During puberty, the growth of the bones is faster than that of the muscles, which may result in muscular tightness. Muscular tightness and asymmetry have been associated with an increase in injury incidence. The assessment of a joint range of motion (ROM) could help to identify athletes classified as high injury risk. The objectives of the present study were to describe the lower-extremity flexibility profile (LEFP) of youth competitive inline hockey players using the ROM-SPORT battery (I) and to identify muscular tightness and asymmetry (II). Seventy-four young players were examined for maximum passive ankle, knee, and hip ROMs. Muscle asymmetry or tightness was classified according to cutoff scores previously described. The LEFP of the 74 players was 10.8° for hip extension, 26° for hip adduction, 33.6° for ankle dorsiflexion, 38.6° for ankle dorsiflexion with knee flexed, 36.7° for hip abduction, 46° for hip internal rotation, 60.6° for hip external rotation, 65.1° for hip abduction with the hip flexed, 66.3° for hip flexion with the knee extended, 119.7° for knee flexion, and 133.7° for hip flexion. The individual analysis of the flexibility values identified tightness in all players for one or more movement, except for hip abduction. A low prevalence of asymmetries was observed (range: 5.4% to 17.6% of players) depending on the ROM. Inline hockey (IH) has become an increasingly popular sport in Spain during the last decade, with the senior women’s and men’s national IH teams placed in the top ten in the IH World and European Championships . IH is aMuscle flexibility is one of the key components of athletic performance together with strength, endurance, speed, and coordination ,9,10. SeOne of the mechanisms that may contribute to muscle tightness in young athletes is the growth spurt . This phThe assessment of the muscular lower-extremity flexibility profile (LEFP) of elite IH players is essential to aid physical trainers to design, adapt, control, and monitor the physical conditioning and manage the injury risk of players . The LEFTherefore, the objectives of the present study were to describe the LEFP of young IH players using the ROM-SPORT battery (I) and to identify players with muscular tightness and asymmetry in each movement (II). n = 24) “Alevin” , and “Infantil”  were included in the study  [ID: 1702/2017].Before data collection, players completed a questionnaire about their IH-related background , and characteristics of sports participation . The information obtained in the questionnaire was cross-referred with the trainer and parents to increase the objectivity. Data from the questionnaires indicated that the sample was homogeneous in potential confounding variables, such as age, body weight, stature, body mass index, training and game participation (a competitive match and 2–3 days of training per week), climatic setting, competitive level, rest periods, and sport/training experience/age. In addition, none of the players were involved in regular strength and flexibility programs during the season. Players did not regularly perform stretching exercises either in the warm-up or in the cool-down of training and competition. Data were collected 3 days into the competitive season and this time frame was selected to ensure that there was stability in the players recruited. Anthropometric measurements  were obtained by the lead researcher at the start of the assessment session.The 11 maximum passive ROM tests of the ROM-SPORT battery were used to assess the participants ,42. For These tests were selected because they have been considered appropriate by the American Medical Association  and are 95) were calculated from the subsequent measurements. Results of pre-measurement and post-measurement sessions displayed a high ICC in all the tests (0.94 to 0.97). The MDC95 for each ROM measure ranged from 3.7° to 6.9° [Before the main data collection, the absolute reliability coefficient was evaluated on 20 healthy athletes. The ROM tests were measured twice 2-weeks apart. An intraclass correlation coefficient (ICC) and the minimal detectable change at a 95% confidence interval  was used to measure the hip abduction ROM and lumbar support  was used to standardize the lumbar curvature. Before each assessment session, the inclinometer was calibrated to either 0° with the vertical or horizontal. The angle between the longitudinal axis of the mobilized segment was recorded (following its bisector) with the vertical or horizontal plane. The endpoint for each test was determined by one or both of these criteria: (1) the athlete´s feeling a strong but tolerable stretch, slightly before the occurrence of pain; (2) one or both examiners (main and assistant) detected a palpable compensatory movement that may increase the ROM [Three weeks before the study, all the IH players performed a familiarization session to learn the correct technical execution of each movement. The dominant side was defined as the participant’s preferred kicking leg ,51. The  the ROM . The strAt the beginning of the testing session, IH players completed a standardized warm-up consisting of 5 min jogging at moderate intensity  and 15 repetitions of dynamic stretching of the evaluated muscles . The warThe distribution of each variable was examined using the Kolmogorov–Smirnov normality test and homogeneity of variance was verified with a Levene test. Both tests confirmed that not all data was normal distribution and homoscedastic.Descriptive statistics including mean and SDs were generated for the 11 ROM measurements. The Wilcoxon test was used to assess the relationship between the values of the dominant and non-dominant sides. The magnitude of the effect size was classified as previously described by Hopkins et al.  as triviAn individual analysis of the ROM values was performed to identify the number of players with muscle asymmetry and tightness. The asymmetry was established according to reference scores previously published in the scientific literature ,52,53,54p < 0.05).Data were analysed using SPSS version 24 . For all analyses, statistical significance was accepted at the 95% confidence level for all statistical parameters (Seventy-four IH players satisfied the pre-specified inclusion and exclusion criteria. p ≤ 0.009) was observed in the HAD (Dom 25.4° vs. No Dom 27.3°), ADF-KF (38.2° vs. 39.1°), and HAB (37.4° vs. 36.1°); however, the effect size was categorized as trivial or small (d ≤ 0.59). Although there were statistically significant differences between the mean ROM values of the dominant and the non-dominant side for HAD, ADF-KF, and HAB, these differences are not clinically relevant  for the physical-sport practice [Asymmetry (practice . This isn = 13), HAD-HF (n = 9), HE (n = 7), ADF-KE (n = 5), HER (n = 4), ADF-KF (n = 4), HIR (n = 2), HAB-HF (n = 1), HF-KE (n = 1), HF-KF (n = 1) ROMs. Concerning the muscular tightness, limited ROM was observed in the HF-KE (n = 74), HAB-HF (n = 72), KF (n = 70), HAD-HF (n = 65), ADF-KF (n = 64), HE (n = 55), HIR (n = 51), HF-KF (n = 40), ADF-KE (n = 20), and HER (n = 2) (p ≤ 0.016) with a “moderate” or “high” effect size (r ≥ -0.411), except for HAB (adductors) and HF-KE (hamstrings). The individual analysis of each player detected asymmetry for HAB ( (n = 2) . The ManTo the best of our knowledge, this is the first study reporting the LEFP in youth IH players. The analysis of the ROM values has shown some negative sport-derived adaptations in these athletes, such as tightness and asymmetries that should be taken into account by coaches and physical trainers to design long-term training models for IH players. When the LEFP of youth competitive IH players was compared with the reference values of the general population (from 18 to 60 years old), a decrease in ROM was found in most hip movements  and for the knee flexors (KF). This decrease in ROM may be due to lower extensibility of the gluteus, pyramidal, fasciae latae tensor, hamstrings, quadriceps, and adductors among other joint tissues . Most ofOn the contrary, IH players displayed higher HE, ADF-KE, and HIR values than the general population. It seems that the dynamic movements performed in the push-off (HE) and gliding (HIR and ADF-KE) phases increase the extensibility of the iliopsoas, external rotator muscles, and gastrocnemius ,68. For 95), the only differences based on practical significance are those obtained for HIR and KF. These higher ROM in both movements is possibly the result of accumulated years of experience of the senior IH players (mean of 13.55 years of experience). Comparing the LEFP of youth IH players with those recently published for elite IH players , we obseInterestingly, our results differ from those described for college ice hockey players . We obseIn support of this assumption, Hogg et al.  found van = 13), HAD-HF (n = 9), HE (n = 7), ADF-KE (n = 5), HER (n = 4), ADF-KF (n = 4), HIR (n = 2), HAB-HF (n = 1), HF-KE (n = 1), HF-KF (n = 1) ROMs. The results of the present study are in accordance with those reported by Cejudo et al. [n = 8), HE (n = 6), HAD-HF (n = 6), KF (n = 4), HAB (n = 2), ADF-KE (n = 2), and HER (n = 2). A push off of both skates against the same ground conditions and the equality of turns in both directions may explain why we observed few cases of asymmetry [Etiological studies have considered asymmetry as an important risk factor for sports injuries ,81,82,83o et al. , who obssymmetry ,70.Individual analysis indicated that a large number of IH players  demonstrated limited ROM in 10 of the 11 assessed movements. Cejudo et al.  showed sThe individual analysis performed in this study to identify muscle asymmetry and tightness is a useful tool to detect athletes with an increased likelihood of sports injury ,37,87. TAn important strategy for minimizing the risk of injury is to acertain the sport’s chronic adaptations to muscle flexibility during growth and maturation in youth IH players. The ROM-SPORT battery used in this study to determine the LEFP could be used by coaches and physical trainers to identify muscular tightness and asymmetry in youth IH players. This ROM assessment would be especially useful during the period of maximal rate of growth, which may result in a growth-related decrease in muscle–tendon flexibility. ROM data can be used as a reference to design stretching exercises or to increase the stretching training load to achieve optimal ROM values for this sport, and consequently, decrease the risk of injury. This stretching training should be established at a young age (6 years) to train general motor abilities including flexibility and aid movement competency, which can both enhance performance and manage injury risk . The range of motion values that define the lower-extremity flexibility profile of youth IH players are 10.8° for HE, 26° for HAD-HF, 33.6° for ADF-KE, 38.6° for ADF-KF, 36.7° for HAB, 46° for HIR, 60.6° for HER, 65.1° for HAB-HF, 66.3° for HF-KE, 119.7° for KF, and 133.7° for HF-KF. The individual analysis of the lower-extremity flexibility profile identified limited ROM in all IH players for one or more of the analyzed movements, except for HAB. A low prevalence of asymmetries was observed .


