Alternaria alternata is an ascomycete fungus that causes leaf spot diseases on various crop plants. In this study, we identified a novel ssRNA mycovirus infecting an A. alternata f. sp. mali strain isolated from an apple orchard in China. Sequence analyses revealed that this virus is related to hypoviruses, in particular to Wuhan insect virus 14, an unclassified hypovirus identified from insect meta-transcriptomics, as well as other hypoviruses belonging to the genus Hypovirus, and therefore this virus is designed as Alternaria alternata hypovirus 1 (AaHV1). The genome of AaHV1 contains a single large open-reading frame encoding a putative polyprotein (∼479 kDa) with a cysteine proteinase-like and replication-associated domains. Curing AaHV1 from the fungal host strain indicated that the virus is responsible for the slow growth and reduced virulence of the host. AaHV1 defective RNA (D-RNA) with internal deletions emerging during fungal subcultures but the presence of D-RNA does not affect AaHV1 accumulation and pathogenicities. Moreover, AaHV1 could replicate and confer hypovirulence in Botryosphaeria dothidea, a fungal pathogen of apple white rot disease. This finding could facilitate better understanding of A. alternata pathogenicity and is relevant for development of biocontrol methods of fungal diseases.Mycoviruses are wide spread throughout almost all groups of fungi but only a small number of mycoviruses can attenuate the growth and virulence of their fungal hosts.  Hypoviridae, Narnaviridae, Barnaviridae, Alphaflexiviridae, Gammaflexiviridae, Deltaflexiviridae and Mymonaviridae, and one floating genus, Botybirnavirus  are widely distributed across all major groups of phytopathogenic fungi . Until rrnavirus , but manHypoviridae contains a sole genus Hypovirus with four assigned virus species. Its members  infect a phytopathogenic fungus, Cryphonectria parasitica, the causal agent of chestnut blight disease , has been a world-wide issue in apple production for decades . We also showed that AaHV1 produces defective RNA (D-RNA) during fungal culture in the laboratory. In addition, we demonstrated that AaHV1 confers hypovirulence in other plant phytogenic fungi.In this study, we identified and characterized a novel hypovirulence-inducing hypovirus from A. alternata strains were isolated from apple leaves with blotch disease randomly collected from a variety of apple orchards in Yangling County, Shaanxi Province of China in 2016. The Botryosphaeria dothidea strain, YL5, and F. graminearum strain, PH-1, were gifted from Dr. Zhonghua Ma  while the C. parasitica strain, EP155 (ATCC 3875)   was a geCC 3875) . The ampMalus domestica cv. Gala) subjected to pathogenicity tests were provided by Dr. Qingmei Guan .All fungal strains were grown on PDA medium for 3–6 days at 24–26°C for morphological observation or on cellophane-covered PDA medium for RNA, DNA and protein extractions. Apple leaves , picked from the edge of a 3-day-old culture colony, were placed on newly grown apple leaves. The petioles of inoculated leaves were wrapped with water-wet cotton for moisturizing. Inoculated apple leaves were kept at 25°C, 70 to 80% humidity and with a photoperiod of 16 h/8 h (day/night). All inoculations were repeated three times.For inoculation of Extraction of ssRNA and dsRNA from fungal mycelia followed the procedure described previously . DsRNA-eA. alternata mycelia cultured on PDA covered with cellophane as described by For viral dsRNA quantification, total RNA was prepared from ® UltraTM RNA Library Prep Kit for Illumina  and sequenced on the Illumina HiSeq 4000 platform . Raw reads were cleaned by removing adapter sequences, and low-quality bases  were trimmed by a Trimmomatic package with default parameters so truncated reads smaller than 35 bp were discarded. All clean reads were then assembled through the de novo assembly program Trinity1 with a K-mer value = 25. Assembled reads were subjected to BLASTX searches with a cut-off of E ≤ 1e-5. To completely sequence the viral genome, RNA ligase-mediated (RLM)-rapid amplification of cDNA ends (RACE) was performed as previously described . The conserved protein domains were predicted by the National Center for Biotechnology Information (NCBI) conserved domain database3. Putative transmembrane domains were determined using the TMHMM server version 2.04 . Potential stem-loop RNA structures were predicted by employing Mfold version 2.3   with each other and replicated across three plates. Mycelium blocks were taken randomly from the fungal pair, donor (virus source) side and recipient side (virus-free strain), to evaluate virus transmission. For viral vertical transmission, the assay was carried out to verify the frequencies of virus transmission through asexual spores. Fungal strains infected with the virus were cultured for 4 weeks on the bench top. Asexual spores were liberated in distilled water and spread on 10-cm PDA plates at appropriated dilutions. Single conidial germ lines were transferred to new PDA plates and cultured for 3 days. The number of infected colonies were scored based on visual observation of distinctive virus infection-associated colony morphologies and dsRNA or RT-PCR detection.The polyethyleneglycol (PEG)-mediated transfection described by The virulence of fungal colonies was assessed on apple leaves. Apple plants were generated from pathogen-free tissue culture and grown in a growth room. Fungi was inoculated on newly developed apple leaves and incubated on a bench top (25–27°C). Lesions were measured at days 5 and 7 post-inoculation. All inoculations were repeated three times.A. alternata, 43 field fungal strains were collected from leaves of apple (M. domestica cv. Fuji) showing symptoms typical of Alternaria blotch disease  while also exhibiting slow growth . PAGE anMK189193. Based on BLAST-P analysis, the large viral-encoded protein has the highest identity  with polyproteins encoded by Wuhan insect virus 14 (WhIV14), a hypo-like virus identified from insect meta-transcriptomics  to those of Macrophomina phaseolina hypovirus 1 (MpHV1)  in length consisting of a long ORF coding for a polypeptide of 4,228 amino acid (∼479 kDa) with a 5′-untranslated regions (UTR) of 491 nts, a 3′-UTR of 868 nts and an adenine tail (poly A) on the 3′-sequence end . The seqiptomics . Additio (MpHV1) , CHV1 (S (MpHV1) , Fusariu (MpHV1)  and CHV2 (MpHV1) . Owing tquencing   , suggestA. alternata strain, YL-3C, and the presence of D-RNA does not affect AaHV1’s ability to attenuate fungal virulence.AaHV1-free isogenic strain was obtained from YL-3C through single asexual spore isolation (YL-3C/dsF). We observed that AaHV1 was vertically transmitted through conidia with approximately 94–95% efficiency regardless of the presence of AaHV1 D-RNA . MoreoveTo obtain a general view surrounding what kind of stresses AaHV1 elicits in fungal hosts, we cultured YL-3C/dsF, YL-3C/AaHV1, and YL-3C/AaHV1-D on different stress-inducing mediums. The presence of 1 M NaCl or 1 M Sorbitol in PDA provides hyperosmotic stress conditions for fungal growth, while 1% SDS and Congo red treatment were used to mimic cytoplasmic membrane and cell-wall stresses . Vogel’sA. alternata host backgrounds, we introduced AaHV1 into a YL-1P strain that contains no detectable dsRNA . We then introduced AaHV1 to YL-1P by spheroplast transfection with total RNA isolated from YL-3C mycelia. The conidial isolates from AaHV1-transfected YL-1P were re-generated and subjected to four rounds of repeated subculture on PDA plates. It is interesting that all AaHV1 detected in the YL-1P host background did not produce D-RNA (data not shown). This result was consistently confirmed after further subculture (six rounds). As seen in To investigate AaHV1 pathogenicity in different le dsRNA . SequencA. alternata f. sp. mali strains. Moreover, AaHV1 accumulation is lower in YL-1P than YL-3C strains, suggesting that different A. alternata strains have varying defense strengths against virus infection.AaHV1-infected YL-1P grew slightly slower than dsRNA-free YL-1P . MoreoveB. dothidea, F. graminearum and C. parasitica, which are the fungal pathogens of apple white rot (or ring rot), wheat head blight and chestnut blight diseases, respectively , but not in F. graminearum PH-1  and C. parasitica EP155   are associated with conferring hypovirulence-associated traits to fungal hosts  to ectively . AaHV1 ad apples . At presal hosts , 2016. OHypoviridae family from a phytopathogenic fungus, A. alternata. Although a number of mycoviruses have been identified from A. alternata, this is the first report of a hypovirulence-inducing mycovirus in A. alternata. Therefore, the finding with this virus provides a valuable experimental system to study the molecular aspects of virus-fungus interactions and fungal pathogenicity in A. alternata. Hypoviruses have a prominent place in mycovirology, in particular owing to long and intensive studies on CHV1 in the fungal host, C. parasitica (C. parasitica pathosystem (In this study, we characterized a novel mycovirus related to members of the rasitica . CHV1 prrasitica . In addihosystem , 2019. LNo datasets were generated or analyzed for this study.LSu designed the experiments. HL, RB, QL, LY, TP, and LSa performed the experimental work. IBA, HK, and LSu analyzed the data and wrote the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Passiflora edulis in China. The infection of Telosma mosaic virus (TeMV), a potyvirus, and its effects on the phytochemical components of P. edulis remain largely unknown in China.Viral disease has become the most severe constraint for the cultivation and production of P. edulis plants showing distorted leaves and severe mosaic skin on green fruit were identified with TeMV infection through traditional transmission electron microscopy, RT-PCR and modern small RNA sequencing (sRNA-seq) platform. The contents of phytochemical components and the activities of antioxidative enzymes were compared between virus-infected and virus-free P. edulis to confirm the effects of TeMV infection on host plant.P. edulis fruits and leaves with Electron microscopy. Partial coat protein genes of TeMV were successfully amplified by RT-PCR in infected P. edulis leaves and fruits but not in healthy plants. Abundant small interference RNAs (siRNAs) sequences, showing several characterizations, were specifically generated from the TeMV genome in infected plant fruits by sRNA-seq platform. Furthermore, fruit length, fruit thickness (wideness) and fruit weight decreased significantly due to TeMV infection. The levels of total protein and total sugar increased significantly; however, the level of total fat, total acid and vitamin C decreased obviously after TeMV infection. The level of total phenols, a secondary metabolite, was obviously higher in TeMV-infected than TeMV-free P. edulis fruit. The activities of superoxide dismutases (SOD) and catalases (CAT) obviously increased in TeMV-infected in comparison with healthy P. edulis fruit.Firstly, approximately 700 nm linear virus particles, representing TeMV, were detected in infected P. edulis fruits, differently and selectively modulated the phytochemical components of P. edulis fruits. In turn, P. edulis plants enhanced their tolerance to the stress of TeMV infection by increasing the secondary metabolite level and the antioxidative capacity. This is of significant importance to understand the effects of TeMV infection on the biochemical changes and the antioxidant defense mechanism in P. edulis.TeMV infection adversely affected the development of  Passiflora edulis, an important Passifloraceae fruit, is cultivated throughout tropical and subtropical regions of the world, including China. It has been widely used in folk medicine to treat anxiety, insomnia, asthma, bronchitis and urinary infection. The constituents of different functional extracts include phenols, proteins, flavonoids, alkaloids, cyanogenic