Tepovirus (Betaflexiviridae), for which the name olive virus T (OlVT) is proposed. A reverse transcription–polymerase chain reaction assay was developed which detected OlVT in samples collected in olive growing regions in Central and Northern Greece, showing a virus prevalence of 4.4% in the olive trees screened. Sequences of amplified fragments from the movement–coat protein region of OlVT isolates varied from 75.64% to 99.35%. Three olive varieties  were infected with OlVT via grafting to confirm a graft-transmissible agent, but virus infections remained latent. In addition, cucumber mosaic virus, olive leaf yellowing-associated virus and cherry leaf roll virus were identified.Field surveys were conducted in Greek olive orchards from 2017 to 2020 to collect information on the sanitary status of the trees. Using a high-throughput sequencing approach, viral sequences were identified in total RNA extracts from several trees and assembled to reconstruct the complete genomes of two isolates of a new viral species of the genus  Olea europaea L.) have been cultivated in the Mediterranean area since prehistoric times. Greek olive cultivation ranks third in the EU, after Spain and Italy [Alphanecrovirus, Tombusviridae), tobacco necrosis virus D (TNV-D) , cucumber mosaic virus (CMV) , arabis mosaic virus (ArMV), cherry leaf roll virus (CLRV), olive latent ringspot virus (OLRSV) , tobacco mosaic virus (TMV) , olive latent virus 2 (OLV-2) , olive latent virus-3 (OLV-3) , strawberry latent ringspot virus (SLRSV) (Secoviridae) and olive vein yellowing associated virus (OVYaV) (Closteroviridae) are officially recognized [Ricinus communis L. [Olives  for which the name olive virus T (OlVT) is proposed. RT–PCR tests identified OlVT, CMV, CLRV and OLYaV infection in several olive trees.This study is the first nationwide survey to investigate the presence of viruses in Greek olive orchards. Visual inspections during the survey did not reveal trees with conspicuous symptoms indicating viral infection and thus confirmed the health status of the trees. To support the survey, a global analysis of olive transcriptomes was performed following a high-throughput sequencing (HTS) approach and subsequently a screening by RT–PCR. HTS permitted the discovery of a new viral species assigned to the genus Tepovirus members, in particular to the prunus virus T (PrVT) isolate Aze239 [Tepovirus , 2OG-Fe(II) oxygenase , viral RNA helicase , RNA-dependent RNA polymerase  in the REP protein, the viral movement protein domain  in the MP protein, and the tricho-coat domain  in the CP protein. Analysis of sample GR170 . Alignment of the two OlVT isolates revealed 96.6% and 84.4% identity in the 5’ and 3’ untranslated regions (UTRs), respectively. At the ORF nt/aa level, the highest values were observed for the CP, with 87.8% and 91.4% identity at nt and aa level, respectively . Phylogeepovirus A showed epovirus B showed OlVT was transmitted by grafting scions from GR168 infected olive field tree to Koroneiki, Arbequina and Frantoio varieties; however, they remained symptomless. Eight months after grafting, OlVT was detected by RT–PCR , and theIn total, six sequences from six trees were obtained by the Sanger sequencing of RT–PCR products (GenBank accessions MΖ020972-MΖ020977), in addition to the sequence of isolate GR170, which was afterwards subjected to HTS. Nucleotide sequence similarity in the partial MP_CP region was in the range of 75.6–99.3%, with the highest diversity between the isolates obtained in two successive years from the same tree in Fthiotida (GR135OL and GR170) and the OlVT isolate from Korinthos (Peloponnese) (GR258OL) .Visual inspections of olive trees in Greek orchards did not reveal conspicuous symptoms indicating viral infection. Viral testing by specific RT–PCR tests revealed viral infections in 13 out of the 158 samples  shared a 91.55–100% sequence identity. One CMV isolate  was grouped in subgroup IA of CMV; the isolates from Korinthia and Argolida in the Peloponnese  were identical and highly similar (99.16%) with that of GR45OL from Agrinio, and they were all clustered in CMV subgroup IB. The Greek olive isolates were 85.55–89.1% similar at the nt level, with the only three olive isolates available in the GenBank, from Albania (LR865402-LR865404).The amplification of the HSP70 homologue was successful in only one out of the two isolates of OLYaV (GR80OL) and resulted in a 339 nt sequence when excluding the primers (GenBank accession MW805382). This isolate showed higher sequence similarity (98.8%) with an isolate from the Ruget variety of French olive material kept in the USDA germplasm collection in California (HQ286488). Our isolate showed only 75.89% sequence similarity with the available Greek olive isolate (LR593885).Plantago major (KF779205) and an Actinidia chinensis (KF779202) isolate, both from New Zealand.Using the CLRV specific primers, we amplified an identical 341 bp long fragment from the 3’ UTR region for the two Greek olive isolates . These sequences showed only 91.9% similarity with two other identical Greek olive isolates already publicly available . Higher sequence similarity (93.84%) was observed with a Tepovirus [Betaflexiviridae family [Tepovirus includes potato virus T [Virus discovery studies in Greek olive orchards using HTS analysis enabled the assembly of the whole genome of a novel species of the genus epovirus  accordine family . In addi virus T  and prun virus T ,10, whil virus T  