Solenopsis invicta is an invasive ant that has quickly spread worldwide causing significant ecological and economic impacts. Its virome has begun to be characterized pertaining to potential use of viruses as natural enemies. Although the S. invicta virome is the best characterized among ants, most studies have been performed in its native range, with less information from invaded areas.Advances in sequencing and analysis tools have facilitated discovery of many new viruses from invertebrates, including ants. S. invicta, in two introduced areas, U.S and Taiwan. The data set used here was obtained from different stages  of S. invicta life cycle. Publicly available RNA sequences from GenBank’s Sequence Read Archive were downloaded and de novo assembled using CLC Genomics Workbench 20.0.1. Contigs were compared against the non-redundant protein sequences and those showing similarity to viral sequences were further analyzed.Using a metatranscriptome approach, we further identified and molecularly characterized virus sequences associated with S. invicta transcriptomes. Sequence comparisons revealed extensive divergence across ORFs and genomic regions with most of them sharing less than 40% amino acid identity with those closest homologous sequences previously characterized. The first negative-sense single-stranded RNA virus genomic sequences included in the orders Bunyavirales and Mononegavirales are reported. In addition, two positive single-strand virus genome sequences and one single strand DNA virus genome sequence were also identified. While the presence of a putative tenuivirus associated with S. invicta was previously suggested to be a contamination, here we characterized and present strong evidence that Solenopsis invicta virus 14 (SINV-14) is a tenui-like virus that has a long-term association with the ant. Furthermore, based on virus sequence abundance compared to housekeeping genes, phylogenetic relationships, and completeness of viral coding sequences, our results suggest that four of five virus sequences reported, those being SINV-14, SINV-15, SINV-16 and SINV-17, may be associated to viruses actively replicating in the ant S. invicta.We characterized five putative new viruses associated with S. invicta in introduced areas with potential to be used as biological control agents, which will require further biological characterization.The present study expands our knowledge about viral diversity associated with  Insects are the most abundant and diverse group of animals on earth . High thSolenopsis invicta, is an invasive pest causing significant ecological impact and economic loss in invaded areas [S. invicta was accidentally introduced into the southern region of the United States (U.S.) almost a century ago, becoming a serious problem [S. invicta in introduced areas [S. invicta populations may reach sizes even greater than those observed in its native range, making control difficult and even more costly [S. invicta populations in the U.S. have facilitated its dispersal across the world, contributing to repeated introduction events in several countries, such as China, Taiwan, and Australia [S. invicta infestation. Highly competitive ability, generalist feeding habits and high populations densities make S. invicta a successful invasive species causing huge disturbance in biodiversity by displacing native ants and other arthropods in introduced regions [S. invicta [The red imported fire ant, ed areas , 16. Ori problem . Since t problem . Limiteded areas –21. There costly . High deustralia . Morrisoustralia  demonstr regions . Current invicta . Low effS. invicta virome pertaining to potential use in biological control [S. invicta virome is composed of mainly positive-sense single-strand RNA (+ ssRNA) viruses in the order Picornavirales. These include eleven viruses from families Dicistroviridae, Polycipiviridae, Iflaviridae, Soliniviridae and two unclassified viruses [Totiviridae and Parvoviridae, respectively, have been characterized [S. invicta in its native range, only the species Solenopsis invicta virus 1, Solenopsis invicta virus 2, Solenopsis invicta virus 3, Solenopsis invicta virus 6 and Nylanderia fulva virus 1 have been reported in the United States [S. invicta virome, the effect of these viruses and their potential use in biological control will require additional investigation. Interactions among S. invicta and Solenopsis invicta virus 1 (SINV-1), SINV-2 and SINV-3, have been the only ones previously characterized [S. invicta colonies, with greatest potential as a biological control agent [Classical biological control has been one strategy used in an attempt to control this pest in the U.S., with viruses considered a promising resource to be used as biopesticides –26. Over control , 27–30.  viruses , 31. Addcterized , 32. Whid States , 31. Altcterized , 33, 34.ol agent , 35.S. invicta in two introduced areas, U.S and Taiwan. These investigations utilized existing publicly available RNA sequences deposited in NCBI GenBank as Sequence Read Archive (SRA) data files  virus sequences included in the orders Bunyavirales and Mononegavirales reported.In addition, two + ssRNA viruses sequences, included in the family Iflaviridae and an unassigned species, and a partial ssDNA virus sequence, were also characterized.Here using a metatranscriptome approach, we identified and molecularly characterized five new virus genome sequences associated with es Table . Five neS. invicta collected in Mississippi, U.S. [S. invicta, six libraries [−2 were downloaded and further analyzed. The final data set analyzed here consisted of 13 libraries, all from S. invicta transcriptomes. Detailed information on the samples are presented in Table Contigs corresponding to putative new virus genome sequences were initially obtained from a transcriptome project studying differential gene expression between larvae and pupae stages of pi, U.S. . To furtibraries  were dowS. invicta were first filtered by mapping them back to the S. invicta genome (GenBank accession: GCA_000188075.2) and only unmapped reads were used for de novo assembling . Multiple sequence alignments of deduced amino acid sequences were prepared using the MUSCLE option in MEGA7 . Alignmecox1), that presented high transcriptional levels, ribosomal protein L18 (rpl18) and translation elongation factor 1 (eif1-beta), both with lower transcriptional levels. Viral and internal genes abundance were calculated as percentage of viral or housekeeping reads per total number of reads in each library  and dinucleotide composition bias has been used to infer putative host-virus association and taxonomic placement for a given virus sequence , 44. To  package  in R sof package , both imS. invicta transcriptomes collected from three geographic locations in the U.S. and one location from Taiwan, representing different stages of the S. invicta life cycle , the only species within the genus Horwuvirus , suggests that they are putative new members of this genus within the family Nyamiviridae.The SINV-15 genome comprises a linear negative-sense, single strand RNA varying from 10,113 to 10,135 nt  including RdRp, helicase, and the canonical picornavirus capsid protein domain . These results indicate that SINV-16 is a new iflavirus reported from S. invicta.SINV-16 has a positive sense single-stranded RNA genome varying from 10,355 to 10,369 nt , isolated from a pool of marine crustaceans within the genus ia fulva .Parvoviridae , predicted to encode three ORFs on the positive strand, with typical organization of viruses within the family dae Fig. F. The ORrus Fig. D. While cox1, rpl18 and eif1-beta  will be challenging with invasive ant systems.S. invicta. Curiously, while a great diversity of positive and negative sense viruses have been reported from ants [S. invicta virome has been composed mainly of   ssRNA viruses in the order Picornavirales, with no negative sense RNA viruses previously reported. One factor that could be responsible for this bias is the selection of polyadenylated RNA for library preparation in previous studies [Bunyavirales [Mononegavirales associated with S. invicta. Although we did not perform any amplification step of viruses genomes characterized here, the presence of untruncated ORFs carrying intact functional domains , associated with a pool of horseflies (family Tabanidae), and proposed that it may represent a transitional form between plant-infecting virus and arthropod-specific viruses. In addition, a partial genome sequence of another tenui-like, FCTenV1, has been reported associated to Culex annulirostris [S. invicta transcriptome (Order Hymenoptera). Interestingly, in contrast with WhHV, which lacks an ambisense coding strategy, the SINV-14 genome sequence exactly mirrors the genomic structure of typical plant tenuivirus, predicted to encode proteins using ambisense strategy, and also has the conserved sequences at the ends of all four segments identical to those found in tenuivirus genomes  related to plant tenuiviruses. Further identity analysis showed that sequence is 99.8% identical to SINV14 RNA4. They suggested that the sequence would be likely a contamination due to the ant diet feeding either plant or infected insect. Tenuiviruses are typical plant viruses that replicate in the insect vector [S. invicta. Furthermore, the highest virus abundance was found in a sample prepared from a dissected ant brain, which rules out the possibility of contamination due to feeding on plants or association with insect vectors infected with a tenuivirus. The Maize stripe virus (MSpV), a typical plant tenuivirus, was detected in the brain of its vector Peregrinus maidis, providing evidence of replication of tenuiviruses in this tissue [Valles et al.  using ant vector , 54, ands tissue . Tenuivis tissue , 57; this tissue . Althougs tissue , our resS. invicta, suggest an intimate and long-term codivergence process between virus-host.While SINV-14 is evolutionarily closely related to insect tenui-like viruses and plant tenuivirus, the synonymous codon usage and dinucleotide analyses demonstrate a distinct compositional bias compared to FCTenV1 and WhHV, and all other viruses analyzed here, indicating that the virus may be actively replicating in ants rather than plants and other insects. The active replication may have driven the virus genome to distinct compositional bias at the nucleotide level, while