compounds, glycosides, vitamins, minerals and terpenoid compounds [P. edulis in their diet and hence the demand for P. edulis has increased. On the other hand, the cultivation and production of P. edulis are severely affected by several diseases caused by fungal, bacterial and viral pathogen [P. edulis. Viruses infecting P. edulis included members of the genus Potyvirus, Cucumovirus, Begomovirus, Tymovirus, Cilevirus, Cilevirus, Carlavirus, Tobamovirus and Nepovirus [Telosma cordata plants in Vietnam, subsequently patchouli plants in Indonesia and recently passion fruit in Thailand, Haikou and Fujian of China [P. edulis showed severe symptoms, such as mosaic and distorted leaves, mosaic skin on green fruit and decreased fruit size. The effect of viruses infection on the chemical composition of host plants has been widely reported. Indian cassava mosaic virus (ICMV), Plum pox virus (PPV) and Tomato leaf curl Palampur virus (TLCPV) infection modified the composition of nutritive and bioactive compounds of plant host, Momordica charantia, Prunus domestica and Cucurbita moschata, respectively [Passion fruit woodiness virus (PWV), another potyvirus, the most economically important viral disease of passion fruit plants in Brazil, can affect the content of phenolic compounds, an antioxidant molecule, in rinds [P. edulis fruit.ompounds , 2. Due pathogen . Among wepovirus –12. Teloof China –17. TeMVectively , 18, 19.P. edulis in Zhangzhou City of Fujian Province by traditional electron microscopy, RT-PCR and modern small RNA deep sequencing, (b) to examine the effect of TeMV infection on contents of phytochemicals in the fruits, (c) to investigate the role of individual antioxidative enzymes (SOD and CAT) in protection of P. edulis plants against oxidative damage caused by virus infection.The aims of the present study are (a) to identify the TeMV infection in P. edulis samples (n = 50) showing severe mosaic and distorted leaves and mosaic skin on green fruit after 7 days were respectively collected from Zhangzhou  and Longyan (Xinluo district and Wuping County) in Fujian. Total RNA was extracted from 0.1 g symptomatic leaves and fruits with Trizol reagent  following manufacturer’s instruction. The cDNA was prepared from total RNA using M-MLV reverse transcriptase and gene-specific primers (available upon request) in a final volume of 20 μL. Total RNA and primer were denatured at 70 °C for 10 min, then 10 μL of Master Mix  was added before incubating at 42 °C for 1 h, then at 70 °C for 10 min. The about 500-bp fragment for TeMV was amplified using 2 μL cDNA as the template in a final volume of 50 μL with InvitrogenTM SuperScriptTM IV VILOTM kit  [P. edulis samples were used as a control.P. edulis leaves and fruits were investigated for virus particles with the negative staining method as described by Brenner with moderate adjustment [After confirmation by RT-PCR, infected and healthy justment . Virus pP. edulis fruits (n = 3) were performed by Novogene technology co. LTD  as described previously [k-mer of 17 or 19. Assembled contigs were used to search the GenBank/EMBL/DDBJ database using BLASTn or BLASTx. Coverage and distribution of virus specific contigs by siRNAs were determined using the program MAQ under default parameters.Library construction and sequencing of small RNA extracted from infected and healthy eviously , 23. Brin = 9) positive for the presence of TeMV infection were uses for phytocheical analysis. Fruit juice obtained from healthy and infected fruit respectively was homogenized with a homogenizer for phytochemical analysis. To determine of total protein, 100 g fruit juice were digested with 5.0 ml of 2 M NaOH at 100 °C for 60 min. After centrifugation at 160000 g for 10 min, the protein content in the supernatant was detected with the method of the Folin-Phenol reagent using bovine serum albumin  as a standard [Nine samples  was assayed as described by Misra with moderate modification [The SOD activity of healthy and infected fruit juice (fication . In brie2O2 was followed at 240 nm by observing decrease in absorbance. Enzyme specific activity is expressed as μmol of H2O2 oxidized min− 1 (mg protein)− 1.Also, the CAT activity of fruit juice obtained above was assayed as described by Beers with moderate modification . In briep < 0.05 using least significant difference (LSD) test with SPSS statistics software 17.0.All data were expressed as mean and standard deviation (σ) of at least three replicate samples. The data were analyzed using one-way analysis of variance (ANOVA). Significant differences were calculated at P. edulis samples (n = 300) showing severe mosaic and distorted leaves and mosaic skin on green fruit . Furthermore, TeMV siRNAs were predominantly 21-nt and 22-nt long . Thus, sRNA-seq was used to investigate the production of siRNAs in ong Fig. . TeMV siong Fig. . TeMV siong Fig. . TeMV siP. edulis, physical properties  of P. edulis (n = 9) were evaluated in TeMV-infected and TeMV-free plants fruits. Our results revealed that fruit length, fruit thickness (wideness) and fruit weight decreased significantly in virus-infected P. edulis plants when compared with that of virus-free P. edulis plants  into H2O2 and O2. In turn, CAT break down H2O2 in the living. Thus, the activities of antioxidative enzymes (SOD and CAT) were analyzed in virus-infected and virus-free P. edulis fruits. Our results demonstrated that there was an obviously increase in the activities of SOD by 63.0% and 27.3% increase in CAT  specifically from TeMV, including the amount (0.44%), the length distribution (mainly 21-nt and 22-nt), the bias of first nucleotide (mainly G and C), the polarity distribution  and frequency distribution (hot and cold spots) of vsiRNAs along the TeMV genome, the coverage (73.2%) and the average depth (9.5) , which in turn could defend against invading pathogens at moderate level [2−) into H2O2 and O2. In turn, CAT break down H2O2 in the living. In this study, a significant decrease of Vc (30%)  Fig.  but an oP. edulis plant in Zhangzhou City of Fujian Province in China at molecular level through traditional and modern bio-technologies and for the first time assessed its impacts on phytochemicals components and antioxidative enzymes activities of diseased plants. This is of significant importance to understand the effects of TeMV infection on the biochemical changes and the antioxidant defense mechanism in plants after virus infection.The studies presented here confirmed the infection of TeMV in


Preterm labour and birth (PTL/PTB) is characterised by major health and developmental risks for children, life–changing consequences for their families, and substantial healthcare and economic challenges for wider society. While it is known that PTL/PTB impacts infant healthcare costs in the short and long term in Germany, maternal costs have not been described in detail. The aim of this study was to comprehensively describe costs and resource use among PTL/PTB mothers during pregnancy, at hospitalisation for delivery, and up to three years after delivery—overall and according to gestational age (GA) at delivery.This study used data from the Statutory Health Insurance (SHI) sample of the AOK Hessen database in Germany. Mothers aged 12–44 years with deliveries between 2009 and 2013 and > 9 months of medical history prior to delivery were included. PTL/PTB mothers were defined by an International Classification of Diseases, 10th Revision (ICD-10) code for PTL during pregnancy, a diagnosis-related group (DRG) code indicating birthweight < 2500 g, or delivery of an infant < 37 weeks GA. Inpatient and outpatient resource use and total direct medical costs were examined during pregnancy, at delivery hospitalisation, and up to three years post-delivery.Of all mothers, 2147 (20%) experienced PTL/PTB. During pregnancy, median costs for PTL/PTB mothers were €2130. During delivery hospitalisation, the mean length of stay for all PTL/PTB mothers was 6.0 days, and median costs were €2037. Length of stay and costs declined with increasing GA. Long term, PTL/PTB mothers’ total median costs were €607 in Year 1, €332 in Year 2, and €388 in Year 3 post-delivery. In each year after delivery, median costs appeared to be greater for mothers who delivered at lower GAs.In this description of costs and resource use among PTL/PTB mothers in Germany throughout the pregnancy and up to three years after delivery, the greatest costs were noted prior to delivery. Costs appeared to decrease with increasing GA, particularly during the delivery hospitalisation and the first year after delivery.The online version of this article (10.1186/s12884-018-1912-0) contains supplementary material, which is available to authorized users. Preterm labour (PTL) is defined as regular uterine contractions, accompanied by cervical change, that occurs before 37 weeks gestation . ApproxiPTB is a leading cause of neonatal morbidity and mortality . PTB hasThe impact of PTL/PTB on maternal outcomes, resource use, and costs is less well described. In a US study, pregnancies with a PTL admission were shown to lead to significantly poorer outcomes compared with pregnancies without a PTL admission, with higher rates of maternal intensive care unit (ICU) admission, inpatient maternal mortality, and 30-day maternal mortality . In a stThe aim of this analysis was to describe resource use and costs among PTL/PTB mothers during pregnancy, during delivery hospitalisation, and up to three years after delivery—overall and stratified according to GA of infants at delivery—using data from a German health insurance fund. Our analysis was descriptive in nature, and aimed to estimate the absolute rather than excess costs incurred by PTL/PTB mothers.This study utilised administrative insurance claims data from the Statutory Health Insurance (SHI) sample of AOK Hessen (Versichertenstichprobe AOK Hessen/KV Hessen) . Hessen We included mothers in the SHI sample who had a recorded diagnosis-related group (DRG) delivery code and a German procedure classification (Operationen- und Prozedurenschlüssel [OPS]) delivery code  code indicating PTL during pregnancy and/or PTB  and the actual delivery date (defined by OPS codes) of less than 37 weeksA DRG code indicating infant’s birthweight < 2500 g  were used to define GA. Mothers with missing GA were assigned to the > 37 weeks of GA based on the distribution of GA in the rest of the population.