and cher virus T  have beein-house bioinformatics pipeline optimized for long reads. The number of raw reads obtained per sample was in the range of ca. 0.8 × 106–2 × 106 and was sufficient to generate contigs that covered most of the genome of the two assembled OlVT isolates, whose complete sequence was determined by the complementary Sanger sequencing of amplification products. Analysis of the assembled OlVT genomic sequences clearly indicated the presence of two distinct isolates of the same viral species, with an identity percentage of 81% at nt level, similar to that observed for the PrVT isolates previously described [Total RNA was used as the input for library preparation, rather than dsRNA or sRNAs, which can be used in alternative HTS approaches  and haveThe RT–PCR assay we developed allowed for the detection of OlVT in 4.4% of the tested trees , which were scattered in different areas in Central and Southern Greece. There are no known vectors for tepoviruses, and thus, the only known means of dissemination is infected vegetative propagation material. Nevertheless, similar to PVT , OlVT maThe virus survey results indicate that the infection rate in Greek olive orchards is rather low (8.2%) compared to that in other countries ,22,23,24Besides OlVT, only CMV, OLYaV and CLRV were detected in olive trees, but at low prevalence. CMV is endemic in many economically important crops in Greece . CMV in Closteroviridae [The olive-specific OLYaV, an unassigned species, possibly belonging to a new genus in the family oviridae , was detoviridae ,26,30, coviridae , or woodoviridae , no suchoviridae ,26,32. Hoviridae . In Greeoviridae ; howeverCLRV was also detected in Greek olive trees. The virus is known to cause severe disease in several of its numerous woody hosts . CLRV isFrom our olive virus survey, we can report the identification of a new virus species, OlVT, and the presence of a few known olive-infecting viruses in Greek orchards. None of the viral infections were associated with symptoms and the incidence was low. Contrasting the generally high virus incidence in “in situ” germplasm collections, the good health status of the Greek olive orchards can be stated. The fact that hardwood cuttings, the vegetative propagation materials, are the main means of viral spread, as well as the propagation of Greek autochthonous varieties by local nurseries and the, until recently, only very limited introduction of new materials from abroad may all be reasons for the low virus incidence. We found the new virus OlVT the most frequently, and this virus, which is not associated with symptoms, may have been present for quite some time in our country.In spring 2017, 42 samples were collected from olive trees from different areas in Greece , for virTotal RNA was extracted from leaf tissues using an RNeasy Plant Mini Kit , quality-checked by agarose gel electrophoresis and subsequently processed in the RNA-seq pipeline established at the DSMZ Plant Virus Department. The 42 samples collected in 2017 were processed in 11 pools (MiV300-310). An additional sample collected in 2019 that tested positive for OlVT in field surveys by RT–PCR was sequenced as a single . RibosomTrees were individually tested for the presence of ArMV, CLRV, CMV, OLYaV, OlVT, SLRSV, TMV and TNV-D using RT–PCR tests, with the primers reported in Total RNA was extracted from 0.1 g of material from a mixture of cortical scrapings and leaves (including midrib) from each tree, which was powdered in liquid nitrogen prior to further processing using an RNeasy Mini Kit . RNA was quantified in a NanoDrop 1000 Spectrophotometer (Thermo Fisher Scientific) and examined for integrity by agarose gels electrophoresis. The cDNA was synthetized from 1μg of total RNA using a RevertAid H Minus Reverse Transcriptase (Thermo Fisher Scientific) in the presence of 1.5 μg of random hexamers (Promega), in a 20µl reaction. Total RNAs extracted from freeze dried leaves infected with CMV, TMV, ArMV, TNV-D and SLRSV (DSMZ Plant Virus Collection), and OLYaV and CLRV RNA samples kindly provided by the Istituto per la Protezione Sostenibile delle Piante  were used as positive controls. For OlVT, a partial genome fragment cloned in a pDrive vector  was used as positive control.2 so as to have a final concentration optimized for each primer set  and 1.25 units of DreamTaq DNA Polymerase, in 1X Buffer (Thermo Fisher Scientific). NoRT reactions and cDNA synthesized from healthy plants served as controls. PCR conditions were as follows: initial denaturation at 95 °C for 3 min, followed by 35 cycles of denaturation at 95 °C for 30 s, annealing for 30 s at the appropriate temperature for each primer set and extension at 72 °C for 1 min, with a final elongation step of 72 °C for 5 min. PCR products were separated in 1.5% agarose gels, stained with ethidium bromide, purified with a Nucleospin Gel extraction kit (Macherey Nagel), and Sanger-sequenced in both directions by a commercial sequencing service provider (Applied Biosystems).RT–PCR was carried out in a total volume of 50 μL, containing 1μL of cDNA template, 0.2 µM of each primer , 0.2 mM https://www.ebi.ac.uk/Tools/msa/clustalo/ (accessed on 18 March 2021).Pairwise similarities of OlVT with other tepoviruses were calculated according to the MUSCLE alignment on Sequence Demarcation Tool software (SDTv1.2) . Phylogehttp://www.ncbi.nlm.nih.gov (accessed on 25 March 2021). Subsequently, sequences were aligned using MUSCLE with a percentage Identity Matrix (PIM) calculated using the SIAS tool (http://imed.med.ucm.es/Tools/sias.html (accessed on 25 March 2021)). MEGA version 7.0.26 [The molecular variability of the other viral sequences was investigated by retrieving related sequences from GenBank using BLASTn . Scions were cleft-grafted onto two or three plantlets each of Koroneiki, Arbequina, Arbosana, Mastoidis, Mavrolia Messinias , Frantoio and Coratina . All recipient trees used as rootstocks were previously tested and found to be virus-free. Grafted plants were subsequently kept in insect-proof greenhouses at ambient temperatures ranging from 10 °C to 30 °C. Samples from the newly developing vegetation of the rootstock were taken after 3 months (in May) and 8 months (in October) and tested by RT–PCR for the presence of OlVT.