maintaining protein integrity at the amino acid level and close relationship with those of other tenui-like viruses, as observed through phylogenetic analysis. The fact that most sequences examined here are probably from viruses that replicate in plant or vertebrate hosts and also in the insect vector could be the reason driving such difference between SINV-14, most likely associated only with ant, compared to other viruses. Furthermore, phylogenetic congruence across different segments that mirror the genetic structure of invasive We presented strong evidence that the sequences from SINV-14 may be from a virus that has been associated long-term and may actively replicate in ants. However, the possibility of this virus, and other tenui-like viruses, replicating in plants is unknown. The presence of the protein carrying an NS4 domain, that is related to cell-to-cell and long-distance movement in plants  in insecS. invicta. Our results suggest fluid shifts in the virome of this invasive species. Further research describing this virome in native and invasive regions and ecosystems could provide insight on virus evolution and invasion mechanics.Altogether, based on virus abundance compared to housekeeping genes, abundant viruses with viral reads higher than 0.01%, phylogenetic relationship, complete viral coding sequence regions recovered, and compositional bias for SINV-14, our results suggest that four out five viruses reported here, those being SINV-14, SINV-15, SINV-16 and SINV-17 are truly replicating in S. invicta in introduced areas. In addition to revealing new virus-host interactions, contributing to better understanding of viral evolutionary history and emergence, the discovery of new viruses expands the range of agents with potential to be used in biocontrol programs, which will require further biological characterization. By understanding these interactions, we may be better equipped to cope with ongoing global changes and introductions of invasive organisms and their associated viruses.The present study expands our knowledge about viral diversity associated with Additional file 1.  Table S1: Descriptive statistics of fire ant transcriptome datasets analyzed in this study.Additional file 2.  Table S2: Open reading frames (ORFs) characterization from putative new viruses based on ORFinder followed by BLASTp analyses, as implemented at National Center for Biotechnology Information Search database (NCBI).Additional file 3.  Table S3: Viral conserved domains detected in ORFs from putative new viruses based on Conserved Domains Database (CDD).Additional file 4.  Table S4: Data set used in the compositional analyses.Additional file 5. Figure S1: Neighbor-Joining phylogenetic tree (1000 bootstrap replications) showing the clustering of the SINV-14 segments. Isolates are highlighted according to sampled place as indicated at the bottom of the figure.Additional file 6.  Figure S2: Alignment of non-structural protein 4 (NS4) amino acid sequence of SINV-14 with those from typical plant tenuiviruses. Positions with single conserved amino acid are highlighted in black, groups of similar properties are highlighted in light gray and positions in SINV-14 that shares the same amino acid with at least one plant tenuivirus sequence, are highlighted in dark gray. Sites containing residues essentials for cell-to-cell movement and long-distance movement are indicated by asterisk and filled circle, respectively. These sites were mapped using Maize stripe virus (MSpV) as a model (59). Matrix of pairwise identity of NS4 amino acid sequence are shown at the bottom of the figure. Multiple sequence alignment and pairwise identity were generated in Clustal O v. 1.2.4. European wheat striate mosaic virus (EWSMV), MN044345; Rice hoja blanca virus (RHBV), MG566077; Echinochloa hoja blanca virus (EHBV), L48441; Rice stripe virus (RSV), D10979; Maize stripe virus (MSpV), L13438.


A simple reordering of the reaction sequence allowed the improved synthesis of EIDD‐2801, an antiviral drug with promising activity against the SARS‐CoV‐2 virus, starting from uridine. Compared to the original route, the yield was enhanced from 17 % to 61 %, and fewer isolation/purification steps were needed. In addition, a continuous flow procedure for the final acetonide deprotection was developed, which proved to be favorable toward selectivity and reproducibility. John Wiley & Sons, Ltd.An improved protocol was developed for the synthesis of EIDD‐2801, which is currently in Phase II clinical trials for the treatment of COVID‐19. The process employs uridine as starting material and proceeds through triazolation, one‐pot acetonide protection/esterification and telescoped hydroxyamination/continuous flow acetonide deprotection. In view of the severity of the COVID‐19 pandemic, there is a continuous quest for new drugs for the treatment of this disease. EIDD‐2801, a NHC (β‐d‐N4‐hydroxycytidine) prodrug, has shown potent activity against multiple CoVs in animal studies and is currently in Phase II clinical trials on symptomatic patients with COVID‐19. This antiviral candidate is structurally similar to remdesivir, but blocks RNA polymerase differently, which possibly makes them complimentary. Since EIDD‐2801 can be taken orally in the form of a pill, the potential treatment against COVID‐19 would be made more accessible compared to remdesivir that currently has to be administered intravenously.The current medical crisis caused by the SARS‐CoV‐2 virus, has impacted people's lives and economy worldwide, with presently more than 35 million confirmed cases.1 In v This was the only reported synthesis in the open literature until recently, when routes toward EIDD‐2801 were reported that replaced uridine for cytidine. In the original route, first an acetonide protection followed by selective esterification of the 5'‐hydroxy group is performed in a one‐pot fashion. Then, the molecule is activated by introduction of the 1,2,4‐triazole moiety which is further displaced by hydroxylamine. After removal of the acetonide protecting group, EIDD‐2801 is obtained via a complex crystallization procedure. As no isolated yield for the final deprotection step has been disclosed in the patent, an overall yield (17 %) can only be stated for the synthesis of protected EIDD‐2801. The main disadvantage of the reported route is the low yield (29 %) in the triazole coupling step.EIDD‐2801 was developed by Emory University researchers, and the disclosed route consists of a five‐step synthesis starting from uridine , because of the associated health risks. Unfortunately, less triazole also leads to lower isolated yields of 1 . Compared to the original route, we were able to increase the yield for the triazolation step from 29 % to 88 % by introducing the triazole moiety as first step. Importantly, also column chromatographic work‐up could be avoided.Following the original procedure of Reese and co‐workers, which involves a one‐pot TMS protection of uridine and phosphorus oxychloride‐promoted triazole coupling using Et as base,81 wa% Scheme . Product2SO4 in acetone), no conversion of 1 to the desired acetonide 2 was observed. Higher concentrations (0.5 equiv.) of H2SO4 lead to hydrolysis of the triazole moiety. Addition of 2 equiv. of 2,2‐dimethoxypropane (DMP) did not significantly improve the reaction. However, when exchanging acetone for MeCN as solvent in combination with the use of DMP, a 73 % conversion to 2 was achieved. Further optimization revealed, that the amount of H2SO4 could be reduced to 5 mol‐% ,5 no c2. The excess of anhydride was necessary, because it was quenched by the 2 equiv. of MeOH that were released from DMP. Therefore, after stirring of 1 with DMP and H2SO4 in MeCN for 30 min at room temperature, a stepwise azeotropic distillation was performed to remove MeOH. Applying this protocol resulted in only 1.1 equiv. of isobutyric anhydride being necessary for full conversion of 2 (Scheme 3 was obtained in quantitative yield and ≥ 99 % HPLC purity after extractive work‐up in this one‐pot procedure.Upon attempting the acetonide formation and esterification in a one‐pot fashion similar to the patented route (see Scheme 1a3 can be readily converted to hydroxylamine 4 by stirring in iPrOH at room temperature with 1.5 equiv. of hydroxylamine (50 wt.‐% in H2O) for 20 min  revealed that H2SO4 in combination with iPrOH as solvent furnished the highest conversion (80 %) to EIDD‐2801 after heating at 60 °C for 30 min. The main side reaction was found to be the ester hydrolysis yielding N‐hydroxycytidine 5  was employed in the hydroxyamination and acetonide deprotection, we envisioned that these two steps could be combined to a one‐pot procedure. A similar process has been recently demonstrated in an alternative route toward EIDD‐2801. During batch optimization studies (see Table S3) we experienced reproducibility issues that were mainly related to the exothermic nature upon H2SO4 addition in the deprotection step. Therefore, we decided to transfer the batch process to continuous flow, where better mixing and temperature control allows a superior control of exotherms and in return would provide reproducible results.Since the same solvent system . During the deprotection optimization studies, ester hydrolysis and hydroxylamine–hydroxyl exchange occurred as most prominent side reactions yielding compounds 5 and 6 . Because of precipitation issues in iPrOH, MeOH was selected as solvent. The highest conversion to EIDD‐2801 (79 %) was obtained at only 5 min of residence time using 2.75 equiv. of H2SO4 at 100 °C, while 5 (11 %) and 6 (8 %) were at a minimum. Importantly, the flow process under optimum conditions not only permitted higher yields, but it also enabled a significant chemical intensification and a proper reproducibility as compared to our initial batch attempts. By employing these flow conditions for scale‐out (16 min), pure EIDD‐2801 was obtained in 69 % isolated yield (307 mg) after chromatographic purification.A simple flow set‐up was therefore established using a heated reaction coil together with individual streams for introducing solutions of the substrate and the acid reagent, which were combined in a Y‐shaped mixer Figure A. TriazoIn conclusion, an improved protocol was developed to access EIDD‐2801 from uridine as starting material Scheme . By straSupporting InformationClick here for additional data file.