< 28 weeks (extremely preterm infants)28–36 weeks (other preterm infants)≥37 weeks (term infants)Driven by the GA groups, as defined by ICD-10 codes, mothers were subsequently classified into three groups based on their infant's GA:Data on demographics and clinical characteristics were assessed at delivery and during the nine-month baseline period. Characteristics of interest included age at delivery, plurality of births (multiple or singleton), infant GA, and maternal risk factors for PTL,  which coResource use and total direct medical costs (in Euros) were examined during pregnancy, at delivery hospitalisation, and up to three years post-delivery. During each period, outpatient resource use, outpatient prescription data, inpatient resource use, and other services (defined below), which included all services not reimbursed in the inpatient or outpatient setting, were considered. Specifically, we considered the following resources in the outpatient setting: laboratory tests, preventative procedures (such as cancer screening and vaccinations), basic procedures , prescribed medications, general practitioner [GP] visits, gynaecologist/paediatrician visits, and any other specialist visit. It should be noted that due to the nature of the reimbursement system in Germany, which reimburses outpatient physicians on a quarterly basis, visits to physicians are recorded as one per quarter irrespective of how many encounters took place within the same quarter. Other services considered were: remedies , medical devices, midwifery services, driving services, and other (such as household help or home care). Outpatient costs were estimated as total costs for each service used or drug prescribed. In the inpatient setting we considered the total number of all-cause hospitalisations, length of stay (LOS), pregnancy/labour procedures, diagnostic tests, or therapeutic procedures (such as operations) performed during hospitalisation. Inpatient costs were estimated based on DRG codes per hospital stay. Costs were estimated from the third-party payer perspective, corresponding to the SHI fund, which is in accordance with Institute for Quality and Efficiency in Health Care (IQWiG) guidelines for cost of illness analyses in the German setting .As not all mothers were insured for 365 days in the respective years of follow-up (lost to follow-up or reached the end of the observation period), costs for the first, second, and third year of follow-up were evaluated for those mothers who had sufficient follow-up and were continually insured in the respective year. It should be noted that we allowed women to enter the cohort up until the end of the study period (31 December 2013)—this means that only women enrolled prior to the 1 January 2011 could accrue the full three years of follow-up, and among these, only those who did not die or were lost-to-follow-up (LTFU) were observed for the full three years. We nonetheless chose to include women who could not be followed for the full three years to maximise the study cohort available for analysis.P-values comparing the characteristics of PTL/PTB mothers to non-PTL/PTB mothers were calculated using the chi-squared test/Fisher’s Exact test in those instances when expected cell counts < 5. Univariable negative binomial models were used to estimate the rate of resource utilisation during follow-up as the number of events per person-year with 95% confidence intervals (CI). Costs were presented using summary statistics, which were estimated and included mothers without any resource use .Continuous variables were described using average values (median and mean) and measures of data dispersion . Categorical variables were described using frequencies and percentages. All data programming and analyses were carried out using Microsoft SQL server 2008  and SAS for Windows Release 9.3 .N = 10,678) and 2.3% to multiples (N = 247). Approximately 19.7% (N = 2147) of the mothers had a diagnosis of PTL during pregnancy or had a PTB. Most mothers (88.2%) had an expected date of delivery variable, which allowed us to directly calculate the GA. A further 1.7% could be categorised into a GA group based on ICD-10 codes, and the remaining 10.1% were assigned to the ≥ 37 weeks GA group as described in the methods section. All mothers who were identified based on giving birth to a low-birth-weight (LBW) infant also had an estimated GA indicating a PTB. Overall, most PTL/PTB mothers delivered at term . Approximately 32.3% delivered between 28 and 36 weeks GA, and 2.1% delivered before 28 weeks GA. The proportion of PTL/PTB mothers who delivered at term differed depending on whether the mother gave birth to singletons or multiples .In total, we identified 10,925 mothers in the SHI sample that were eligible for inclusion. An illustration of the cohort selection process can be seen in Fig. The characteristics of PTL/PTB mothers are described in Table p < 0.0001). PTL/PTB mothers were also more likely to have a CCI score greater than 0 (p = 0.0142), as well as hypertension (p = 0.0002) and diabetes (p = 0.0004). Compared with non-PTL/PTB mothers, those with PTL/PTB were more likely to deliver by Caesarean section (p < 0.0001) and to give birth to twins (p < 0.0001).There were some significant differences between PTL/PTB mothers and non-PTL/PTB mothers Table . AlthougA breakdown of the frequency of resource use during pregnancy among PTL/PTB mothers can be seen in Table The median total cost among PTL/PTB mothers during pregnancy was €2130 (mean: €2925). Costs per sector and resource use are presented in Table During delivery hospitalisation, the mean LOS for PTL/PTB mothers was 6.0 days . Mean LOS declined with increasing gestational age, from 13.3 days for < 28 weeks to 8.3 days for 28–36 weeks and 4.7 days for ≥37 weeks Fig. . Trends Median total costs during delivery hospitalisation were €2037 . Stratified by GA, costs declined with increasing GA; while the cost associated with delivery hospitalisation was €5158 (IQR: €3711–€7488) among mothers delivering with a GA of < 28 weeks, it declined to €2699 for mothers delivering at a GA of 28–36 weeks and €1815 (IQR: €1576–€2536) for mothers giving birth at ≥37 weeks Fig. . Trends Overall, the most common inpatient service used was ‘monitoring and management of a high-risk delivery’ (31%) followed by ‘monitoring and management of a normal birth’ (22%). However, this varied according to the GA. In the < 28 weeks GA group, the most common procedures were Caesarean section (33%) and post-partum instrumental removal of the placenta (16%). In contrast, the most common procedure in the ≥37 weeks group was episiotomy (13%).The rate of resource use after delivery was found to decline with time since delivery for several of the resources considered Tables , and 5. The rate of resource use across settings of care in the three years after delivery, split by GA, can be seen in Tables During the follow-up period, PTL/PTB mothers’ total median costs were €607  in the first year, €332  in the second year, and €388  in the third year . To maximise the sample and increase the generalisability of results, we reduced the length of minimum medical history required for inclusion to nine months prior to delivery; this meant we were unable to characterise the obstetric history of women in detail. In addition, we allowed mothers to enter the cohort until the end of the study period, with the trade-off that we would not have long-term follow-up data for women entering toward the end of the study period. While these strategies allowed us to maximise the starting cohort sample size, the sample size decreased with time, as only mothers with recorded deliveries prior to 1 January 2011 (38%) could be potentially observed in the database for the entire three years of follow-up. Although the potential impact of loss to follow-up should be born in mind when interpreting the results, we do not expect significant bias to be introduced, as the main cause of the reduction in the sample size over time was women entering the study close to the end of the study period. The resulting lack of precision and wide CIs, however, is a limitation. A further limitation is the lack of an ability to link data between mothers and infants, which could lead us to miss instances of PTB, and prevented any assessment of combined mother-infant costs. As with any administrative database study, missing or potentially erroneously recorded diagnostic codes is also a limitation.Our manuscript aimed to assess the total costs associated with all PTL/PTB mothers. However, it is likely that mothers of multiples or mothers with complications during birth have greater resource use and costs compared with mothers of singletons without complications. Further research on how resource use and costs vary not only according to GA, but to the parity and medical history of the mothers, would be of value. In addition, although our aim was to provide a descriptive overview of costs associated with PTL/PTB in Germany, future studies utilising an appropriately constructed control group of non-PTL mothers who deliver at term—to quantify the excess costs associated with PTL/PTB—would also be of interest. We found no published cost estimates of pregnancies leading to term birth in Germany to contrast our estimates of costs associated with PTL/PTB; however, prior comparative studies have found that mothers who experience PTL/PTB have significantly worse health outcomes,  longer LDespite these limitations, our analysis provides up-to-date data on the prevalence and maternal PTL/PTB costs and resource use in a country where limited information on these exist. The apparent trend of declining costs with increasing GA we observed is interesting, and highlights the importance of considering maternal costs, in addition to infant costs, in burden of illness studies of PTL/PTB. Future studies investigating the causes of the increase in costs at lower GAs, particularly over the longer term, would be of great interest. Our analysis is also strengthened by reflecting direct medical costs from the third-party payer perspective, and a long follow-up, relative to previously published studies.Our findings suggest that estimates of national costs of PTL/PTB should take the contribution of maternal short and long-term costs into account. Although analyses by GA were descriptive, costs appeared to be greater for mothers who delivered infants at lower GAs. This gradient suggests that PTL/PTB may also have an impact on maternal resource use and costs. Further studies utilising appropriate control groups and methods to control for confounding are needed to adequately assess this. Although data on absolute costs cannot determine the most efficient allocation of resources alone,  our findAdditional file 1:Table S1. Drg and ops codes used to identify deliveries. Diagnosis-related group and operationen- und prozedurenschlüssel codes used while examining the statutory health insurance (SHI) sample of aok hessen (versichertenstichprobe AOK Hessen/KV Hessen) to identify deliveries. (DOCX 21 kb)Additional file 2:Table S2. Codes used to identify ptl/ptb deliveries. International classification of diseases, 10th revision and diagnosis-related group and operationen- und prozedurenschlüssel codes used while examining the statutory health insurance (SHI) sample of aok hessen (versichertenstichprobe AOK Hessen/KV Hessen) to identify preterm birth/preterm labour deliveries. (DOCX 15 kb)


Cryptococcus gattii is an etiologic agent of cryptococcosis and a serious disease that affects immunocompromised and immunocompetent patients worldwide. The therapeutic arsenal used to treat cryptococcosis is limited to a few antifungal agents, and the ability of C. gattii to form biofilms may hinder treatment and decrease its susceptibility to antifungal agents. The objective of this study was to evaluate the antifungal and antibiofilm activities of an ethanolic extract of Cochlospermum regium (Schrank) Pilger leaves against C. gattii. The antifungal activity was assessed by measuring the minimum inhibitory concentration (MIC) using the broth microdilution technique and interaction of the extract with fluconazole was performed of checkerboard assay. The antibiofilm activity of the extract was evaluated in 96-well polystyrene microplates, and the biofilms were quantified by counting colony forming units. The extract showed antifungal activity at concentrations of 62.5 to 250 μg/mL and when the extract was evaluated in combination with fluconazole, C. gattii was inhibited at sub-MIC levels. The antibiofilm activity of the extract against C. gattii was observed both during biofilm formation and on an already established biofilm. The results showed that the ethanolic extract of the leaves of C. regium shows promise for the development of antifungal drugs to treat cryptococcosis and to combat C. gattii biofilms. Cryptococcosis is a fungal infection that occurs worldwide. Despite the predominant result of opportunistic infections of immunocompromised patients, the incidence of infections among immunocompetent individuals is increasing –4. Recen Cryptococcus neoformans and C. gattii [ C. gattii is not only more clinically aggressive but also more difficult to control. Species of the C. gattii complex can compromise immunocompetent individuals and cause severe diseases of the central nervous system, such as meningitis, encephalitis, and meningoencephalitis [ C. gattii complex than those caused