Rare diseases are difficult to diagnose. Due to their rarity, heterogeneity, and variability, rare diseases often result not only in extensive diagnostic tests and imaging studies, but also in unnecessary repetitions of examinations, which places a greater overall burden on the healthcare system.Diagnostic decision support systems (DDSS) optimized by rare disease experts and used early by primary care physicians and specialists are able to significantly shorten diagnostic processes. The objective of this study was to evaluate reductions in diagnostic costs incurred in rare disease cases brought about by rapid referral to an expert and diagnostic decision support systems.Retrospectively, diagnostic costs from disease onset to diagnosis were analyzed in 78 patient cases from the outpatient clinic for rare inflammatory systemic diseases at Hannover Medical School. From the onset of the first symptoms, all diagnostic measures related to the disease were taken from the patient files and documented for each day.The basis for the health economic calculations was the Einheitlicher Bewertungsmaßstab (EBM) used in Germany for statutory health insurance, which assigns a fixed flat rate to the various medical services.For 76 cases we also calculated the cost savings that would have been achieved by the diagnosis support system Ada DX applied by an expert.The expert was able to achieve significant savings for patients with long courses of disease. On average, the expert needed only 27 % of the total costs incurred in the individual treatment odysseys to make the correct diagnosis. The expert also needed significantly less time and avoided unnecessary examination repetitions.If a DDSS had been applied early in the 76 cases studied, only 51–68 % of the total costs would have incurred and the diagnosis would have been made earlier. Earlier diagnosis would have significantly reduced costs.The study showed that significant savings in the diagnostic process of rare diseases can be achieved through rapid referral to an expert and the use of DDSS. Faster diagnosis not only achieves savings, but also enables the right therapy and thus an increase in the quality of life for patients.The online version contains supplementary material available at 10.1186/s12913-021-06926-y. In Europe diseases are considered rare when they affect fewer than 1 in 2.000 persons . BecauseSurveys conducted in the USA and Great Britain revealed that the average patient visits eight physicians, four outpatient departments and four specialists until the correct diagnosis is found while getting two to three false diagnoses on the way to it .A further problem is that there is often no interdisciplinary exchange between the treating physicians who see the patient in the course of their diagnostic odyssey .Extensive tests and imaging examinations are thus unnecessarily repeated resulting in a delay in treatment and as a consequence in higher health care costs . CompareTo provide a point of contact for patients and physicians who are at a loss and to find the right diagnosis in a timely manner, interdisciplinary special centers for rare diseases have been established. On the one hand, experts for a wide range of diseases work in these centers who exchange ideas in an interdisciplinary way and have a large base of knowledge and experience, and on the other hand the centers offer all the necessary structures and resources to diagnose and treat patients with rare diseases .Diagnostic decision support systems promise to make diagnosis even easier. Through the use of artificial intelligence, physicians should be supported in their diagnostic considerations -12. The A retrospective study conducted at the Outpatient Clinic for rare inflammatory systemic diseases at the Hannover Medical School reveals that there is a great potential for diagnostic cost savings through the use of expert diagnostic decision systems. The study shows that diagnostic decision systems can achieve a significant acceleration of the diagnostic process .Based on these findings it is the aim of this study to evaluate the reduction in diagnostic costs incurred, resulting from rapid referral to an expert and from the use of a diagnostic support system for rare diseases.A retrospective study was conducted in the outpatient clinic for rare inflammatory systemic diseases at the Hannover Medical School.The outpatient clinic for rare inflammatory systemic diseases is an expert center. The cases we studied  were assFirst, we determined all diagnostic costs incurred in these cases. Secondly, we investigated the potential reduction in diagnostic delay that could be achieved by the use of an DDSS. From these data, we calculated the potential reduction in incurred costs that could be achieved.The rationale was that if a diagnosis is correctly suggested by a DDSS, targeted diagnostics will enable a rapid diagnosis thus leading to a reduction in costs.Patient cases from a previous study  were selCases with a particularly severe course and diagnostic odysseys were prioritized according to a subjective assessment by the head of the Rare Inflammatory System Diseases Clinic . The preFirst, the date on which the patient first presented to a physician with disease-specific symptoms was extracted from all files.All data from subsequent diagnostic examinations related to the disease were also documented in tabular form until the final diagnosis was made. In the tabular overviews it was noted for each date whether it was a physician’s appointment or an inpatient hospital stay. In addition, all diagnostic examinations carried out on the individual dates were recorded in the tables down to the smallest detail. For patients diagnosed by the expert for rare diseases at the Hannover Medical School, the date of first presentation to the expert was also recorded.There are three ways in which patients come to the expert center. Either as an acute emergency, by referral from a specialist for diagnostic clarification, or patients with an already confirmed diagnosis of a rare disease.For this reason, the patients were divided into three groups for the economic analyses:Diagnosis by the expert after a long diagnostic odyssey:This group included patients who had particularly long courses of disease with diagnostic odysseys. Specialists reached their diagnostic limits with patients in this group and therefore referred the patients to the expert, who was finally able to make the correct diagnosis.2.Diagnosis by the expert in an acute course:Patients who had particularly severe, life-threatening courses and were referred to the expert as an emergency in a period of less than one month or had to be treated directly by the expert were assigned to this group.3.Diagnosis by external medical specialists:If a diagnosis was made outside of Hannover Medical School and the patient was referred to the expert at Hannover Medical School for further treatment due to lack of expertise, the patient was assigned to this group.The prototype of the knowledge-based, probabilistic diagnostic support system Ada, named Ada DX, was used in the study .First, the point of time with the first disease-relevant findings was recorded for each patient. For each patient the data of all the documented visits at health care providers were extracted from the medical record. Data from written and dated documentations were taken into account, such as medical discharge letters, referral forms and documented test results from laboratories and pathologists. All data were recorded on a year and month basis . From thThe findings were assigned to the time of the respective visit, but not to previous visits based on medical history information. Previously collected findings were retained for future visits if they were not refuted by other documented information . While aWith the entry of first symptoms Ada DX constructs two lists:On the one hand, a list is created in which diseases are arranged according to the fit of the symptom constellations to the specific disease phenotypes and, on the other hand, a “probability disease list” in which diseases are assigned to the symptom according to probabilities taking into account epidemiology .The lists are adjusted with each visit at which new diagnostic findings occur.To assess the possible impact on the time of diagnosis the disease suggestions in the “fit”- disease list are included. The “fit”- disease list was chosen because it is not expected that rare diseases are diseases with a high probability of occurrence .They are rather diseases which are only considered as a tentative diagnosis which are suggested due to the fit of the constellation of findings .As Ada DX is still a prototype, the system does not yet cover a sufficient number of diseases from the broad spectrum of rare diseases.At the time of the previous study , Ada’s kAll included patient cases were evaluated individually. This means that medical services provided in connection with the disease were extracted from all patient records since the first signs of the disease appeared. The medical services were tabulated by days and years.Since our entire patient collective had statutory health insurance, the “Einheitliche Bewertungsmaßstab (EBM)” was used as the basis for our health economic analyses.EBM is a remuneration system for statutory health care in Germany. With the help of billing numbers, each service provided by a physician is assigned to a specific remuneration.In the case of patients with statutory health insurance, physicians do not receive their salaries directly from the patient, but bill the Association of Statutory Health Insurance Physicians  for their services according to EBM. The “Kassenärztliche Vereinigungen (KVs)” are corporations under public law that organize medical care through statutory health insurance funds .For our analyses, we used the current online version of EBM as of 01.01.2021. To be able to retrospectively determine the costs incurred, we assigned the corresponding cost parameters based on the EBM to the medical services we tabulated from the individual patient records.Depending on the particular analyses, single or multiple EBM numbers were required to represent the specific level of costs incurred. For the calculation of treatment costs, physician fees for medical services were added according to the billing numbers.However, there are certain rules for billing. For example, EBM numbers can mean exclusions for other EBM numbers that are billed in the same session. In this case, these cannot be added together. If a particular examination could not be assigned to a single billing number in the EBM pool, a flat rate was calculated based on similar examination methods.Using the method described above, all direct costs incurred could be calculated retrospectively. Data cleaning, sorting, and manipulation, as well as calculations, were primarily accomplished using R and Excel.To determine the potential savings of diagnostic decision support systems, the timestamps for the times when AdaDX displayed the correct diagnosis among the top-5 or as the top-1 suggestion were transferred from the study by Ronicke et al.  and inteTo calculate the total costs that would have incurred if the treating physicians had used a diagnostic decision support system, the fees of all physician services were added that were actually provided up to the date of the hypothetical diagnosis.“diagnosis by the expert after a long diagnostic odyssey”), 8 cases to group two (“diagnosis by the expert in an acute course”) and 16 cases to group three . Table 93 patient cases were analyzed. 15 cases were excluded due to missing data. Of the remaining cases, 54 cases were assigned to group one  showed that the patients had to visit an average of 8 different physicians/clinics until they received a correct diagnosis. This was also reflected in the economic analyses:The economic analysis of group one showed that the total costs incurred until a correct diagnosis was made averaged €8.756,49 . This represents the average amount of total direct costs incurred until a successful diagnosis could be made. Dividing total costs into two distinct sections, subdivided by the date a patient was referred to the expert center, yields a mentclass2pt{minimBased on our calculations, we estimate the cost reductions enabled by a DDSS to be up to 50 %. By the time the correct diagnosis was displayed among the top 5 suggestions, an average of €4.439,39 in physician costs would have been incurred with the help of the DDSS. Until the DDSS suggests the correct diagnosis first, an average of €5.851,01 incurred, which is ~ 67 % of the total costs. During the analysis of the data, it was noticed that certain analyses were repeated several times and at different times in the patient’s medical history.The distribution shown in Fig. Genetic examinations turned out to be conspicuously cost-intensive in the analyses. For this reason, a sub-analysis of the costs incurred by genetic analyses was created. Genetic testing was almost exclusively initiated by the expert. These sub-analyses of the diagnostic costs of the expert showed that in cases (n = 15) where genetic analyses are performed Fig. , signifiWith an average of €12.234,49 in diagnostic costs, cases in which genetic analyses were performed were significantly higher in total costs than the average of study participants.The small group 2 (n = 8) which included all the patients that had particularly severe, life-threatening courses and had to be diagnosed by the expert as quickly as possible showed a slightly different distribution of the costs.As a rule, these patients visited on average only 2–3 different physicians/clinics until the correct diagnosis was made, but they still caused relatively high costs.It is noticeable that the majority of the costs incurred occurred after the patients were transferred to the expert center Fig. . This coGroup 3 consisted of 16 patients who were correctly diagnosed outside of MHH and visited an average of 3–4 different physicians/clinics prior to diagnosis. Due to lack of expertise, patients in this group were referred to the expert at MHH for further treatment.The total costs of €8.000,75 were almost as high as in group 1 and there incurred obviously no costs at MHH  of 2020 showed that every third German had to wait more than three weeks for an appointment with a specialist .In comparison a more structured and targeted approach was noticeable in the treatment by the expert. Directly for the presentation all documents about the examinations carried out so far had to be submitted by the patient. Through the review of the documents, it was possible to gather first conclusive, diagnostic information.Also, the performance tables we prepared for the analyses clearly showed that the expert conducted more extensive investigations. More laboratory parameters were collected than in most of the previous examinations and there was an interdisciplinary exchange. In addition to his/her expert knowledge the location is undoubtedly an advantage for the expert. The connection to the clinic makes imaging examinations possible relatively fast and long waiting times cease through rapid internal consultations.As 80 % of rare diseases are of genetic origin  and a coThe results of the economic analyses point to a saving potential through DDSS. On average, DDSS Ada DX would have caused less costs in diagnostics in all the three groups. The cost savings can be attributed to two reasons:The first causal reason is that unnecessary repetitions of examinations can be avoided through the use of DDSS as all the examinations are stored in the system.The second even more important cause of savings is a quicker diagnosis through DDSS. Ronicke et al.  could shThe explanation for a quicker diagnosis and thus cost reduction lies in the diagnostic quality improvement through DDSS , 18, 20.With the awareness that a patient may be suffering from a rare disease, the GP, for example, as the first contact could consult an expert and thereby increase the diagnostic quality , and if It can be concluded that every medical professional can qualitatively improve their diagnostic skills with DDSS  and therThe condition necessary to benefit from the above advantages of DDSS is to record as many diseases as possible and to permanently develop the knowledge base further according to the latest scientific findings. However, should a decision-making tool not be sufficiently developed and use outdated literature, it represents more a security risk than a benefit .Also, an optimal integration into the stressful clinic and practice every day work is of great importance for fully utilizing the efficiency of DDSS . A simplAfter the saving potential through DDSS has now been widely explained, it must finally be mentioned that in addition to the possible training costs also costs occur for the integration and installation of DDSS .Taking an overall view of the most analyses that were examined in this study, the median costs tended to be below the mean value indicating that a small proportion of the patients with high costs have driven up the average costs resulting in a positive imbalance. The typical (median) patient caused in all likelihood lower costs than indicated here while patients with especially long odysseys caused above-average costs, what was to be expected.In principle, a retrospective approach is suitable for the studies of economic benefits through expert knowledge and DDSS. A disadvantage of the retrospective approach is that an evaluation of the results is only explorative.The patient cases we studied are only a small grouping of special rare diseases, which is why the results cannot be applied to all rare diseases.There are some disadvantages in data entry due to the retrospective study design:Although great care was taken to ensure the completeness of the patient files, it must be assumed that in some cases, due to the overall very long course of the disease documentations of medical examinations that were made before presentation to the expert, are missing. It can thus be presumed that the costs can even be higher before expert presentation.Although case entry was based on chronological entry of written documented information from medical records to reduce retrospective bias and retrospective misinterpretation, it was not blinded because patient cases were already known .Another limitation due to the retrospective study design is that the effects of the diagnostic support system were not measured in clinical practice. Some authors are concerned that there may be increased clinical testing in inexperienced clinicians in clinical practice . HoweverAs the evidence increases, the probability differences between the diseases in the lists grow. However, it must be noted here that the responsibility for determining when a diagnosis can be considered correct lies with the treating physician. Accordingly, the suggestion of a correct diagnosis by the system does not mean that it will be diagnosed. Therefore, diagnostic time gains and cost savings from the system may vary by physician.As far as the economic analyses are concerned, our results are limited to costs incurred in Germany. Viewed internationally, for example in comparison with the USA, the diagnostic costs can be many times higher. An international comparative study would certainly be of interest.The patient collective comprised patients from all over Germany, some of them who travelled far to be examined by the experts. This offers potential for a study on indirect costs, such as those caused by journeys to physicians or productivity restrictions that arose in the course of the treatment odysseys. Angelis et al. showed that depending on the type of rare disease massive indirect costs can also arise, which can even exceed the direct costs . In addiIt would also be of interest to determine the impact of a correct diagnosis on patients’ quality of life in a potential study.The study showed that in case of rare diseases high diagnostic costs incur. The later the referral to an expert the more extreme became the economic burden. With the help of expert knowledge, the diagnostic processes could be accelerated and unnecessary examinations avoided.Ada DX provided correct suggestions for rare diseases before clinical diagnosis in most patient cases which was reflected in the economic analyses with a high potential for savings.DDSS not only saves costs through earlier diagnosis, but also by avoiding unnecessary repeat examinations. It must be considered that system implementations require expenditure - but this should be compensated for by the expected benefits.In conclusion, based on this study, rapid referral to an expert and the use of a DDSS promise a cost-effective, faster diagnosis in rare disease patients.Additional file 1Additional file 2Supplementary Table 2


Buteo buteo) has a considerable chance of acquiring the infection and therefore may function as bio-sentinel for the presence of virus in wildlife. This study aimed to determine the virus distribution and associated pathological changes in the tissues of Common buzzards that died with HPAI H5 virus infection during the 2020–2021 epizootic. Eleven freshly dead, HPAI H5 virus-positive Common buzzards were necropsied. Based on RT-PCR, all birds were systemically infected with HPAI H5N8 virus, as viral RNA was detected in cloacal and pharyngeal swabs and in all 10 selected tissues of the birds, with mean Ct values per tissue ranging from 22 for heart to 32 for jejunum. Based on histology and immunohistochemistry, the most common virus-associated pathological changes were necrotizing encephalitis (9/11 birds) and necrotizing myocarditis (7/11 birds). The proventriculus of two birds showed virus-associated necrosis, indicating tropism of this virus for the digestive tract. Our advice is to collect at least a miniset of samples including brain, heart, liver, and spleen, as these tissues were positive both by RT-PCR and for virus-antigen-associated lesions.Highly pathogenic avian influenza (HPAI) in wild birds is a major emerging disease, and a cause of increased mortality during outbreaks. The Common buzzard ( The 2020–2021 epizootic of HPAI H5 virus in Europe was the biggest on record in both wild birds and poultry. Based on the animal disease notification system of the affected countries, over 1000 detections of HPAI H5 virus were reported in wild birds4. The subtype H5N8 was the first and most widespread genotype circulating during the first months of the epizootic 4. Nevertheless, the virus reassorted multiple times, so that sixteen distinct genotypes co-circulated. Early surveillance showed that the first incursions in Europe of the HPAI H5N8 virus occurred in the fall of 2020; however, the epizootic season extended into winter–spring–summer of 2021, with the outbreaks mainly clustered in two peaks: in November 2020 and March 20214. The highest number of detections in wild birds occurred in waterfowl species (order Anseriformes). The second highest number of detections occurred in raptors ; nine different raptor species were found positive, and Common buzzard  accounted for the highest number of detections in raptors4. These birds are infected predominantly by ingesting infected prey. Medium-sized raptor species like the Common buzzard, that can hunt and feed on sizeable birds and ingest a high quantity of infected meat, are considered to be at high risk of becoming infected with HPAIV, and to die of related disease11. In this context, the investigation of Common buzzard mortality for HPAI virus could be used in addition to waterfowl mortality as a passive-surveillance system for early warning and duration of the presence of the virus in wild bird populations.Highly pathogenic avian influenza (HPAI) is a major emerging disease, and a cause of mass mortality in wild birds during outbreaks15. Studies on those birds showed that HPAI viruses in wild raptors are primarily neurotropic, and that infected raptors usually present with neurologic signs due to encephalitis, often resulting in death15. The condition of some of the carcasses in those reports was suboptimal due to a combination of autolysis, scavenging and freezing, all factors that may impede detailed pathological assessment, in particular identification of specific cell types and evaluation of autolysis-prone tissues such as enteric epithelium.Wild raptors also have been affected by severe HPAI disease during earlier HPAIV epizootics6. Updated knowledge of the virology and pathology of infections with more recently circulating viruses is necessary in order to better understand the current pathogenesis of HPAI in Common buzzards; to provide updated guidance regarding the key tissues to collect for HPAI diagnosis at autopsies; and to compare the presence of virus in cloacal and pharyngeal swabs, routinely tested for HPAIV surveillance, with the presence of virus in the main tissues.Despite the high number of reported infected Common buzzards, we are not aware of any studies on the pathogenesis of infection with recent HPAI H5 viruses in this species. This is particularly relevant because, since the 2005–2006 HPAI H5N1 outbreak, HPAI H5 viruses have evolved rapidly and currently are thought to persist longer in wild bird populationsTo determine the virus distribution and associated pathological changes in the tissues of Common buzzards that died with HPAI H5 virus infection in the Netherlands during the 2020–2021 HPAI epizootic, a collaboration was established among citizen scientists, animal organizations and researchers, aiming to retrieve and examine freshly dead Common buzzards. Eleven Common buzzards were examined and necropsied. All the birds tested positive for HP H5N8. This study documents the virological and pathological findings associated with 2020–2021 HPAI H5N8 infection in Common buzzards and discusses its implications.All birds tested negative for antibodies against avian influenza virus NP.http://www.gisaid.com, A/Common Buzzard/Netherlands/4/2020, isolate EPI_ISL_1575129). Based on virus culture in MDCK-cell culture, three birds in total tested positive in pharyngeal (B9 and B11), and cloacal (B3 and B9) swabs.Pharyngeal and cloacal swabs were positive for HPAI H5N8 virus by RT-PCR, with comparable viral RNA levels in pharyngeal and cloacal swabs Table . SequencAll tissues tested were virus-positive by RT-PCR Table , indicatBirds did not present external lesions, and were in moderate to good state of nutrition. The crop of the birds was empty (an indication that they had not fed at least for 24 h prior death), and four birds had scarce food remains from a recent meat meal in the ventriculus. At internal examination only two birds presented gross abnormalities. When present, the main lesions were multifocal, well-delimited hemorrhages in the heart and in the brain of one bird. The liver of a different bird appeared swollen and with rounded margins. The other organs appeared grossly normal.Several organs, and in particular the brain, heart, liver, and spleen, showed histological lesions that colocalized with moderate to abundant presence of viral antigen Fig. . The braThis study describes the virological and pathological findings of Common buzzards infected with the 2020–2021 HPAI H5N8 virus. These analyses showed that the main lesions were HPAI virus-associated inflammation and necrosis in multiple tissues including brain and heart, confirming HPAI as cause of death or severe disease.16. The Common buzzard is mainly a resident bird, which generally inhabits woodlands but is adaptable to wetlands17. Its feeding behavior as an opportunistic predator and scavenger has the potential to expose it to HPAIV-infected prey. Given these predisposing biological traits, it is not unexpected that Common buzzards accounted for the highest number of HPAI virus detections in raptors during the 2020–2021 epizootic.The Common buzzard presents with several characteristic traits that make it a valuable bioindicator of HPAIV presence in wildlife. It is a medium-sized raptor, present almost throughout Europe. In the Netherlands, its population has been stable since 1970 with an estimated maximum winter population of 30,000–50,000 individuals19. This study also supports those findings, as the most consistent lesion in Common buzzards was viral encephalitis, with confirmed presence of viral antigen in affected neurons. In addition to the nervous system, all the tissues tested of the Common buzzards were positive for virus based on RT-PCR and showed infection-related, histological lesions, indicating that HPAI H5N8 virus infection in the Common buzzard causes systemic disease.Previous studies showed that HPAI viruses in raptors are highly neurotropic and cause severe neurological diseaseThis study showed that HPAI H5N8 virus is also highly cardiotropic, as the myocardium of the Common buzzards contained the highest amount of virus based on RT-PCR Table , and vir20. Interestingly, the proventriculus of two birds in our study showed necrotic lesions with viral antigen. This finding further supports the oral route of infection, although we cannot exclude the possibility that the proventriculus was infected via the hematogenous route. It also provides new records of HPAIV enterotropism in wild birds. The adaptation to the intestinal tract is a mechanism recently reported for HPAI H5N8 virus, that may allow a more efficient fecal–oral transmission in wild birds5.The Common buzzard is considered to be infected via the oral route by ingesting HPAIV-infected preys. Transmission of HPAIV from ingesting infected chicken meat has been experimentally confirmed in raptors9. In this study, cloacal and pharyngeal swabs had comparable RNA-levels, and both were adequate for the detection of the virus. The tissue analysis by RT-PCR showed that heart, brain, and air sac had highest viral RNA concentrations compared to other organs. Although not confirmed by a quantitative real time PCR, the results obtained by RT-PCR are well supported by histopathology and immunohistochemistry. Our advice for diagnostic pathologists is to collect at least a miniset of samples including brain, heart, liver and spleen, as these tissues are relatively easily sampled and were positive by both RT-PCR and for virus-antigen-associated lesions. For virus diagnosis of Common buzzards found dead , it is enough to collect pharyngeal and cloacal swabs, because they were positive by RT-PCR with Ct values that were comparable to those in most tissues .Real time PCR (RT-PCR) is the preferred test for HPAI virus detection for active and passive bird surveillance19. All the birds in our study were females. Females are larger than males , thus it is possible that female raptors are easier to find during surveillance or that there are sex-associated differences in feeding patterns.We did not detect antibodies against avian influenza virus NP in the sera of the Common buzzards in this study. Most of the birds (8/11) were juveniles in their first year of life, and likely they did not have protective antibodies from previous infections, as this was the first time in their lives that they experienced a HPAI epizootic. The absence of antibodies indicates also that the Common buzzards died acutely soon after infection, similarly to experimentally infected raptors that did not seroconvert before early deathThis study showed that HPAIV infection in Common buzzards produced severe systemic disease, and subsequent acute death based on the stage of the pathological changes and absence of serum antibodies. Cloacal and pharyngeal swabs were comparable in detecting the infection. Many organs contained viral RNA; with heart, brain and air sac containing the highest amount of viral RNA. The proventriculus of two birds showed virus-associated lesions, implying a possible adaptation of the virus to the gastro-intestinal tract.In November 2020, during the first peak of the 2020–2021 HPAIV epizootic, 11 wild Common buzzard carcasses were presented at Erasmus MC, