, clinical, or experimental studies. Determining which bioaerosol sampler to use has been historically difficult, but moving forward, this study provides valuable evidence to guide bioaerosol sampler selection. Click here for additional data file. Click here for additional data file.


A great challenge of research is the utilization of natural or synthetic zeolites, in place of natural pozzolans, for manufacturing blended cements. The difficulties of interpretation of the pozzolanic behavior of natural zeolite-rich materials and the role played by their nature and composition can be overcome by studying more simple systems, such as pure synthetic zeolites. This study aims at investigating the pozzolanic ability of isostructural zeolites with different framework compositions, such as three sodium zeolites of the faujasite (FAU) framework type: LSX, X, and Y. The pozzolanic activity has been estimated by thermogravimetry and X-ray diffraction analysis. The overall outcome of the investigation is that the zeolite structure affects its pozzolanic activity, as zeolites with similar framework densities exhibit distinct abilities to fix lime. Moreover, the framework composition is effective either from a kinetic point of view or on the total amount of fixed lime. Zeolite X appears to possess the best average features.  A pozzolanic material is generally defined as a natural, synthetic, or secondary raw material, containing high percentages of active SiO2 and Al2O3. These can react, in the presence of water, with lime or lime-releasing materials , giving products such as hydrated calcium aluminates and hydrated calcium silicates, having a hydraulic character, i.e., displaying mechanical resistances. Among the many pozzolanic materials, zeolites have attracted increasing interest in recent years.The raising attention to environmental problems  and extra-framework, poorly bonded, alkaline, or alkaline earth cations, balancing the negative charge of the AlO4 tetrahedra. The extensive inner surfaces and the resulting adsorption properties, combined with molecular sieving and cation exchange abilities, give these materials unique and valuable possibilities of application in dozens of processes of industrial relevance .A great deal of research has been carried out on the utilization of either natural ,6,7,8,9 In a lime/zeolite system, different simultaneous reactions and equilibria occur, e.g., for a lime/Na–zeolite system: 2 and related dissociation equilibria:(a) Dissolution of solid (s) Ca(OH)2+ and Ca(OH)+ in solution and Na+ in zeolite (z):(b) Ion–exchange equilibria involving Ca2+, tend to give mixtures of silicates and aluminates, instead of aluminosilicates (framework silicates), as it happens in environments where the alkaline cations are predominant  range, under the following mathematical formulation:A quantitative evaluation may also be worked out from the Fratini plot. The UNI EN196–5 standard  provides2 blend at t = 0, i.e., before the addition of water and therefore before the pozzolanic reaction starts. The zeolite is in this case zeolite X. Three major thermal effects are observed in the curve: (i) the weight loss in the region I (100–300 °C) is connected to the release of “zeolitic” water; (ii) the weight loss in the region II (350–550 °C) is attributed to the dehydration of Ca(OH)2; and (iii) the small weight loss in the region III (550–850 °C) is attributed to the decarbonation of CaCO3 .The progress of the pozzolanic reaction between lime and zeolites has been evaluated by measuring the free lime content in the pastes at different curing times. 2 pastes. Note that the presence of zeolite hides, especially in the early stages of the reaction, the possible dehydration effects due to CSH and CAH. Analogous curves, as those in After water addition, upon increasing the curing time (t > 0), regardless of the zeolite type, a progressive decrease of lime content in the pastes is observed due to the progress of the reaction between silicoaluminate species, coming from zeolites, and lime, to form calcium silicate hydrate (CSH) and calcium aluminate hydrate (CAH). L(r) and mL(i) are the reacted and initial amounts of lime in the paste, respectively, mw is the amount of released water, and 4.11 is the stoichiometric conversion factor from water to lime in the reaction of lime dehydroxylation. The estimation of lime amount in the pastes was performed by thermogravimetric analysis, using the following equation:2 dehydroxylation. Calculated data, concerning the residual unreacted lime, were estimated from the region II of the various thermograms and processed with Equation (6).2+ is removed but not fixed. Accordingly, zeolites L and X, having a higher aluminium content and therefore a higher cation exchange capacity, resulted as more effective in subtracting Ca2+ from the system compared to Y, which was in accordance with the data reported by Mertens et al. [At short curing times (3 hours), the zeolites showed different behaviors: the less siliceous L and X fixed roughly 22% and 18% of the initial lime amount, respectively, whereas the most siliceous Y reacted with only 8% of lime. As