by species of the C. neoformans complex [ C. neoformans [Despite the importance of this disease, the treatment of cryptococcosis is limited to the antifungals fluconazole and amphotericin B, which are used alone or in combination with 5-flucytosine . The pri. gattii , which r. gattii . C. gattphalitis . In addi complex . Despiteoformans . C. gattii complex have some attributes that can hinder antifungal therapy, such as heteroresistance to fluconazole [ C. gattii, helps fungi escape the immune system [ Cryptococcus spp. to antifungal drugs and host defense mechanisms [Species of theconazole . In thesconazole . Anothere system  and decre system . In addichanisms .Cryptococcus activity to aid in the treatment of cryptococcosis and alleviate the limitations of the current options. A promising alternative is the use of medicinal plants. Recently, Kumari et al. [ Cryptococcus species, but these authors did not include isolates of C. gattii.In this context, it is of great importance to identify compounds with anti-i et al.  evaluateCochlospermum regium (Schrank) Pilger, also known as yellow cotton tree, is a shrub found in the Brazilian cerrado, Paraguay, and Bolivia. It is a medicinal plant that is popularly indicated for the treatment of various diseases [ C. regium from roots presents antimicrobial action against Staphylococcus aureus and Pseudomonas aeruginosa and studies report the presence of various compounds such as phenolic, gallic acid, and ellagic acid [ C. regium [ C. regium leaf extract against Escherichia coli and Candida tropicalis [ C. regium leaves (Schrank) Pilger against C. gattii.diseases . The extgic acid –21, as w. regium , 22. Rec C. regium (Schrank) P. were collected in the cerrado of Mato Grosso do Sul state  under authorization number 57730-1 of the Biodiversity Authorization and Information System (SISBIO). The plant was identified in the herbarium of the Faculty of Biological and Environmental Sciences of the Federal University of Grande Dourados by Professor Dr. Zefa Valdivina Pereira. A sample of the species was deposited in the herbarium under DDMS registration 5001.The leaves of C. regium (ECR) was obtained by mixing 200 g of the powdered plant material with 1000 mL of 95% ethanol and maintaining the mixture at 25 °C for 72 h with shaking every 12 h. The obtained vegetal extract was rotoevaporated  at 35 °C until complete volatilization of the solvent and then lyophilized using an EC MicroModulyo system coupled to a Savant VLP80 ValuPump vacuum pump.The leaves were dried in a circulating air oven at 30 °C and pulverized in a blade mill. The crude extract of the leaves of C. gattii strains were assayed, including two isolated from sputum specimens (476 and 2164) and one from cerebrospinal fluid , obtained from the Microbiological Research Laboratory of the Federal University of Mato Grosso do Sul, Campo Grande, Mato Grosso do Sul, Brazil. A reference strain, C. gattii , was included in all assays.For the antifungal susceptibility and checkerboard assays, threeIn each experiment, the yeasts were subcultured in Sabouraud Dextrose Agar  for 48 h at 35 °C. For antifungal susceptibility and checkerboard assays, the density of yeast cells suspended in a saline solution (0.85%) was adjusted to a transmittance of 88% at 530 nm using a spectrophotometer. Next, 1:50 and 1:20 cell dilutions were made in RPMI-1640 medium  containing L-glutamine, buffered with 0.165 M MOPS (3-(N-morpholino) propanesulfonic acid, Sigma-Aldrich) and supplemented with 2% glucose. Clinical and Laboratory Standards Institute  [The antifungal activity of ECR was evaluated using the microdilution technique in broth, according to the standards of the M27-A3) , with soμg/mL, and the microplates were incubated for 48-72 h at 35 °C.The ECR was dissolved in 80% dimethyl sulfoxide  and serial dilutions of the extract were performed in 96-well flat-bottom microplates (Kasvi) containing RPMI-1640 medium. The ECR concentrations assayed were 1.95, 3.90, 7.81, 15.62, 31.25, 62.50, 125, 250, 500, and 1000  C. gattii after incubation. Fluconazole was used as a control, and the assay was performed in duplicate at two different times.Minimum inhibitory concentrations (MICs) were determined as the lowest ECR concentration that prevented visible growth ofμg/mL for ECR and 0.5-64 μg/mL for fluconazole. Together, the drugs formed a matrix of combinations of different concentrations in 96-well flat-bottom microplates, which were incubated for 48-72 h at 35 °C.An assay to evaluate the combined effect of ECR and fluconazole was performed based on the CLSI M27-A3 . The finThe interpretation of results was based on the fractional inhibitory concentration index (FICI), defined as follows: FICI=+. The antifungal activity of the extract and the antifungal combination was interpreted as synergistic (FICI≤0.5), additive effect (0.5<FICI<1), indifferent (1≤FICI<4), or antagonist (FICI≥4) .Growth Conditions for C. gattii ATCC 56990. For the ECR activity assays on biofilms, C. gattii ATCC 56990 was cultured in Sabouraud dextrose broth (HiMedia) for 24 h at 30 °C with shaking. The cells were collected by centrifugation at 5000 g for five min, washed three times with phosphate-buffered saline (PBS), and resuspended in RMPI-1640 medium. Using a Neubauer chamber, the cell density was adjusted to 1x108 colony forming units (CFUs)/mL [CFUs)/mL .Activity of ECR on Biofilm Formation by C. gattii ATCC 56990. The effect of ECR on biofilm formation was assessed according to Martinez and Casadevall [ C. gattii biofilms were washed three times with 0.05% Tween 20 in trisphosphate buffer (TBS) to remove the nonadherent cryptococcal cells. The biofilms were characterized by the C. gattii viability assay to determine the CFUs, and the experiment was conducted in triplicate.sadevall , with miActivity of ECR on Preformed Biofilms. The effect of ECR on preformed biofilms was evaluated according to Martinez and Casadevall [ C. gattii ATCC 56990 added in RPMI-1640 broth. To form biofilms, the microplates were incubated at 35 °C for 48 h with shaking. C. gattii biofilms were carefully washed three times with 0.05% Tween 20 in TBS to remove the nonadherent cryptococcal cells. After washing, the biofilms were treated with 2.5, 5, or 10 mg/mL ECR. The microplates were incubated again at 35 °C for 48 h with shaking. Next, the biofilms were washed three times with 0.05% Tween 20 in TBS to remove the nonadherent cryptococcal cells. A positive control  and a negative control (broth only) were included. The biofilms were characterized by the C. gattii viability assay to determine the CFUs, and the experiment was conducted in triplicate.sadevall , with miViability of C. gattii Cells in a Biofilm after Being Treated with ECR. The viability of C. gattii strains was determined in each of the biofilm assays described above. After the biofilms were scraped from the wells of the microplates, the resulting cell suspensions were vigorously vortexed for 5 min to disaggregate the cells. Serial dilutions (in PBS) of each cell suspension were prepared and plated by the ASD drop plate technique [10 CFU/cm2) of microtiter plate well were enumerated [echnique . Plates umerated . p<0.05 was considered significant for all evaluations. Statistical and graphical analyses were performed using GraphPad Prism® 7.0 .The results were evaluated by ANOVA, and the means were compared by Tukey's posttest. A value of C. gattii ATCC 56990 and three clinical C. gattii isolates (two from sputum and one from CSF) are presented in μg/mL.The MIC results for ECR against C. gattii was inhibited at sub-MIC levels. However, according to the observed FICI values (1 to 1.25), the combination of the two antifungal drugs was not different from the drugs alone . When comparing C. gattii ATCC 56990 biofilm formation in the presence and absence of ECR, the extract reduced the amount of biofilms formed by 25.33 to 42.58% in a dose-dependent manner, with a maximum observed reduction of 2 log10. For preformed biofilms, ECR reduced the amount of biofilms by 7.13 to 15.44% , ECR could be used to prevent biofilm formation. It is important to highlight that our previous study showed that ECR is innocuous for animal cells (92% cell viability for VERO lines) and did not show mutagenic potential in an Ames assay [Considering our previous findings on the antimicrobial activity of ECR against other pathogenic microorganisms , we belies assay . in vitro. Although this could be viewed as an unfavorable result, the concomitant use of both drugs in the same patient without one interfering with the antifungal activity of the other could be allowed. Thus, fluconazole could be used to treat cryptococcosis in parallel with ECR, avoiding the formation of biofilms on cell surfaces and preventing reinfections. Complementary studies could also validate the use of ECR as a coadjuvant in the treatment of cryptococcosis caused by C. gattii species.In the present study, we also evaluated the combination of ECR with fluconazole and observed that these antifungal drugs did not show a satisfactory interaction Cryptococcus are endowed with a polysaccharide capsule composed primarily of glucuronoxylomannan, which is an important structural component of Cryptococcus biofilms [ Cryptococcus spp. may form biofilms on polystyrene plates and medical devices [ C. gattii biofilm formation, since an ECR concentration of 10 mg/mL resulted in a reduction of 42.58% of viable C. gattii cells compared to the control, indicating its potential use in prophylaxis.The formation of biofilms contributes to the permanence of microorganisms in a host and helps protect against antimicrobials . The yeabiofilms . Cryptoc devices , 33 and  devices . The eva devices , 34. The C. regium leaves showed antifungal activity and especially antibiofilm activity against C. gattii. Thus, the results of the present study expand the known antifungal properties of the leaves of this plant. However, additional tests are necessary to characterize the chemical compounds responsible for this antifungal activity. In addition, trials aimed at assessing the applicability of the extract at promoting positive health outcomes should be performed.The results of our study showed that an ethanolic extract of