Malassezia species represent the most common fungal inhabitant of the mammalian skin microbiome and are natural skin commensal flora. However, these fungi are also associated with a variety of clinical skin disorders. Recent studies have reported associations of Malassezia with Crohn’s disease and pancreatic cancer, further implicating this fungal genus in inflammatory and neoplastic disease states. Because M. sympodialis has lost genes involved in RNA interference (RNAi), we hypothesized Malassezia could harbor dsRNA mycoviruses. Indeed, we identified a novel mycovirus of the totivirus family in several Malassezia species and characterized the MsMV1 mycovirus of M. sympodialis. We found conditions that lead to curing of the virus and analyzed isogenic virus-infected/virus-cured strains to determine MsMV1 genetic and pathogenic impacts. MsMV1 induces a strong overexpression of transcription factors and ribosomal genes, while downregulating cellular metabolism. Moreover, MsMV1 induced a significantly higher level of beta interferon expression in cultured macrophages. This study sheds light on the mechanisms of pathogenicity of Malassezia, focusing on a previously unidentified novel mycovirus. Saccharomyces and Ustilago strains exhibit a killer phenotype conferring a growth advantage over uninfected strains and other competing yeast species, whereas hypovirus-infected Cryphonectria parasitica displays defects in growth, sporulation, and virulence. In this study, we identify a double-stranded RNA (dsRNA) mycovirus in five Malassezia species. Sequence analysis reveals it to be a totivirus with two dsRNA segments: a larger 4.5-kb segment with genes encoding components for viral replication and maintenance, and a smaller 1.4-kb segment encoding a novel protein. Furthermore, transcriptome sequencing (RNA-seq) of virus-infected versus virus-cured Malassezia sympodialis revealed an upregulation of dozens of ribosomal components in the cell, suggesting the virus modifies the transcriptional and translational landscapes of the cell. Given that Malassezia is the most abundant fungus on human skin, we assessed the impact of the mycovirus in a murine epicutaneous infection model. Although infection with virus-infected strains was not associated with an increased inflammatory response, we did observe enhanced skin colonization in one of two virus-infected M. sympodialis strains. Noteworthy, beta interferon expression was significantly upregulated in bone marrow-derived macrophages when challenged with virus-infected, compared to virus-cured, M. sympodialis, suggesting that the presence of the virus can induce an immunological response. Although many recent studies have illuminated how widespread mycoviruses are, there are relatively few in-depth studies about their impact on disease caused by the host fungus. We describe here a novel mycovirus in Malassezia and its possible implications in pathogenicity.Mycoviruses infect fungi, and while most persist asymptomatically, there are examples of mycoviruses having both beneficial and detrimental effects on their host. Virus-infected  Viruses are widely distributed in nature and are found in a variety of hosts, ranging from single-celled organisms to plants and mammals. The presence of mycoviruses, viruses that infect fungi, can be benign but is also associated with toxin secretion in yeast, hypovirulence in pathogenic plant fungi, and hypervirulence in dimorphic fungi –5. Mycov–8–Saccharomyces cerevisiae. This toxin-producing dsRNA virus was characterized in the 1970s , impairs growth of the fungus (Cryphonectria parasiticahypovirus (CHV). Five different viral segments isolated from C. parasitica have been shown to confer a high level of hypovirulence, although the level of impact on fungal growth observed in the infected fungal strain appears to be virus strain dependent , argonaute (AGO), and RNA-dependent RNA polymerase (RDP) -induced RNA silencing, and when the virus was cleared from ascospores, IRT silencing was restored  that suppresses hairpin RNA-triggered silencing in the fungus  is an important pathway conserved in many organisms and serves to regulate gene expression, suppress transposon movement, and maintain genome integrity. Additionally, a functional RNAi pathway has been shown to suppress mycovirus proliferation , 21. These (RDP) . It was se (RDP) . To thisse (RDP) . In Asperestored . Other ee fungus . In R. nof dsRNA . Most re mycelia . ExampleMalassezia is a monophyletic group of lipophilic yeasts that colonize sebaceous skin sites and represent more than 90% of the skin mycobiome. Despite being a commensal yeast, Malassezia is associated with a number of common skin disorders, such as seborrheic dermatitis, atopic eczema, and pityriasis versicolor, and also systemic infections  and pancreatic cancer , and proteomics -binding capabilities have been identified in extracts of Malassezia and have been named MalaS allergens. Ten allergens have been identified in M. sympodialis, and three in M. furfur. Eight of these allergens are conserved proteins that share high similarity with the corresponding mammalian homolog, suggesting that the induction of autoreactive T cells by Malassezia allergens could play a role in sustained inflammation  in cultured macrophages was associated with all virus-infected strains tested. These findings reveal a novel mycovirus harbored by Malassezia with possible implications for interactions with the host in both the commensal and disease states. pathway . Given tMalassezia species have lost the genes required for a functional RNAi pathway , one of three M. pachydermatis isolates (33.3%), and no M. furfur isolates. dsRNA species were also found in one of two M. globosa isolates, one M. obtusa isolate, and one M. yamatoensis isolate  and M. furfur (B) strains was analyzed by electrophoresis on a 0.8% agarose gel with 0.5 μg/ml ethidium bromide. Download FIG S1, TIF file, 0.5 MB.Copyright © 2020 Clancey et al.2020Clancey et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the M. sympodialis as a representative species of the Malassezia genus. To unveil the biological and physiological roles of the putative Malassezia mycovirus, a crucial first step was to define conditions that allow elimination  of the mycovirus. In other fungi, mycoviruses can be cured by exposure to stressful conditions or certain drugs. In S. cerevisiae, exposure to the fungicide cycloheximide cures killer cells of the killer phenotype  medium and subjected to various stresses. We found that six passages on mDixon medium with no additives at high temperature (37°C) resulted in a complete loss of both dsRNA segments in four M. sympodialis virus-infected strains . In parallel with the cDNA synthesis, cloning, and sequencing, the congenic virus-infected and virus-cured strains KS012 and SEC494 of M. sympodialis, respectively, were subjected to rRNA-depleted RNA sequencing. The rationale of the RNA-seq analysis was 2-fold: first, allowing a more facile identification of the entire viral genome, and second, elucidating possible transcriptomic changes evoked by the mycovirus in the host cells.To determine if the dsRNA moieties isolated from M. sympodialis virus-infected and virus-cured isolates were assembled with Trinity. The resulting transcripts were searched using the sequences previously obtained through TA cloning as a query, and two perfect hits  were obtained following a BLASTn search against the virus-infected strain KS012. Both hits are part of two larger independent contigs . Combined with nucleic acid analysis based on agarose-ethidium bromide-stained gels, this sequencing further supports that the dsRNA had been completely eliminated in the virus-cured strain.For the identification of the viral genome, adapter-free RNA-seq reads derived from 10.1128/mBio.01534-20.1TEXT S1M. sympodialis MsMV1. Note that the designations “TRINITY_DN8321_c1_g1_i1 len = 4613 path=[4591:0-4612] ” and “TRINITY_DN4986_c1_g1_i1 len = 4601 path=[4579:0-4600] ” are automatically generated by the software Trinity. We arbitrary used the designations DN8321 and DN4986 to indicate the contigs that represent the M. sympodialis MsMV1 mycovirus genome, as identified by BLAST analyses. Considering “TRINITY_DN8321_c1_g1_i1 len = 4613 path=[4591:0-4612] ,” according to the software instructions, TRINITY_DN8321_c1_g1_i1 indicates Trinity read cluster ‘TRINITY_DN8321_c1’, gene ‘g1’, and isoform ‘i1’. ‘Len’ indicates the length of the cluster (contig) in base pairs, and path information indicates the path traversed in the Trinity compacted de Bruijn graph to construct that transcript. Download Text S1, DOCX file, 0.02 MB.Identification of the large dsRNA segment of the Copyright © 2020 Clancey et al.2020Clancey et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the M. sympodialis KS012 genomic DNA, suggesting that the viral sequence is not integrated in the genome but is rather cytoplasmic. This finding is corroborated by in