reported in the introduction, the first stage of the whole pozzolanic reaction in a zeolite–lime system consists of cation exchange, which is a fake pozzolanic reaction, because Cas et al. . It shou2 and Y still longer, fixing some 47% of lime after 90 days. After the first stages, in fact, where cation exchange prevails, both the higher specific surface of the zeolite and its higher silica-to-alumina ratio (see Zeolite L–lime pastes reached the maximum amount of fixed lime (around 30%) after 3 days, whereas zeolites X kept reacting until 28 days, fixing roughly 44% of Ca(OH)atio see  become tThe above findings were confirmed by the XRD analysis of the three zeolite–lime systems cured at different times . In all 2O3·CaCO3·11H2O, ICDD ref. code: 00–041–0219) was detected in all three systems. This product originates from the carbonation of calcium aluminate hydrate, which is the first to form in the reaction between zeolite and lime.The appearance of a calcium aluminum oxide carbonate hydrate (3CaO·Al2(CO3)2(OH)4·6H2O, ICDD ref. code: 00–030–0222), together with traces of calcium silicate hemihydrate   . 5Si5Al(OH)O17·5H2O, ICDD ref. code: 00–041–0219), and calcium silicate monohydrate   .5Si6O16(OH)2·8H2O, ICDD ref. code: 00–029–0331)   was obse(t) and Le are the fixed lime percentages at time t and at equilibrium, respectively, and k (w% h−1) is the rate constant of the reaction. Such reaction can be linearized as follows:The data of TG analysis (lime dehydroxylation) were further processed to investigate the mechanism of the lime–pozzolan reaction. It was assumed that the process of CSH and CAH formation, governed by the interaction between zeolite and the ionic calcium species (see Equations (1) and (2)), can be described by a second-order kinetic equation:An inspection of (i) The systems with the two less siliceous zeolites reach the equilibrium values rather rapidly (after some 7 days for zeolite L and after 28 days for zeolite X), as the experimental data are very close to the equilibrium data, which were obtained theoretically from the model (second column in (ii) The zeolite L–lime–water system has the fastest reaction kinetics, while the zeolite Y–lime–water system has the slowest one;2 from the system, compared to the more siliceous X and Y, as already observed in the preliminary pozzolanicity test, which was carried out according with the European standards (see (iii) On the contrary, zeolite L appears to be the least effective in subtracting Ca(OH)ards see .3 (where T stands for tetrahedra), respectively, but the LTA framework appears to be more easily attacked by an alkaline, Ca2+-rich solution than a FAU framework [Comparing these evidences with our previous results, it appears that the two main parameters examined, the structure and framework composition, i.e., siliceousness, act in a distinct way. It is generally known that zeolites react as more readily as their framework densities are lower, but it has been demonstrated that each structure with their structural peculiarities can diversely affect the reaction kinetics. In fact, the framework densities of LTA and FAU are very close to each other, 12.9 and 12.7 T/1000 Åramework . On the The best compromise appears to be zeolite X, which exhibits a reasonable reaction rate in the early stages of the reaction together with a good effectiveness at fixing lime at longer curing times.The results of this study, dealing with the pozzolanic activity of isostructural sodium FAU zeolites with different framework compositions, together with those of the previous investigation on isochemical sodium zeolites with different structures (LTA and FAU) , outlineDuring the first few hours, the main active factors are the Si/Al ratio  and the ion exchange capacity of the participating zeolite: both factors, which are in fact correlated, increase the amount of calcium removed from the contacting solution. Accordingly, zeolites L and X were able to subtract more calcium than Y zeolite, even if the concurrent effect of their smaller particle size could also have played a role.As the reaction proceeds, the specific surface and the silicon content of the zeolite become the dominant factor governing the reaction kinetics: the L zeolite fixed the lower lime amount (30%) in the shortest time (3 days), while the Y zeolite fixed about 47% of the lime in 90 days.Concerning the reaction products, while all three systems developed calcium aluminium oxide carbonate hydrate, which formed at first as a reaction product between zeolite and lime, at longer reaction times, the more aluminium-rich zeolites showed the presence of paraalumohydrocalcite, while the silicon-rich Y zeolite produced tobermorite as a reaction product.The kinetics of the hydration process, monitored up to 90 days, were successfully modelled with a pseudo-second-order reaction equation. The results showed that:Zeolite X (FAU) has demonstrated, in this study, the best overall pozzolanicity, because it couples reasonable reaction kinetics with a relevant ability to fix lime, which means that it also has a good capacity to increment the mechanical resistances of the hardened mortars.


Different measurements of white matter signal abnormalities (WMSA) are often used across studies, which hinders combination of WMSA data from different cohorts. We investigated associations between three commonly used measurements of WMSA, aiming to further understand the association between them and their potential interchangeability: the Fazekas scale, the lesion segmentation tool (LST), and FreeSurfer. We also aimed at proposing cut-off values for estimating low and high Fazekas scale WMSA burden from LST and FreeSurfer WMSA, to facilitate clinical use and interpretation of LST and FreeSurfer WMSA data. A population-based cohort of 709 individuals  was investigated. We found a strong association between LST and FreeSurfer WMSA, and an association of Fazekas scores with both LST and FreeSurfer WMSA. The proposed cut-off values were 0.00496 for LST and 0.00321 for FreeSurfer -corrected values). This study provides data on the association between Fazekas scores, hyperintense WMSA, and hypointense WMSA in a large population-based cohort. The proposed cut-off values for translating LST and FreeSurfer WMSA estimations to low and high Fazekas scale WMSA burden may facilitate the combination of WMSA measurements from different cohorts that used either a FLAIR or a T1-weigthed sequence. Alsord.edu/)  is increrd.edu/) –13. To oRegarding the association between automatic segmentations of hyperintense and hypointense WMSA, studies are scarce but some data show a close correlation between the two , 14. HowUnderstanding how these three measurements relate to each other, endorsing their potential interchangeability, is of utmost importance because WMSA are frequently assessed with different methods and MRI sequences across studies. This makes it difficult to combine data from different cohorts. Hence, our primary aim was to investigate the association between the Fazekas scale, hyperintense WMSA based on the LST software, and hypointense WMSA based on the FreeSurfer software, in order to understand their association and potential interchangeability, in a large population-based cohort. Our secondary aim was to propose reliable cut-off values for automatic segmentations of WMSA data from LST and FreeSurfer to identify low and high Fazekas scale WMSA burden. These cut-off values might potentially facilitate combination of WMSA data from different cohorts, software, and MRI sequence types (FLAIR and T1-weigthed), as well as facilitate clinical use and interpretation of LST and FreeSurfer WMSA data.In our population-based cohort of 709 individuals , 15.1% had high Fazekas WMSA burden (i.e. scores 2 and 3) and the rest had low WMSA burden (i.e. Fazekas scores 0 and 1) . After a1=0.563; p<.001; β2=0.413; p<.001). The paired-sample t-test revealed that LST WMSA volumes were significantly larger than FreeSurfer WMSA volumes (t(708)=-15.9;p<.001). Visual inspection of (109.1) = −12.1;p<.001) and between Fazekas and hypointense WMSA (t(107.1)=-10.1;p<.001) were also significant, revealing larger volumes for the high Fazekas scale WMSA burden category  curve in order to obtain reliable cut-off values for automatic segmentations of WMSA data from LST and FreeSurfer and identify low and high Fazekas scale WMSA burden. The ROC analyses separating low and high Fazekas WMSA burden categories revealed an AUC value of 93% for LST WMSA, and an AUC value of 94% for FreeSurfer WMSA . The proIn a large population-based cohort of 70-year old individuals, 15.1% had a high Fazekas WMSA burden. This frequency is, as expected, higher than that reported in younger population-based cohorts (frequency of 10%)  and loweRegarding the ROC analyses, both automatic segmentation methods could separate low and high Fazekas scores with high sensitivity and specificity. Since the proposed cut-offs values for low and high Fazekas WMSA burden are adjusted for the TIV, we believe that these values may be more easily transferred to volume estimations from other MRI scanners, when similar MRI scanning protocols are used. There may still be some source of error related to inter-rater variability for the Fazekas scale. However, inter- and intra-rater reliability is usually higher for the Fazekas scale than for other visual rating scales .Some limitations should be mentioned. Despite that Fazekas visual rating does not seem to be influenced by the experience of the rater , future In conclusion, we provide data on the association between Fazekas scores, hyperintense WMSA, and hypointense WMSA in a large population-based cohort. Validating our proposed cut-off values in other cohorts is of great importance for future studies combining WMSA measurements based on different MRI sequences (FLAIR and T1-weigthed).As part of the Gothenburg H70 Birth Cohort Studies all men and women born 1944 on dates ending with 0, 2, 5, or 8, and registered as residents in Gothenburg according the Swedish Tax Agency were invited to a comprehensive examination on ageing and age-related factors . IndividFor the current study, inclusion criteria were: (1) availability of Fazekas visual ratings; and (2) outcomes for both LST and FreeSurfer suitable for analysis , giving a sample of 709 individuals . MMSE ranged between 20 and 30 . The majority of participants (99.3%) were cognitively normal or had mild cognitive impairment (no dementia). CDR distribution was CDR 0= 81.8%; CDR 0.5= 18.1%; and CDR 1= 0.1%.MRI data were acquired in a 3.0T Philips Achieva system , using a 3D T1-weigthed Turbo Field Echo (TFE) sequence ; and a 3D FLAIR sequence .The Fazekas scale was applied on FLAIR MRI data on the axial plane and was scored following standard guidelines . Brieflyhttps://www.fil.ion.ucl.ac.uk/spm/). LST utilizes a lesion prediction algorithm (LPA) based on FLAIR images intensity distribution (hyperintensities) that builds a lesion probability map for each individual. The T1-weighted images were processed with the FreeSurfer image analysis suite. FreeSurfer detects hypointensities and automatically labels WMSA volumes for each participant using a probabilistic procedure as well [WMSA were also automatically segmented with LST 2.0.15 and FreeSurfer 6.0.0. LST is an open source segmentation toolbox implemented in the SPM software ( as well . MRI dat as well . WMSA vo as well .http://www.r-project.org). A p-value <0.05 (two-tailed) was deemed significant in all the analyses.Associations between LST and FreeSurfer WMSA estimations were tested using the Pearson correlation. Quadratic simple regression was also used to test for the association between LST and FreeSurfer WMSA. Paired-sample t-test was used for the comparison of LST and FreeSurfer WMSA mean volumes. Two independent-sample t-tests were used for the comparison of low (scores = 0 and 1) and high (scores = 2 and 3) Fazekas scale WMSA burden categories in LST and WMSA mean volumes. The cut-off value for estimating low and high Fazekas scale WMSA burden from LST and FreeSurfer WMSA was derived as follows. First, Fazekas scores were categorized as low WMSA burden or high WMSA burden . Second,The H70 study was approved by the Regional Ethical Review Board in Gothenburg , and by the Radiation Protection Committee  in concordance with the 1964 Helsinki declaration and its later amendment.