Magnaporthe oryzae and designed as Magnaporthe oryzae ourmia-like virus 4 (MOLV4) is reported. MOLV4 has a genome that is 2497 bp long and possesses a single open reading frame (ORF), which encodes the product RNA-dependent RNA polymerase (RdRp). Sequence similarities were found between the MOLV4 encoded RdRp and the counterparts of a few previously reported ourmia-like mycoviruses. Virus-curing and biological comparison indicate that the virus has no or mild effects on the morphology and mycelium growth rate of the host fungus. Phylogenetic analysis using the RdRp aa sequences was performed. The results show that MOLV4 is clustered with the ourmia-like mycoviruses, forming a clade closely related to ourmiaviruses but distinct from narnaviruses. In addition, database searches revealed that several MOLV4-related sequences are present in the transcriptome shotgun assembly (TSA) library, expressed sequence tag database (ESTdb), whole-genome shotgun (WGS) library, and genomic survey sequences (GSS) libraries of a few other species of eukaryote organisms. Our results show that MOLV4, together with other similar ourmia-like mycoviruses, might represent a virus clade that links the plant ourmiaviruses and fungal narnaviruses and has a wide range of hosts.Here, the molecular characterization of a novel mycovirus that was isolated from a phytopathogenic fungus  Mycoviruses  are ubiquitous and can infect and replicate in all major taxa of fungi, including plant-pathogenic fungi . AlthougThe genomes of most mycoviruses are double-stranded (ds) RNA or positive single-stranded ((+)ss) RNA , and sevOurmiavirus, whose genome is composed of three ssRNA segments encoding proteins of RdRp, CP, and MP [Ourmiavirus genus are recombinants of narnavirus-like viruses and plant viruses, such as tombusviruses [Up until now, viruses in the Narnaviridae family were the simplest viruses that infected filamentous fungi, yeasts, and oomycetes . Viruses, and MP . It has sviruses . The CP Magnaporthe oryzae is the causal agent of rice blast, the most destructive disease of rice that could result in great yield loss in rice fields annually. Polyhedral virus particles suggestive of virus infection were previously reported in this fungus, the first plant-pathogenic fungus from which viruses were isolated [Magnaporthe oryzae virus 1, 2, and 3  were assigned to the family Totiviridae and two viruses, Magnaporthe oryzae chrysovirus 1A and B (MoCV1-A and MoCV1-B) were assigned to the family Chrysoviridae. The MoCV1-A and MoCV1-B have the ability to impair vegetative and invasive growth, as well as to alter the colony morphology of their host fungus [Magnaporthe oryzae virus A (MoVA) [Magnaporthe oryzae ourmia-like virus 1 (MOLV1) [Ourmiavirus genus, respectively.isolated ,16. Recet fungus ,18,19. IA (MoVA)  and Magn (MOLV1) . The RdRMagnaporthe oryzae ourmia-like virus 4 (MOLV4), from a M. oryzae strain HNDW-6. Viral genome organization and phylogenetic analysis indicate that this virus is closely related to the ourmia-like mycoviruses and the plant viruses of the Ourmiavirus genus. Bioinformatic analyses led to the finding that related viral sequences were present in the database, such as the transcriptome shotgun libraries (TSA) and expressed sequence tag database (ESTdb), suggesting a wide prevalence of these kinds of viruses across kingdoms. In addition, biological function of the MOLV4 have been discussed.In this study, we characterized a novel mycovirus, named M. oryzae strain HNDW-6 was originally isolated from rice leaves infected with rice blast disease in Hunan province, China. It was grown on potato dextrose agar  at 27 °C. For dsRNA extraction, mycelial plugs were cultured in potato dextrose (PD) broth  liquid medium at 27 °C, in an orbital shaker at 110 rpm for 4 to 7 days.w/v) electrophoresis and visualized under an AlphaImager HP gel imaging system  after being stained in a 0.1 mg/mL ethidium bromide solution.DsRNAs were extracted from fungal mycelium using the methods described by Morris and Dodds with minor modifications . DNA andThe dsRNAs were purified and used as templates for cDNA synthesis. cDNA libraries were constructed using random hexadeoxynucleotide primers  based on the methods described previously . The intDatabase searches were conducted against the EST and TSA databases available from NCBI using the tBLASTn, as described previously . For thihttp://mfold.rna.albany.edu/?q=DINAMelt/Quickfold) [Potential open reading frames (ORFs) of the full-length cDNA sequence were deduced and their homologous amino acid sequences were searched in NCBI by ORF Finder and BLASTp programs, respectively. Multiple sequence alignment was carried out using the CLUSTALX 1.8 program . Phylogeickfold) .In order to determine the effects of the virus infection on the host fungus, we used compounded methods of thermal treatment and cycloheximide to eliminate the virus from strain HNDW-6 as described previously with minor modifications . Hyphal t test at α = 0.05.Morphology and growth rates were assessed by culturing mycelial plugs, collected from the 4-day-old actively growing plate, on PDA for 3–7 days at 27 °C in the dark. Mycelial growth rates were tested by measuring the diameters of each colony. The test of each strain had three repetitive PDA plates. The data for the growth rate test were analyzed by a one-way analysis of variance in the SPSS software (IBM SPSS statistics 20). Treatment means of mycelial growth rates between different fungal stains were compared using Student’s M. oryzae strains, a dsRNA segment of approximately 2–3 kb in size was detected from the strain HNDW-6 when extracted with CF-11 cellulose chromatography method and electrophoresed on 1% agarose gel ssRNA mycovirus named MOLV4. The genome organization of this virus is illustrated in Phomopsis longicolla RNA virus 1 (PlRV1), followed by other ourmia-like mycoviruses, including Sclerotinia sclerotiorum ourmia-like virus 3 (SsOLV3) , Rhizoctonia solani ourmia-like virus 1 (RsOLV1) , Botrytis ourmia-like virus (OuMV) , and other plant ourmiaviruses  residues with a calculated molecular weight of 76.7 kDa. This protein showed 57%  aa identity to the RdRp of aviruses . Conservaviruses .The genome of MOLV4 had a 5′-UTR of 413 bp and a 3′-UTR of 29 bp. The potential secondary structures of the termini of the positive-strand MOLV4 were predicted using Mfold RNA structure software . The firOurmiavirus genus. These aa sequences were multiple aligned and used for phylogenetic tree generation by the NJ method in MEGA 7 [Ourmiavirus, but distinct from the mycoviruses in the family Narnaviridae , plants , vertebrates (Sardina pilchardus), and arthropods . Matches from the TSA libraries of fungi, plants, vertebrata, and arthropods are listed in Homology searches of the public database such as the genomes and transcriptomes of cellular organisms have been reported to be an available approach for the detection of novel viral sequences and other unraveled non-retroviral RNA and viruses integrated into eukaryotic genomes ,32,33,34Botryotinia fuckeliana, B. tulipae, Magnaporthe grisea, and Blumeria graminis. The accessions were then assembled into seven MOLV4-like sequences (contigs or singletons) i.e., MoESTc1_OL and MoESTc2_OL from M. grisea, BtESTc1_OL and BtESTc2_OL from B. tulipae, BfESTc1_OL from B. fuckeliana, and BgESTc1_OL and BgESTc2_OL from B. graminis. Reverse BLAST analysis of the viral-like EST sequences on the complete NR database revealed 30% to 86% aa identities to the RdRps of ourmia-like mycoviruses and ourmiaviruses.In addition, we searched the EST database for MOLV4-like sequences. We found 100 MOLV4 related accessions derived from the EST libraries in several fungal strains including Symbiodinium minutum genomic clone SYB2F-146E05 (GA560351) and the uncultured marine RNA virus genomic (DX421080.1) showed moderate levels of aa sequence identities .Interestingly, using the MOLV4 sequences as a query, we also found two MOLV4 like sequences in the GSS library by a tBLASTn search. The two GSS sequences from the Calonectria naviculata (NAGG01000037.1), C. leucothoes (NAJI01000289.1) and C. pseudonaviculata (JYJY01001043.1) were found, which were termed as Calonectria naviculata MOLV4-like sequence 1 (CnMLS1), Calonectria leucothoes MOLV4-like sequence 1 (ClMLS1), and Calonectria pseudonaviculata MOLV4-like sequence 1 (CpMLS1). BLASTX searches in the NR database, using the three hit sequences as queries, revealed 24% to 55% aa sequence identities to the ourmia-like viruses.Several previous studies have demonstrated the presence of non-retroviral RNA viruses and DNA viruses in the eukaryotic genome. During BLAST searching for the MOLV4 like sequences in the whole-genome shotgun (WGS) library, three significant matches of chromosomes of the M. oryzae strains (HNDW-8 and HNDW-10) were compared in biological traits including colony morphology and growth rate. Aside from the HNDW-10, which has a relative lighter color, the HNDW-6, HNDW-6VF, and the HNDW-8 showed similar morphological characteristics. In addition, the four M. oryzae strains had no significant difference in growth rate. Strains HNDW-6, HNDW-6VF, HNDW-8, and HNDW-10 grew at the average rates of 7.33, 7.36, 7.27, and 7.41 mm/d, respectively  still has the ability to replicate in cells of plant leaves [Olpidium, Polymyxa, or Spongospora. This appeared when these viruses were transmitted repeatedly between plants without the involvement of fungi or protists [Since the ourmia-like mycoviruses were similar to the ourmiaviruses and narnaviruses according to RdRp-based phylogenetic analysis, elucidating the origin and evolution of the ourmia-like mycoviruses will have important implications in the understanding of the complex relationship between fungal narnaviruses and plant ourmiaviruses. Previous studies proposed that the ourmiaviruses might have evolved from a narnavirus  via acquisition of the genome segments, encoding MP and CP, from a plant virus, tombusvirus . Howeveriviridae . The DiaNA virus  and MoVANA virus , which it leaves . In addiprotists ,40,41. Tprotists . Of courSaccharomyces cerevisiae [Colletotrichum acutatum was also confirmed [Rhizoctonia solani, providing valid evidence for the transfer of a plant virus between plant and fungus [Olpidium or merely through soil without fungal vectors [Tenuivirus are transmitted by insect vectors [Actually, studying the evolution of some viruses is complex, as the presence of virus transmission between different hosts was considered to be important power for virus evolution. Nerva et al.  reportedrevisiae  and lateonfirmed . Recentld fungus . It is k vectors ,48, whil vectors ,50. Rece vectors . CollectB. graminis, B. fuckeliana, B. tulipae, and M. grisea were found to be similar with the MOLV4 sequences. This indicated that, with the exception of recorded fungus, such as M. grisea and Botrytis sp., the ourmia-like mycoviruses could infect more host organisms, extending the diversity and possible host range of the ourmia-like mycoviruses. In conclusion, the finding of these new viral sequences will help to provide new insights into the origin and evolution of the ourmia-like viruses. Of course, it is yet to be determined if some of the found viral sequences from plants or the other TSA database are truly derived from these annotated host organisms. Since many plants could carry endophytic fungi, which might be infected by mycoviruses [It is possible that some plant or fungi materials used for cDNA library construction might carry viruses that often render virus detection difficult, thus, it is possible to screen viral-like sequences from the TSA and EST databases. In this study, using the MOLV4 sequence as a query, we found many MOLV4-like sequences in the TSA database of many eukaryotes, including plants, fungi, vertebrata, and arthropods. In addition, several EST sequences from the fungus oviruses , we coul


Over the years, technological and process innovations enabled active case finding (ACF) programs to expand their capacities and scope to have evolved to close gaps in missing TB patients globally. However, with increased ACF program’s operational complexity and a need for significant resource commitments, a comprehensive, transparent, and standardized approach in evaluating costs of ACF programs is needed to properly determine costs and value of ACF programs.Based on reviews of program activity and financial reports, multiple interviews with program managers of two TB REACH funded ACF programs deployed in Cambodia and Tajikistan, we first identified common program components, which formed the basis of the cost data collection, analysis, reporting framework. Within each program component and sub-activity group, cost data were collected and organized by relevant resource types . Total shared, indirect and overhead costs were apportioned into each activity category based on direct human resource contribution (e.g. a number of staff and their relative level of effort dedicated to each program component). Capital assets were assessed specific to program components and were annualized based on their expected useful life and a 3% discount rate. All costs were assessed based on the service provider perspective and expressed in 2015 USD.Over the two program years (April 2013 to December 2015), the Cambodia and Tajikistan ACF programs cumulated a total cost of $336,951 and $771,429 to screen 68,846 and 1,980,516 target population, bacteriologically test 4,589 and 19,764 presumptive TB, diagnose 731 and 2,246 TB patients in the respective programs. Recurrent costs were the largest cost components (54% and 34%) of the total costs for the respective programs and Xpert MTB/RIF (Xpert) testing incurred largest program component/activity cost for both programs. Cost per screening was $0.63 and $0.10 and cost per Xpert test was $25 and $18; Cost per TB case detected (Xpert) was $373 and $343 in Cambodia and Tajikistan.Results from two contextually and programmatically different multi-component ACF programs demonstrate that our tool is fully capable of comprehensively and transparently evaluating and comparing costs of various ACF programs. The ambitious End TB Strategy aims for reduction in tuberculosis (TB) incidence and mortality rates to 90 and 95% of 2015 estimates by 2035; yet, current estimated annual rate of decline for TB incidence (2%) and mortality (3%) is far below the rate needed (10% or more) to achieve these targets.In the past decade, multiple technological and process innovations enabled ACF programs to expand the capacity and scope beyond community symptom screening. Some examples of these innovations include involving mass media, educational programs, community engagements to increase awareness of the TB problem –7; use oWhile there is growing evidence demonstrating the public health impact of ACF programs, evidence on costs of ACF programs is far too scarce , 15. ThiTherefore, the overall aim of our study was to develop a generalizable and comprehensive cost data collection, analysis, and results reporting framework that can be adopted for retrospective cost assessment of a wide range of ACF programs. The use of such a tool can facilitate systematic and transparent comparison of costs–total program costs, service  unit, and program yield costs–of ACF programs. To demonstrate the data categorization, analysis, and reporting process using this framework, we report costs of two operationally different multi-component ACF programs targeting different geographical areas and populations that operated in Cambodia and Tajikistan supported by the TB REACH  wave 3 funding cycle.The TB REACH initiative of the Stop TB Partnership (UNOPS) is a multi-lateral funding mechanism supported by the Global Affairs Canada and the Bill and Melinda Gates Foundation with specific aims to improve TB case detection and care cascade in high burden countries by funding innovative projects. During the wave 3 funding cycle, TB REACH funded a total of 32 projects across 24 countries. In this study, we used two TB REACH wave 3 funded programs in Cambodia and Tajikistan to demonstrate the development, data collection and analysis processes of our costing framework. These two programs were one of the more complex, multi-component programs that targeted widely different geographic areas , population and health systems , and epidemiological settings .. Fir. Fir29].Results from our study should be interpreted with the following notion of limitations. First, we were only able to evaluate program costs and activity outputs retrospectively, limiting our analytic option to the top-down method. Therefore, we were not able to capture uncertainties in cost estimates that may arise due to variabilities in operations and workloads that may depend on operational settings conditions., 26, 30 Our evaluation of costs of two contextually and programmatically different ACF programs in Cambodia and Tajikistan demonstrate that our comprehensive costing data collection, analysis, and reporting framework can be used to evaluate and compare costs of a wide range of ACF programs in a highly transparent manner. Furthermore, in efforts to standardize costing practices, a similar approach can be adapted to evaluate other TB control programs  and services. However, retrospective top-down assessment of costs restricts the scope of how costs can be assessed and do not allow evaluation or monitoring of factors that influence the costs of operations. More research is needed in this regard, early and periodic (e.g. quarterly or monthly) assessment of costs using the proposed framework is highly encouraged.S1 File(DOCX)Click here for additional data file.S2 File(DOCX)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.


Ultrastructural studies revealing morphological differences between intact and photodynamically inactivated virions can point to inactivation mechanisms and molecular targets. Using influenza as a model system, we show that photodynamic virus inactivation is possible without total virion destruction. Indeed, irradiation with a relatively low concentration of the photosensitizer (octacationic octakis(cholinyl) zinc phthalocyanine) inactivated viral particles (the virus titer was determined in Madin Darby Canine Kidney (MDCK) cells) but did not destroy them. Transmission electron microscopy (TEM) revealed that virion membranes kept structural integrity but lost their surface glycoproteins. Such structures are known as “bald” virions, which were first described as a result of protease treatment. At a higher photosensitizer concentration, the lipid membranes were also destroyed. Therefore, photodynamic inactivation of influenza virus initially results from surface protein removal, followed by complete virion destruction. This study suggests that photodynamic treatment can be used to manufacture “bald” virions for experimental purposes. Photodynamic inactivation is based on the production of reactive oxygen species which attack and destroy biomolecules. Thus, the results of this study can potentially apply to other enveloped viruses and sources of singlet oxygen. Photodynamic inactivation (PDI) can be used to destroy pathogenic microorganisms including bacteria, viruses, fungi, and protozoa ,2,3 whicEnveloped viruses  can be inactivated by various photosensitizers, including methylene blue , merocyaPhthalocyanines with intensive absorption at the far red to near-infrared wavelengths are commonly used photosensitizers. Energy migration from the triplet excited state of phthalocyanine to the triplet ground state of molecular oxygen leads to the formation of highly reactive singlet oxygen . Periphe2) completely inactivated H5N1 in all virus titers tested by up to 7.0 lg EID50, which is more effective compared to methylene blue or proflavine acetate. The singlet oxygen induced inactivation of viral fusion suggested hemagglutinin [As previously demonstrated in vitro, octacationic octakis(cholinyl) zinc phthalocyanine (Zn-PcChol8+) is highly efficient in disinfecting water from avian influenza A virus, H5N1 subtype . In thisglutinin , the virglutinin  showed fAvian influenza A virus A/domestic duck/Siberia/103/2016 (H5N8) was grown in 10-day old embryonated chicken eggs. After incubation at 37°C for 3 days, the allantoic fluid was harvested and stored at −80°C until use . All H5Ng for 5 min. PBS was then added up to 6 mL and the concentrators were vigorously mixed and centrifuged again at the same speed and duration as above. This procedure was repeated five times. After the fifth centrifugation step, the suspension was collected from the concentrators (about 1 mL per tube). This method provides high quality purification but leads to a reduction in virus titer (from 8.375 ± 0.42 to 7.125 ± 0.34 in our preparation) because some virions can get stuck in the membrane [Initially, the protocol did not contain a purification step but attempted to observe the effects of PDI using non-purified allantoic fluid. However, the allantoic fluid appears to mask viral particles because of the high concentration of proteins and other biomolecules. Therefore, we used a simple and rapid variation of the purification method developed by  for bactmembrane .2 in Eagle minimal essential medium  and gentamicin 50 μg/mL . After virus inoculation, cells were cultured in Eagle minimal essential medium supplemented with 0.2% bovine serum albumin , gentamicin 50 μg/mL (Biolot) and 2 μg/mL of trypsin . In all experiments, cells that were not infected with the virus were included as controls.Madin Darby Canine Kidney (MDCK) cells were grown at 37 °C with 5% CO2 for 5 days. Virus-induced cytopathic effect was detected and virus titers were determined as the 50% tissue culture infectious dose (TCID50) per ml according to Reed and Muench [For virus titration, MDCK cells were grown to 90% confluency in 96-well plates, then washed with PBS and infected with 10-fold serial dilutions (from 10-1 to 10-8) of the treated or non-treated virus. After incubating for 30 min at 37 °C, the supernatant was removed and cells were incubated with 200 μL of virus growth medium at 37 °C, 5% COd Muench . To prev−1 to 10−8) of treated virus which further minimizes the effect of residual products of photodynamic reaction.Singlet oxygen is a nonradical reactive oxygen species that readily oxidizes aromatic amino acids, unsaturated lipids and other biomolecules, and has the extremely short lifetime (about 1 μs) in biological systems . It seem2. The intensity of light reaching the probes was measured with PM160T Wireless Power Meter  and made up 10 mW/cm2. The vial containing the virus suspension was then placed in a plastic water filled box to prevent overheating. In addition, we included two control treatments: dark (incubated in the dark with photosensitizer) and light (irradiated as above without photosensitizer). Zn-PcChol8+ synthesized in the State Scientific Center “NIOPIK” (Russia) was used as a photosensitizing agent for virus inactivation in two treatments corresponding to concentrations of either 2 or 4 µM. The irradiation system was constructed from 30 W halogen lamp with collimated light beam (12 mm diameter). Virus suspensions (1 mL) were irradiated in opened 1.5 mL “Eppendorf” vials for 20 min at light dose of 12 J/cmw/v) was then added to the grid surface for 15 s. After drying, using filter paper again, the grid was examined with a TEM  at an accelerating voltage of 80 kV. A 200 mesh copper TEM grid  with carbon stabilized ultrathin (invisible on the water surface)  formvar Three TEM grids per sample were prepared independently to avoid random artefacts. At least 100 virions were examined in each sample. To prevent accidental irradiation, the preparation of the dark control was conducted in a dark room.Using a non-purified allantoic liquid with 2 µM of photosensitizer proved to be inadequate because it was difficult to observe any structural features. This is likely due to the high concentration of proteins and other biomolecules within allantoic fluid which mask the virions. In addition, these molecules react with the singlet oxygen produced by the photosensitizer. TEM images revealed that purification of the allantoic fluid dramatically improved the visibility of virion structures . The conpurified suspension without any treatment ;irradiated without photosensitizer  suspension with 4 µM of the photosensitizer ;irradiated with photosensitizer at concentrations of 2 µM ;irradiated with photosensitizer at concentrations of 4 µM .Five samples of H5N8 influenza virus were examined with a TEM under different conditions:There were no differences in viral morphology between untreated virions or light and dark controls .In contrast to the controls, virions irradiated with photosensitizer were damaged . VirionsThe virus suspension inactivated with 4 µM of photosensitizer contained three forms of “bald” virions: smooth spherical virus particles (~25% of examined virions), virions with partially destroyed membranes , and completely  destroyed membranes . These fVirus titer was determined by a virus titration using MDCK cells. The virus titration showed that the titers of non-treated purified virions and controls were similar . The titOn the micrographs of both (2 and 4 µM of photosensitizer) photodynamically treated samples smooth spherical vesicles (30–50 nm) and small (5–15 nm) grain-like structures have been detected . ConcentHere, we investigated the ultrastructural properties of photodynamic inactivation of H5N8 influenza virus. We demonstrate that PDI treatment of the virus induces the removal of viral surface glycoproteins and makes virions non-infectious even without membrane damage. Because proteins are readily oxidized by singlet oxygen , hemaggl2) rendered H5N8 virions unable to infect MDCK cells. Such a strong inactivation effect with this octacationic phthalocyanine photosensitizer is not unique to the influenza virus H5N8 as it has been previously shown for the H5N1 subtype [2). Thus, cationic phthalocyanines are highly active photosensitizers towards influenza viruses.In this study, the treatment with the photosensitizer Zn-PcChol8+ (2 or 4 µM) and 20 min white light irradiation , we observed irregular virion shapes which likely reflect virion membrane damage. One explanation is that the hydroperoxides of cholesterol and other lipids disrupt viral membranes  and induIn conclusion, influenza virus in our experiments was inactivated with singlet oxygen that was generated by the photoexcited phthalocyanine photosensitizer. Furthermore, our results demonstrate that the detachment of the influenza virus surface glycoproteins and the loss of infectivity by “bald” virions was the result of the photodynamic treatment. This type of viral inactivation may be representative for other sources of singlet oxygen. Indeed, the highly reactive ROS produced by chemical reactions in the dark  and even


Cryptococcus gattii sensu stricto from the southeastern United States. Our analysis indicates that C. gattii arrived in the southeastern United States approximately 9,000–19,000 years ago, long before its arrival in the Pacific Northwest.We conducted molecular clock analysis of whole-genome sequences from a set of autochthonous isolates of  Cryptococcus gattii species complex consists of >4 major subtypes (VGI–VGIV), which are now considered to be different species  in the Pacific Northwest of Canada and the United States generated a great deal of interest in the study of C. gattii species complex in this area  patient isolates from the southeastern United States (http://beast.community) to predict the timing of the emergence of C. gattii in the southeastern United States.Historical reports have described the presence of C. gattii species complex, specifically for VGIIa and VGIIc (1.59 × 10−8 SNPs/base/y). We determined the best-fitting clock and demographic model combination by implementing path and stepping stone sampling marginal-likelihood estimators as described previously (We identified single-nucleotide polymorphisms (SNPs) and conducted phylogenetic analyses as previously described  had a coCryptococcus was previously estimated at 2.0 × 10−9  of approximately 9,000–19,000 years. A general recombining population mutation rate for C. gattii emergence and subsequent divergence in the southeastern United States is clearly much older than the Pacific Northwest emergence. The sharp contrast in the time of arrival compared with C. deuterogattii in the Pacific Northwest is considerable. Although C. gattii has apparently been in the southeastern United States for thousands more years than C. deuterogattii has been in the Pacific Northwest, the number of cases detected in the southeastern United States is far fewer. Infections in the South are probably not regularly missed, given that C. gattii in the southeastern United States causes primarily a devastating meningitis or meningoencephalitis (Cryptococcus species that has been in the United States for thousands of years (C. gattii sensu stricto) than we know about the one that has only recently arrived (C. deuterogattii).The clonal mutation rate appears to be more appropriate than the general mutation rate for recombining populations for estimating tMRCA, showing that the timing of C. gattii in the southeastern United States occurred long before industrial shipping. Although humans might have populated the southeastern United States by the time of the dispersal, most of the human movement was north to south rather than the reverse (Cryptococcus species separations (C. gattii sensu stricto has been found in and likely originates from South American locales (C. gattii has been hiding, mostly undetected, in the southeastern United States for millennia.The Pacific Northwest emergence has been hypothesized to be related to the opening of the Panama Canal, enabling more shipping from eastern ports of South America to the West Coast of North America (


Virus-induced oxidative stress plays a critical role in the viral life cycle as well as the pathogenesis of viral diseases. In response to reactive oxygen species (ROS) generation by a virus, a host cell activates an antioxidative defense system for its own protection. Particularly, a nuclear factor erythroid 2p45-related factor 2 (Nrf2) pathway works in a front-line for cytoprotection and detoxification. Recently, a series of studies suggested that a group of clinically relevant viruses have the capacity for positive and negative regulations of the Nrf2 pathway. This virus-induced modulation of the host antioxidative response turned out to be a crucial determinant for the progression of several viral diseases. In this review, virus-specific examples of positive and negative modulations of the Nrf2 pathway will be summarized first. Then a number of successful genetic and pharmacological manipulations of the Nrf2 pathway for suppression of the viral replication and the pathogenesis-associated oxidative damage will be discussed later. Understanding of the interplay between virus-induced oxidative stress and antioxidative host response will aid in the discovery of potential antiviral supplements for better management of viral diseases. Due to their high reactivity, they are often referred to as “reactive oxygen species” (ROS). Typical examples of them include superoxide  is a transcription factor, which has evolved for this purpose. It is one of the best-characterized antioxidative transcription factors with an oxidants/electrophile-sensor function . This baA virus needs to express a number of nonstructural and structural proteins inside a host cell to support the viral genome replication and assembly of a new virion. Several viral proteins have been shown to be responsible for the production of various ROS , 35–40. γ-GT) and catalase [Moloney murine leukemia virus (MoMuLV) ts1 is a mutant retrovirus used for the study of a progressive neurodegeneration induced by a human immunodeficiency virus (HIV) , 55. Acccatalase . Overallcatalase . Based ocatalase .Human immunodeficiency virus type 1 (HIV-1) plays an etiological role in the development of acquired immunodeficiency syndrome (AIDS). Besides its immune-compromising effects, HIV-1 infection is also linked with the development of neurocognitive disorders , 60. Par2O2 [In addition to the gp120, one of three regulatory viral genes, a Tat protein also has been shown to be responsible for induction of oxidative stress upon HIV-1 infection –64. In r2O2 . This, i2O2 . These s2O2 . Based o2O2 .The pulmonary complication is one of the leading causes of death by HIV patients . In partTranscriptional regulation of HIV-1 genes is controlled by a distinct viral enhancer and promoter called “long terminal repeats” (LTRs). A variety of host and viral transcriptional factors bind this region in a concerted manner to ensure a fine-tuning of viral gene expression. Particularly, Tat was shown to play an essential role in a positive regulation of the LTR-dependent transcription of viral genes . TogetheγGCSH [HCV infection is responsible for the development of chronic hepatitis . SeveralγGCSH . ParticuγGCSH –75. BaseγGCSH .Chronic hepatitis C was shown to be in a frequent association with steatosis, accumulation of lipid droplet . In ordeNrf2-dependent metabolic reprogramming and its positive implication in the HCV disease progression were examined by Saito et al. . In thisAs previously noted, HCV associated with oxidative stress leads to the activation of the Nrf2 pathway –75. In cInfluenza viruses have been demonstrated to be a causative agent for oxidative stress and respiratory inflammation by a number of studies , 79–82. Respiratory syncytial virus (RSV) is responsible for viral upper and lower respiratory tract infections in infants and young children . RSV infIn regard to potential mechanisms for inactivation of the Nrf2 pathway by RSV, dysregulation of posttranslational modification of Nrf2 was suggested . BasicalHBV is regarded as one of the major etiological factors in the development of HCC . AccumulROS production and its associated oxidative tissue damage have been shown to play a causative role in herpes simplex virus- (HSV-) 1-induced neuropathology –104. Subα [Dengue virus (DENV) is an arthropod-borne tropical virus responsible for the development of dengue fever and related diseases , 110. Stα .Marburg virus (MARV) is a causative agent for lethal hemorrhagic fever in humans . Two sepSpring viremia of carp virus (SVCV) is the etiological agent of spring viremia of carp . SVCV inWest Nile virus (WNV) infection plays an etiological role in the development of neuroinvasive disease such as a mosquito-borne encephalitis –120. WNVRed-spotted grouper nervous necrosis virus (RGNNV), a pathogenic fish virus, induced oxidative stress, apoptosis, and postapoptotic necrosis in a grouper liver cell line . RGNNV iSome viruses induce oxidative stress on purpose for a successful completion of a virus life cycle. However, uncontrolled virus-induced oxidative stress exacerbates the condition of the infected cells. Therefore, upregulation of the cytoprotective and detoxifying protein seems to be beneficial not only for disruption of the ROS-dependent steps of the viral life cycle but also for the amelioration of the exacerbated conditions of the infected host cells. In this regard, numerous pharmacological agents were shown to activate the Nrf2 pathway and lessen the burden of virus-induced oxidative stress , 40, 123α-Luminol  is an anti-inflammatory drug extensively studied by Russian scientists [α-luminol was able to suppress oxidative stress induced by MoMuLV ts1 infection [α-luminol abrogated upregulation of Nrf2 in MoMuLV ts1-infected astrocytes, resulting in restoration of increased GPx and cell surface cysteine-glutamate antiporter (xCT) to a normal level [2O2 and recuperation of cellular DNA synthesis and the GSH level [α-luminol was able to suppress the neurodegeneration in MoMuLV ts1-infected mice [α-luminol treatment even blocked ts1 replication and expression of the gPr80env protein in the CNS of MoMuLV ts1-infected mice [α-luminol suppresses virus replication and virus-induced cytopathology in the CNS by reducing oxidant stress [α-luminol in the preservation of epithelial cell cytoarchitecture and promotion of thymocyte survival in MoMuLV ts1-infected mice [α-luminol [α-luminol were also observed in the intestines of MoMuLV ts1-infected mice [α-luminol was able to preserve crypt-villus epithelial organization and allows survival of intestinal T cells in the MoMuLV ts1-infected mice [α-luminol treatment [α-luminol seems to be beneficial in retarding the pathogenesis of MoMuLV ts1 infection through Nrf2-dependent neutralization of virus-induced oxidative stress [ientists . Jiang enfection . In addial level . This leSH level . In in vted mice . Particuted mice . Based ot stress . In lineted mice . In part-luminol . Similarted mice . Treatmeted mice . Interesreatment . Supprese stress . On the e stress . In thise stress . Howevere stress .κB [Salvia miltiorrhiza (Danshen) [Epigallocatechin-3-O-gallate (EGCG), the predominant catechin from tea, is known to exert a variety of biological activities –135. In κB . Based oκB . TanshinDanshen) , 139. InDanshen) . In partDanshen) . Based oDanshen) .As previously noted, HIV-1 transgene expression in rats significantly dampens alveolar macrophage phagocytic capacity . In partSulforaphane (SFN) is an isothiocyanate abundant in cruciferous vegetables. It is famous for its cytoprotective effects shown by numerous in vivo and in vitro studies , 141. SFα, and heme-regulated eIF2alpha kinase (HRI) [Lindera erythrocarpa Makino, was also found to suppress HCV replication by Nrf2-mediated HO-1 induction [A. paniculata [Tripterygium wilfordii [HO-1 is one of the best-characterized Nrf2 target genes, readily inducible in response to a variety of oxidative stress and cytotoxic insult. HO-1 catalyzes the oxidation of heme to biliverdin (BV), carbon monoxide, and iron . Zhu et se (HRI) . Lucidonnduction . In thisnduction . Androgrniculata , 152, waniculata . In thisniculata . Particuniculata . In an aniculata –156. Amoniculata . On the niculata . In reganiculata . Celastrilfordii –161, wasilfordii .Although previous data all pointed out inhibitory effects of Nrf2-activating pharmacological agents on HCV replication through different mechanisms, following two studies demonstrated differential effects of the Nrf2 pathway on HCV replication. Sugiyama et al. showed knockdown of Nrf2 neutralized the increasing effect of chronic HCV infection on levels of lipid droplets, resulting in inhibition of HCV replication . Saito eβ, RIG-I, and MxA [S. baicalensis were reported to display an anti-influenza activity through Nrf2 activation [Psoralea corylifolia L. [Artemisia rupestris L., is a sesquiterpene with inhibitory activities against influenza viruses [β production via the HO-1 pathway after influenza virus infection [Kesic et al. showed suppression of Nrf2 gene expression enhanced influenza virus replication; meanwhile, pharmacological induction of Nrf2 via supplementation such as SFN and EGCG suppressed viral replication . This da and MxA . Nrf2 kn and MxA . In addi and MxA . In line and MxA . Carboci and MxA , 165. Ca and MxA . Carboci and MxA . In the  and MxA . Compountivation . Bakuchifolia L. –171. Shofolia L. , 173. Ru viruses , 175. A  viruses , 177. In viruses , 177. In viruses , 177. Em viruses , 179. Em viruses . Curcumi viruses , 182. Cu viruses . This innfection .RSV infection is associated with a severe lower respiratory illness characterized by bronchiolitis and respiratory failure. It is the leading cause of infant hospitalization . Loss ofProtzer et al. showed the antiviral activity of HO-1 in HBV infection . In thisSchachtele et al. reported negative modulation of experimental herpes encephalitis-associated neurotoxicity through SFN treatment . In thisα expressions. This led to an increase in the survival rate in suckling mice during DENV infection [α, OASs, and PKR and downregulation of NS2B/NS3 protease [DENV infection was shown to activate the Nrf2 pathway in mononuclear phagocytic cells . Treatinnfection . HO-1 inprotease , 162. Thprotease , 162.Shao et al. showed pharmacological activation of Nrf2 with SFN and bardoxolone enhanced the cellular total antioxidative capacity by upregulation of SOD1 and HO-1 and dampened SVCV replication . KnockinZhang et al. found that an isatin derivative was able to inhibit coxsackievirus replication through the Nrf2-dependent upregulation of NQO1 and GCLM . MelittiHuang et al. showed that hemin is able to induce HO-1 expression via the Nrf2 pathway and this leads to inhibition of Zika virus replication . This inVesicular stomatitis virus (VSV) is a prototypical oncolytic virus that has demonstrated potent oncolytic activity in preclinical models and is being evaluated in clinical trials –191. SFNIntracerebral injection of Theiler's murine encephalomyelitis virus (TMEV) into susceptible strains of mice causes a chronic demyelinating disease . KobayasRabbit hemorrhagic disease virus (RHDV) is used as an experimental model to study fulminant hepatic failure . MelatonPorcine circovirus type 2 (PCV2), a single-stranded DNA virus, is the primary causative agent of several syndromes collectively known as porcine circovirus disease . Gan et In this paper, the roles of virus-induced oxidative stress in the viral life cycle and the pathogenesis of viral diseases were reviewed. Particularly, the cellular management of this virus-induced oxidative stress by cellular utilization of the Nrf2 pathway and its implications in the viral replication and the progression of the viral diseases were explained. In addition, examples of positive and negative regulations of the Nrf2 pathway by a number of pathogenic viruses were described. Finally, various methods of pharmacological and genetic modulations of the Nrf2 pathway as a potential therapeutic option were listed. Considering the significant impact of the Nrf2 pathway on the pathophysiology of both host cell and virus, Nrf2 modulators may be able to serve as a promising supplement for viral diseases by therapeutic modulation of virus-induced oxidative stress in the near future.


Picornaviruses constitute one of the most relevant viral groups according to their impact on human and animal health. Etiologic agents of a broad spectrum of illnesses with a clinical presentation that ranges from asymptomatic to fatal disease, they have been the cause of uncountable epidemics throughout history. Picornaviruses are small naked RNA-positive single-stranded viruses that include some of the most important pillars in the development of virology, comprising poliovirus, rhinovirus, and hepatitis A virus. Picornavirus infectious particles use the fecal–oral or respiratory routes as primary modes of transmission. In this regard, successful viral spread relies on the capability of viral capsids to (i) shelter the viral genome, (ii) display molecular determinants for cell receptor recognition, (iii) facilitate efficient genome delivery, and (iv) escape from the immune system. Importantly, picornaviruses display a substantial amount of genetic variability driven by both mutation and recombination. Therefore, the outcome of their replication results in the emergence of a genetically diverse cloud of individuals presenting phenotypic variance. The host humoral response against the capsid protein represents the most active immune pressure and primary weapon to control the infection. Since the preservation of the capsid function is deeply rooted in the virus evolutionary dynamics, here we review the current structural evidence focused on capsid antibody evasion mechanisms from that perspective. Picornaviruses have been pivotal in the foundations of virology. Original research on “ultra-filterable infectious agents” such as foot-and-mouth disease virus (FMDV) and poliovirus (PV) began the era of animal virology . This genus contains poliovirus (PV), coxsackieviruses A/B (CVA/B), enteroviruses (EV), echoviruses (E), and rhinoviruses (RV). Further serological classification results in hundreds of serotypes. Hepatitis A virus (HAV) is the sole human-virus species in the genus Hepatovirus. Other human picornaviruses include members of the genera Cardiovirus (Saffold virus—SAFV), Cosavirus (CoSV), Parechovirus (Ljungan virus—LV), Kubovirus (Aichi virus—AiV), and Salivirus   that serve as mRNA for viral protein synthesis  P1, encoding the structural capsid viral proteins (VP4–VP2–VP3–VP1), while, in some picornaviruses, also codifying a short leader L-protein; (ii) P2, encoding the viral non-structural proteins 2A−2B−2C; and (iii) P3 encoding the viral non-structural proteins 3A−3B−3C−3D   .The larger proteins (VP1–3) form the external and internal capsid surface. These proteins have a common fold, the “jelly-roll,” formed by two 4-strand anti-parallel β-sheets and two helices . ConversPicornavirus cell infection starts with its attachment to cell receptors . Therefovβ3 and αvβ6 integrin used by some CVAs  ICAM-1 used by HRV and CVA  the LDL-receptor used by HRV-C  escape mutants Hiding the receptor-interacting surface from Abs surveillance is the hypothetical function of the canyon  codon usage, (ii) mutational robustness, (iii) modification of translational efficiency, and (iv) RdRp fidelity manipulation (Burns et al., Further elucidation of the mechanisms underlying these phenotypes could be used for rational codon rewiring in combination with increasing CpG and UpA frequencies to activate innate host immunity (Kumagai et al., The control and eradication of pathogenic picornaviruses is an ongoing problem. Picornavirus evolution, ruled by high mutation rates and recombination, has made this viral group genetically and antigenically highly variable. Moreover, changes in virulence represent an unpredictable threat. Important lessons drawn from the polio eradication battle indicate the necessity of a new generation of vaccines (Agol et al., JC and GM conceived the review project, designed figures, wrote the manuscirpt, and approved it for publication. They both contributed equally to the work.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


We report unusual persistence of Zika virus RNA in semen, confirmed by sequencing at 515 days after symptom onset and detectable for >900 days, in a patient with immunosuppression.Zika virus RNA has been detected in semen samples collected  Detection of Zika virus RNA in semen was described previously in an immunocompetent man 370 days after symptom onset; envelope and precursor of M protein gene sequencing indicated high genetic stability in semen 3–4 months after symptom onset (The patient has multicentric reticulohistiocytosis (MRH), a rare rheumatologic condition, which was diagnosed in 2015. When MRH was diagnosed, the patient had multiple pruritic, firm papules and nodules on his face and neck. He also had lesions with a characteristic coral bead appearance at periungal sites. In addition, he had severe joint pain and stiffness affecting his hands and knees and drenching sweats. His MRH diagnosis was confirmed by testing of a punch biopsy of a lesion. He was HIV negative, and his immunoglobulin levels and immunoglobulin electrophoresis results were normal. He was initially treated with topical steroids and antihistamines, but he only had limited relief. He was prescribed oral steroids and required high doses to control his symptoms. Clinicians added methotrexate and hydroxychloroquine to his medications as steroid-sparing agents and to reduce the chance his MRH would progress to erosive disease.https://www.euroimmun.com) in initial samples, but we noted seroconversion on day 13 . We did the same for the day 515 sample and sequenced it using MiSeq . We used double indexing to prevent cross-contamination and index misassignment errors. We attempted viral culture on multiple semen samples, as previously described . We consWe also prepared 90 plasma samples from patients infected with hepatitis C, collected for a separate study, in parallel with the day 515 sample. We did this to exclude the possibility of cross-contamination from our patient’s previous samples, particularly the day 13 sample, which were shipped, prepared, and sequenced 6 months earlier. We did not detect Zika virus in any of the hepatitis C samples. We found no evidence of cross-contamination with Zika virus sequences during processing that could explain the near-whole genomes detected in the day 515 sample. Consensus sequences were consistent with all samples having come from the same patient with only 2 mutations, 1 synonymous change at codon 2921 and a K3272E substitution, acquired during the 502 days between the first and last samples sequenced . We depo>3 months rather than >6 months (Counotte et al. systematically reviewed all available evidence on the risk for sexual transmission of Zika virus (In our case, Zika virus RNA might have persisted in semen because of failed immune clearance secondary to the patient’s MRH or his immunosuppressive drug treatment. However, when advising returning male travelers in couples planning pregnancy, clinicians should be aware that Zika virus RNA shedding in semen might be intermittent and persist for longer in patients with immunosuppression.Additional information for study of prolonged Zika virus RNA detection in semen of patient with immunosuppression.