silico BLAST searches that failed to detect either large- or small-segment viral sequences in the de novo genome assembly of M. sympodialis KS012 (described below).Moreover, PCR analyses with primers designed to specific regions of the large and small dsRNA segments failed to produce amplicons on RDP) from Scheffersomyces segobiensis virus L (SsV L) with 52.69% homology. A BLASTp search of the ORF produced by a frame 2 translation identified the capsid protein from the same S. segobiensis virus, in this case with 53.87% homology , distinguished by a −1 ribosomal frameshift, a common feature of totiviruses  33). A . A 33). homology . Phylogee family . Moreoved DN4986 . These rM. sympodialis strains KS012 and SEC494, respectively. Two highly significant hits  were obtained only against the genome of the virus-infected strain KS012. The two hits are part of two independent contigs  hits to the 3′ region of uncharacterized and hypothetical proteins in Sporisorium reilianum and Streptomyces, respectively; thus, no significant hits were obtained from NCBI for the small segment.Similarly, the 374-bp query sequence from the smaller dsRNA segment identified via TA cloning was subjected to a BLASTn search against the assembled transcriptomes of the virus-infected and virus-cured 10.1128/mBio.01534-20.2TEXT S2M. sympodialis MsMV1. For the contig designations, see explanation in the legend for Text S1. Download Text S2, DOCX file, 0.02 MB.Identification of the small dsRNA segment of the Copyright © 2020 Clancey et al.2020Clancey et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the M. sympodialis encodes a previously unidentified, novel protein of unknown function that is likely secreted from the cell. Given the data supporting that these dsRNA segments represent a dsRNA virus in M. sympodialis, we termed this virus MsMV1 .To infer the function of this virally encoded protein, the predicted amino acid sequence was subjected to domain prediction using InterProScan. A 305-amino-acid noncytoplasmic domain was identified at the C-terminal end of the protein, and a short 19-amino-acid signal peptide is predicted at the N terminus . Signal M. sympodialis genome ATCC 42132 using HiSat2. Transcript abundance was estimated through StringTie, and differentially expressed genes with a false-discovery rate (FDR) <0.05 and a log2 fold change ±1 were identified with EdgeR  and 83 genes being downregulated (log2 fold change between −1 and −4.6)  membrane protein, MSYG_1988, a gene whose predicted product is involved in the movement of substances across the ER membrane and/or protein synthesis. The second most downregulated gene is MSYG_0220 (log2 fold change −4.1), which is an ortholog of S. cerevisiaeCDC39. Cdc39 is a component of the Ccr4-NotI complex that, in addition to its role as a global transcriptional regulator, regulates mRNA decay. These data suggest that elimination of aberrant mRNA transcripts may be inhibited in the virus-infected strain. Interestingly, we identified a gene (MSYG_3324) with the lowest log2 fold change (−11.88) that did not pass the statistically significant threshold of FDR <0.05. The gene MSYG_3324 encodes a hypothetical protein that has a guanine nucleotide exchange factor domain, leucine-rich repeats of the RNase inhibitor (RI)-like subfamily, and an N-terminal herpes BLLF1 superfamily domain, which consists of the BLLF1 viral late glycoprotein that is the most abundantly expressed glycoprotein in the viral envelope of the herpesviruses. Strikingly, BLLF1 represents a major antigen responsible for production of neutralizing viral antibodies in vivo  . Among tnd −4.6) . The remNA genes . The chaThe three technical replicates of each strain produced highly similar expression profiles, which are distinct from one another . The larM. sympodialis cured strain SEC494, comparing it to the virus-infected strain KS012. Only two nucleotide changes unique to the virus-cured isolate SEC494 were identified. These include mutations in the coding regions of KAE1, an ATPase, and CRM1, a karyopherin responsible for the transport of molecules between the cytoplasm and the nucleus  calling of the  nucleus . As thes nucleus ; Text S3CC 42132  to C, anCC 42132 .10.1128/mBio.01534-20.3TEXT S3KAE1 and CRM1 in the M. sympodialis strain SEC494. Download Text S3, DOCX file, 0.3 MB.Screenshot of the variants presents in the coding regions of the genes Copyright © 2020 Clancey et al.2020Clancey et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mBio.01534-20.5FIG S2M. sympodialis virus-infected and virus-cured strains compared to the reference strain ATCC 42132. (A to C) Dot plot comparisons of the M. sympodialis strain ATCC 42132 with the virus-infected M. sympodialis KS012 (A) and the virus-cured SEC494 (B), and of the M. sympodialis isogenic strains KS012 and SEC494. (C) Read coverage of the M. sympodialis strains KS012 and virus-cured SEC494 compared to the haploid reference strain ATCC 42132. Download FIG S2, TIF file, 0.6 MB.Genomic comparisons of Copyright © 2020 Clancey et al.2020Clancey et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mBio.01534-20.8TABLE S1M. sympodialis virus-cured SEC494 compared to the virus-infected KS012. Download Table S1, XLSX file, 0.01 MB.Variants present only in the Copyright © 2020 Clancey et al.2020Clancey et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the Malassezia to the mouse dorsal ear following mild tape stripping . *, P < 0.05; ****, P < 0.0001. Black bars below the x axis labels group virus-infected/virus-cured strain pairs. “+” indicates virus infected; “−” indicates virus cured. Download FIG S3, TIF file, 0.4 MB.Select virus-infected strains exhibit increased ability to colonize skin in a murine ear skin infection model. (A) Fungal load, (B) ear thickening, and (C) cytokine expression in the skin of mice that were epicutaneously infected with the indicated virus-infected and virus-cured strain pairs for 4 days, (D) 2 days, or (E) 7 days. The mean + SEM for each group are indicated. Data in panels A to C are pooled from two independent experiments with Copyright © 2020 Clancey et al.2020Clancey et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the Malassezia primarily interacts with keratinocytes and tissue-resident mononuclear phagocytes, including macrophages. Therefore, we assessed the interaction of M. sympodialis virus-infected and virus-cured strains with macrophages. Bone marrow-derived macrophages (BMDMs) are mature, differentiated macrophage that are suitable for host-pathogen interaction studies  in the parasite is associated with parasite dissemination and increased disease severity  BMDMs were infected with the indicated virus-infected and virus-cured strains at an MOI of 1 and analyzed for viability and CD86 surface expression by flow cytometry at 16 h postinfection or infected at an MOI of 5 and analyzed for TNF expression by RT-qPCR at 24 h postinfection. Each bar represents the mean + SEM for four samples, each being the pool of two separate wells. Data are pooled from two independent experiments. The dotted line indicates the expression levels under unstimulated conditions. (B) Flow cytometry gating strategy used to assess BMDM viability (top row) and CD86 expression (bottom row). (C) BMDM viability, CD86 surface expression, and TNF expression of wild-type and −/−Tlr3 BMDMs infected with the indicated virus-infected and virus-cured strains and analyzed as in panel A. Data are from one out of two representative experiments with each value being the pool of two wells. Statistics were calculated using an unpaired Student t test. *, P < 0.05. “+” superscripts indicate virus-infected strains; “−” superscripts indicate virus cured. Download FIG S4, TIF file, 0.7 MB.Macrophage viability, CD86 expression, and TNF expression are not affected by the presence of MsMV1 in Copyright © 2020 Clancey et al.2020Clancey et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the C. parasitica carries the CHV1 virus that causes reduced growth and pathogenicity of the fungus. Therefore, trees infected with virus-infected strains of C. parasitica display smaller cankers than trees infected with virus-free strains . Ac. AcMalase family . Using tM. sympodialis of MsMV1  or to an antimicrobial agent (biphenyl) cures of MsMV1 . These athe cell , and if Sclerotinia sclerotiorum, which harbors the SsHV2-L hypovirus, where rRNA metabolic processes and cellular component biogenesis were the classes of the most upregulated genes in virus-infected strains , is an important pathway in living organisms. NO is an important signaling molecule in bacteria and fungi. Furthermore, NO production is positively correlated with aflatoxin production in pression . Flavohebacteria , 49. A lC. parasitica CHV1-infected cells exhibit an upregulation of silencing-related genes in an effort to reduce the viral burden or clear the virus. Alternatively, a different phenomenon occurs in T. marneffei TmPV1-infected cells where the virus can subsequently dampen expression of silencing-related genes and blunt the cellular RNAi defense mechanism . As described earlier, during the virus replication cycle, a ssRNA intermediate is formed prior to virion assembly (MSYG_03961 (CPR3) in virus-infected M. sympodialis cells. CPR3 is a gene that encodes a mitochondrial cyclophilin .Due to the role of IFN-β in the stimulation of antiviral and antiproliferative products , it is pycovirus , 43. In  strains . We posiMalassezia. Although the target of the putative satellite toxin is unknown, it induces an immune response in macrophages. Coincubation of the virus-infected M. sympodialis with virus-cured M. sympodialis and bacterial species  failed to reveal antifungal or antibacterial properties of the virus-infected M. sympodialis strains. Determining if changes in the expression of metabolic genes in the virus-infected strains result in resistance or susceptibility to certain environmental conditions would be interesting to investigate in the future. More research will be required to determine if this possible mycovirus toxin plays a role in common skin disorders caused by Malassezia by targeting the host, bacteria, or fungi of the skin microbiome.In conclusion, we report for the first time evidence of mycoviruses infecting the fungal genus Malassezia sympodialis strains were stored at −80°C in 25% glycerol stocks and maintained on modified Dixon’s (mDixon) medium at 30°C. mDixon medium is composed of 3.6% malt extract, 1% mycological peptone, 1% desiccated ox bile, 1% Tween 60, 0.4% glycerol, and 2% agar for solid medium. Strains used in dsRNA gel images are listed in M. sympodialis liquid cultures as follows. Five-milliliter liquid mDixon cultures were grown at 30°C until saturation, approximately 3 to 4 days. Cultures were pelleted at 3,000 rpm in a tabletop centrifuge for 3 min and washed with 5 ml distilled water (dH2O), and pellets were frozen at −80°C until lyophilization overnight. Following lyophilization, pellets were broken up with 3-mm glass beads and the RNA extraction was performed using TRIzol  according to the manufacturer’s instructions. RNA was resuspended in 50 μl diethyl pyrocarbonate (DEPC) water. RNA was treated with Turbo DNase  according to manufacturer’s instructions and resuspended in 50 μl DEPC water. ssRNA precipitation/dsRNA enrichment was done overnight at −20°C by combining 25 μl of DNase-treated RNA, with 28 μl 8 M LiCl  and 27 μl DEPC water for a final volume of 80 μl. The mixture was centrifuged at 4°C for 10 min, and the supernatant was recovered and transferred to a fresh Eppendorf tube (the pellet can be saved and resuspended in 25 μl DEPC water as an ssRNA control). To the supernatant, 7.5 μl 3 M NaCl  and 187.5 μl 100% ethanol  were added and placed at −20°C for at least 1 h. dsRNA was pelleted at 4°C for 15 min and resuspended in 25 μl DEPC water. To visualize dsRNA and ssRNA bands, 10 μl was electrophoresed on an 0.8% agarose gel stained with 5 μg/ml of ethidium bromide.Total RNA was extracted from M. sympodialis strains KS012, KS013, KS016, and KS017 as described above. Large and small dsRNA segments were excised from the agarose gel and purified via gel extraction using the QIAquick gel extraction kit . 3′ adapter sequences from the Illumina TruSeq small RNA library preparation kit  were ligated onto the dsRNA species by mixing together 1 μl of adapter with 1 μg dsRNA in a 6-μl reaction mixture and heated at 70°C for 2 min. Following heating, tubes were immediately placed on ice. To the adapter reaction mixture, 2 μl of HML ligation buffer, 1 μl RNasin, and 1 μl T4 RNA ligase 2 (truncated) were added and mixed by pipetting. The solution was incubated for 1 h at 28°C.Large and small dsRNA segments were purified from E. coli with One Shot TOP10 chemically competent E. coli . Plasmids were extracted from purified bacterial colonies and subjected to Sanger sequencing (Genewiz) using universal M13F and M13R primers.cDNA synthesis was achieved by combining 5 μl of adapter-ligated dsRNA with 1 μl RT primer and heated at 70°C for 2 min and then immediately placed on ice. Next, the adapter-ligated/RT primer mixture was used as the template with the AffinityScript cDNA synthesis kit  with the following reaction conditions: 25°C for 5 min, 55°C for 15 min, 95°C for 5 min. cDNA was PCR amplified using the RT primer, cloned into the pCR-Blunt vector using the Zero Blunt PCR cloning kit , and transformed into 2O, and resuspended in 1 ml dH2O. The 1-ml cell suspension was transferred to a bead beating tube with acid-washed 425- to 600-μm beads. Cells were pelleted with beads in a microcentrifuge at 6,000 rpm for 2 min. The supernatant was removed, and the cell pellet was flash frozen in liquid nitrogen. Frozen bead/cell pellets were stored at −80°C until RNA extraction. To prepare RNA, bead/cell pellets were thawed on ice and 1 ml TRIzol  was added. Cells were subjected to bead beating 10 times in 1-min “on,” 1-min “rest” cycles. Following bead beating, 200 μl of chloroform was added and the RNA extraction continued as described in the manufacturer’s instructions. Final RNA pellets were treated with Turbo DNase  according to manufacturer’s instructions, and RNA was resuspended in 50 μl of nuclease-free water . Illumina 150-bp paired-end (PE) libraries were prepared using the TruSeq stranded total RNA-seq kit in combination with the Illumina RiboZero yeast kit. Library preparation and RNA sequencing were performed at the Duke University Center for Genomic and Computation Biology.Five-milliliter liquid mDixon cultures were grown at 30°C for 3 days. Cells were pelleted at 3,000 rpm in a tabletop centrifuge for 3 min, washed with 5 ml dHIn silico functional characterization of the mycovirus sequences was performed through BLAST analyses and InterProScan (https://www.ebi.ac.uk/interpro/search/sequence/). For RNA-seq analysis, the most recent M. sympodialis strain ATCC 42132 genome assembly and annotation were used as a reference  annotation, and InterProScan (http://www.heatmapper.ca/expression/), and gene network interaction were determined using STRING (https://string-db.org/cgi/input.pl) with the S. cerevisiae protein set as reference.After sequencing, Illumina raw reads were trimmed with Trimmomatic to remove Illumina adapters . For theeference . Illuminanalysis . Read con Galaxy . DEGs werProScan . The heaM. sympodialis KS012 and SEC494 were grown at 30°C for 3 days. Cells were pelleted at 3,000 rpm in a tabletop centrifuge for 3 min and washed with 10 ml dH2O, and cell pellets were frozen at −80°C. Cell pellets were lyophilized overnight, pulverized using 3-mm glass beads, and subjected to genomic DNA extraction using the CTAB method .Illumina reads of the sing BWA , and thesing BWA . Bam filThe MsMV1 Rdp1 was aligned with representative viral Rdp1 sequences found on NCBI using the Mega7 software. Phylogenies were built using 100 bootstrap values using the maximum likelihood method based on the Tamura-Nei model . ProteinMalassezia strains were grown for 3 to 4 days at 30°C, 180 rpm, in liquid mDixon medium. Cells were washed in phosphate-buffered saline (PBS) and suspended in native olive oil at a density of 20 ODA600 /ml. A 100-μl suspension (corresponding to 2 ODA600) of yeast cells was applied topically onto the dorsal ear skin that was previously disrupted by mild tape stripping while mice were anaesthetized. Ear thickness was monitored before and after infection using an Oditest S0247 0- to 5-mm measurement device (Kroeplin), and the increase was calculated by subtracting the values measured prior to the experiments from those measured at the endpoint. For each animal, the mean of the ear thickness from both ears was calculated. For determining the fungal loads in the skin, tissue was transferred in water supplemented with 0.05% Nonidet P-40 (AxonLab), homogenized and plated on modified Dixon agar, and incubated at 30°C for 3 to 4 days.All mouse experiments in this study were conducted in strict accordance with the guidelines of the Swiss Animals Protection Law and were performed under the protocols approved by the Veterinary office of the Canton of Zurich, Switzerland (license number ZH168/2018). All efforts were made to minimize suffering and ensure the highest ethical and humane standards according to the 3R principles. Wild-type (WT) C57BL/6J mice were purchased by Janvier Elevage and maintained at the Laboratory Animal Science Center of the University of Zurich, Zurich, Switzerland. Mice were used at 6 to 12 weeks in sex- and age-matched groups. Epicutaneous infection of the mouse ear skin was performed as described previously , 64. Bri−/−Tlr3  and 106 cells/1 ml in 24-well plates . Cells were rested overnight and infected the next day with Malassezia at the indicated multiplicity of infection (MOI) for 16 h  or for 24 h . Poly(I:C) (50 μg/ml) was included as a control.Macrophages were generated from WT C57BL/6J and Tlr3−/−  (Swiss I3. LIVE/DEAD Near IR stain (Life Technologies) was used for exclusion of dead cells. Cells were acquired on a FACS Cytoflex (Beckman Coulter), and the data were analyzed with FlowJo software (FlowJo LLC). The gating of the flow cytometric data was performed according to the “Guidelines for the use of flow cytometry and cell sorting in immunological studies” (Macrophages were gently detached by using cell scrapers and stained with antibodies directed against CD11b (clone M1/70), F4/80 (clone BM8), CD86 (clone GL-1), and major histocompatibility complex class II (MHC-II) (clone M5/114.15.2) in PBS supplemented with 1% FCS, 5 mM EDTA, and 0.02% NaNstudies” , includiIl17a, forward 5′-GCTCCAGAAGGCCCTCAGA-3′, reverse 5′-AGCTTTCCCTCCGCATTGA-3′  of each gene was determined after normalization to Actb transcript levels.Isolation of total RNA from ear skin and from BMDM cultures was carried out according to standard protocols using TRI reagent (Sigma-Aldrich). cDNA was generated by RevertAid reverse transcriptase (ThermoFisher). Quantitative PCR was performed using SYBR green (Roche) and a QuantStudio 7 Flex (Life Technologies) instrument. The primers used for qPCR were as follows: ATTGA-3′ ; Tnf, foAACCC-3′ ; Ifnb, fCTTGG-3′ ; Actb, fM. sympodialis virus-infected strain KS012 and M. sympodialis virus-cured strain SEC494 are available in the NCBI SRA database (BioProject PRJNA593722). The large and small dsRNA fragments of the mycovirus genome have been deposited in GenBank under accession numbers MN812428 and MN831678, respectively.Both sequencing reads for


The study investigated the economic burden of vaso-occlusive crisis (VOC) among sickle cell disease (SCD) patients, through assessment of overall utilization and costs and costs per VOC episode .Using the Medicaid Analytic Extracts database, the first SCD-related diagnosis claim (index claim) between June 1, 2009–December 31, 2012 was identified among eligible adults. Patients were required to have continuous medical and pharmacy benefits for 6 months pre- and 12 months post-index. Discrete VOC claims identified within a 3-day gap were combined as a single VOC episode. Annual all-cause and SCD-related medical resources and costs were identified and stratified by number of VOC episodes during the 1-year follow-up period. Health care costs per VOC episode were also examined, stratified by care setting.Enrollees included 8521 eligible patients with a mean age of 32.88 years (SD=12.21). Of these, 66.5% had a Charlson Comorbidity index (CCI) score of 0 (no comorbidities) and 67.3% were female. The average total medical costs were US$34 136 (median=US$12 691) annually, and SCD accounted for 60% of the total costs . Patients with >3 episodes had the highest annual SCD-related costs (mean=US$58 950) across all settings. Health care resource utilization (HCRU) and costs increased substantially as the number of VOC episodes increased. This study was limited to observation of associations rather than causal inference, and by possible coding and identification discrepancies and the restricted generalizability of the population.VOC has a severe impact on medical resource use and costs among the adult SCD population. Further research among broader study populations is needed to facilitate the reduction of VOC episodes and thereby improve clinical and economic outcomes for SCD patients. Sickle cell disease (SCD) is a complex genetic blood disease in which erythrocytes have the propensity to change into a crescent (sickle) shape. This increases interactions with other endovascular cells and causes endothelium dysfunction, inflammation, and vascular damage.5Vaso-occlusive crisis (VOC) is the hallmark of SCD and sometimes the precursor to many life-threatening complications such as acute chest syndrome (ACS), stroke, splenic sequestration, and multisystem organ failure.In addition to its serious impact on QOL, SCD and its complications incur a significant economic burden. It is estimated that in 2005, the mean annual expenditure for children with SCD was US$11 702 under Medicaid coverage and US$14 772 under employer-sponsored insurance.12SCD management is complicated as well as costly, in part due to the many dimensions of care. For instance, prevention of infectious diseases and SCD-related complications is necessary from early childhood. Such preventive approaches include newborn screening, anti-infection vaccinations, and the use of antibiotics from birth until completion of the initial series of vaccinations for encapsulated organisms. Also, to preempt the onset of neurologic complications, annual transcranial doppler ultrasonography screening is used to monitor stroke risk and assess the need for prophylactic transfusion therapy to prevent primary strokes.13™), voxelotor, and crizanlizumab-tmca have recently been added to the established use of blood transfusion and hydroxyurea (HU). HU has been shown to reduce the incidence of VOC and ACS as well as decrease necessary blood cell transfusions and overall health care resource utilization (HCRU); nonetheless, it remains underutilized.18Among SCD maintenance treatments, blood marrow transplant (BMT), the amino acid L-glutamine  and costs per VOC episode (by care setting) to provide a detailed examination of the association between VOC frequency and SCD economic burden.This was a retrospective cohort analysis of patient-level data extracted from the United States Medicaid MAX databases from January 1, 2009 through December 31, 2013. The most recent 5 years of data were used at the time of the study and data was accessed through the Centers for Medicare and Medicaid Services (CMS).The study included fee-for-service (FFS) patients from all available states and Managed Care enrollees who resided in the following 14 states with complete data: Arizona, California, Indiana, Kansas, Kentucky, Minnesota, Nebraska, New Jersey, New Mexico, New York, Oregon, Tennessee, Texas, and Virginia (managed care information for patients residing elsewhere was incomplete in the CMS MAX database).22Patients were included in the study if they were aged ≥18 years and had an SCD diagnosis in any position  during the identification period . The date of the first observed SCD-related diagnosis during the identification period was designated as the index date. Patients were required to have continuous enrollment with medical and pharmacy coverage for the 6-month baseline and ≥1 year of follow-up after the index date, and were followed until the earliest of disenrollment, death, or end of study. Patients who were enrolled in a clinical trial during the study period were excluded due to unique treatment patterns that could influence study outcomes.Patients with VOC episodes were identified at any time after the index date using ICD-9-CM codes . Because treatments for 1 VOC episode can be administered in multiple settings within a given time window, discrete VOC claims within a 3-day gap were combined and recorded as a single VOC episode.Annual all-cause and SCD-related HCRU and costs for each patient were evaluated during the 12 months after the index date. For HCRU analysis, the length of inpatient stays and the number of visits were computed for the fixed 1-year period .For analysis of health care costs by facility type, only FFS patients were calculated (Managed Care cost data are unavailable in the CMS MAX database). Costs were adjusted to 2013 US dollars using the medical care component of the Consumer Price Index (CPI).HCRU and costs were considered SCD-related if they were associated with an SCD diagnosis, SCD medications , or other SCD management procedures .SCD-related HCRU and costs were also stratified by number of VOC episodes in the 1-year follow-up period. Patients were separated into four cohorts, according to number of VOC episodes .Costs per VOC episode were identified in any setting and classified by place of service using a hierarchical order: inpatient, ER, outpatient, office, and other. The definition of outpatient VOCs included those in the outpatient hospital, ambulatory surgery center, nursing facility, or long-term care settings. Office VOCs included those in the independent clinic, federally qualified health center, state or local public health clinic, or rural health clinic settings.Health care costs during the VOC episodes were identified and included the total medical costs in any setting and outpatient pharmacy costs related to SCD treatments. Average costs per VOC episode were calculated for all VOC episodes and for each setting of care.Numbers and percentages are reported for categorical variables. Mean values and standard deviations are reported for continuous variables.This analysis included 8521 qualifying adult patients with SCD , most ofAmong all eligible patients, 24.6% had ≥1 VOC requiring an inpatient stay during the 6-month baseline period. Infectious disease (19.74%) was the most common baseline comorbidity, followed by asthma (10.9%), fever (9.3%), and neoplasms (7.2%). For baseline SCD management, 44.8% of the patients were prescribed antibiotics, followed by acetaminophen (44.1%), folic acid (29.8%), opioids (28.1%), and NSAIDs (26%); 10.8% used hydroxyurea, and 14% had a blood transfusion. Assessment of baseline all-cause HCRU found 53.6% of the patients had ≥1 outpatient ER visit and 29.8% had ≥1 inpatient visit. Among the 8521 total adult patients with SCD, 49.8% (N = 4247) had an FFS Medicaid plan and were included in the analysis for health care costs. The average total medical costs were US$34 136 (median=US$12 691) in 1 year and SCD accounted for 60% of the total costs . The largest all-cause medical cost driver was inpatient costs, with average annual expenditure at US$17 535 (median=US$1590) per patient; SCD accounted for around 87% of these inpatient costs (mean=US$15 237). SCD also contributed to high proportions of costs in other settings such as pharmacy (67%) and ER (68%).The study population also utilized health care resources frequently. Almost all the enrolled patients had ≥1 all-cause pharmacy visit (95.2%), and 86% of these visits were related to SCD treatments. Large proportions of the patients had ≥1 all-cause outpatient hospital (87.4%) and outpatient office visit (83.6%), with 57% and 75% of these visits due to SCD, respectively. Among the 8521 enrollees, most had no VOC episode during the 1-year follow-up period, but a large number had ≥3 episodes. Specifically, 4452 patients had no episode, 1253 had 1 episode, 570 had 2 episodes, and 2246 had ≥3 episodes.Regarding SCD-related HCRU, patients with ≥3 episodes had the highest mean number of outpatient hospital visits , outpatient ER visits , inpatient visists , inpatient length of stay , and pharmacy visits . As expected, utilizations increased in positive correlation with the number of episodes . Evaluation of SCD-related costs found total costs were highest among patients who had ≥3 episodes, with a mean of US$58 950 (SD=US$93 147). Across the 4 cohorts, the largest cost driver was inpatient costs; the ≥3 cohort had the highest mean inpatient costs of US$45 088 (SD=US$85 112), followed by pharmacy, outpatient hospital, and outpatient ER costs. As with utilizations, costs increased for patients with a higher number of VOC episodes . In addition, the frequency distribution of SCD-related health care costs for patients with FFS coverage was examined. Patients with total cost of US$0 were excluded because the actual cost might not have been captured. The mean total cost for patients with 0 pain crisis to ≥3 crisis ranged from US$5516 to US$62 577 during the 12 months of follow-up. Also, the frequency distribution for the total SCD-related health care costs also showed that patients with ≥3 crisis incurred a maximum total health care cost of US$1 125 371. Among the 4247 total adult SCD patients with FFS Medicaid, 2316 (50.9%) had ≥1 VOC episode after the index date. A total of 40 772 VOC episodes were identified in the following settings : 15 395 from inpatient, 14 779 from ER, 7420 from outpatient, 2365 from office, and 813 from other settings. The mean duration of each episode was 5.5 days (SD=11).The mean total medical cost per VOC episode was US$4861 (SD=US$14 296). The mean costs per VOC episode varied widely across different settings, with inpatient VOCs incurring by far the highest per-episode cost , followed by ER (US$1072 [SD=US$3458]), outpatient (US$695 [SD=US$2437]), and office settings (US$306 [SD=US$1480]). This study explored the medical expenditures of adult SCD patients, detailing the breakdown of utilizations and costs not only by number of VOC episodes but also per-episode, stratified by setting. The results revealed the biggest cost drivers to be inpatient vistis and pharmacy, and shed more light on the economic burden of the disease. Although other studies have explored the economic burden of SCD, most were conducted at the individual state level and relevant only to specific treatments,Previous studies indicate that VOC frequency, more so than pain duration or pain severity, is associated with increased utilization of health services,In addition, the present study showed that VOC episodes were the primary reason for the high medical utilizations and costs among the study population, based on the findings on the ratio of HCRU and costs, stratified by different number of VOC episodes. As expected, the study showed that patients with the highest VOC frequency (the ≥3 episode cohort) incurred the highest health care costs. They accounted for 26% of the total sample, indicating that a large group of adult SCD patients were suffering from high-frequency VOC episodes, and this complication was inflating overall costs for the SCD population at large.Hence, this study confirmed a correlation between episode frequency and HCRU and costs . This could reflect increases in episode severity requiring more resources, which was further suggested by the per-episode cost findings, as inpatient VOC episodes had high mean costs. Moreover, the costs per VOC episode included both VOCrelated costs and all other medical costs incurred by patients with VOC during the episode period, which more precisely reflects the real-world medical costs associated with VOCs.It is also important to note there are some barriers associated with acute VOC management. These include incomplete understanding of the underlying mechanism of vaso-occlusive pathophysiology, sparse access to SCD experts, lack of evidence-based treatment guidelines, and inconsistent adherence to health care provider indications.Further, an adjunct analysis examined the frequency distribution for the total SCD-related health care costs for FFS SCD patients. This analysis showed that patients with frequent crises could be associated with a huge economic burden of over US$1 million annually, as observed in some patients with more than 3 crises in a year. However, we cannot ascertain whether this high cost is mainly because of the high number of crises or the severe complications associated with the crises.While the study findings elucidate medical resource use and expenditures in this population, several limitations should be noted.The study relied on descriptive statistical analysis of outcomes, which requires caution in drawing conclusions from cohort comparisons, as these results are trends without direct experimental testing, such as randomized controlled trials, and therefore imply only correlation, not causation. Moreover, claims data analysis has its own limitations. For example, the data and corresponding outcomes may be affected by administrative diagnosis coding errors, codes entered as rule-out criteria and not actual indications of disease presence, and deliberate misdiagnoses to justify claims for off-label prescriptions.Another limitation of the study is that in order to capture all SCD patients including those with low health care utilizations, patients with ≥1 SCD diagnosis in any setting were identified as SCD patients; this might have inadvertently included some non-SCD patients due to coding discrepancies. Although this study shows a large proportion of patients had high VOC frequency, we believe there is underreporting of the true number of SCD patients who experienced VOC and frequency of the VOC because home-managed VOCs were not capturable in the dataset used for this study.In addition, the dataset was from 2009–2013 and real-world clinical management of SCD may have changed since the end of the study period.The dataset also presented another limitation specific to this study. As the target was HCRU and costs by facilities, only FFS patients were included in the analysis because costs for Managed Care enrollees are not recorded in the Medicaid MAX dataset. This reduced the sample size and limited the generalizability of the study outcomes to other populations.Notwithstanding these limitations, the study does lay the groundwork for further research that can put the analysis into more specific statistical models and examine how the economic outcomes are related to the reduction of VOC episodes, controlling for covariates such as patient demographics and clinical characteristics.The study found VOC episodes are associated with significant economic burden among patients with SCD, as they led to higher frequencies of inpatient utilizations, which were the largest cost driver among the study population. Moreover, HCRU and costs were closely and positively associated with the number of VOC episodes. In particular, among patients who had 3 or more VOC episodes per year, those episodes accounted for over 90% of the SCD-related inpatient visits and costs. Thus, VOC frequency can be seen as a strong indicator for medical resource use and expenditures. These findings warrant future investigations among broader study populations with more sensitive analysis, and ultimately may help clinicians reduce VOCs and thereby mitigate the overall clinical and economic burden of SCD.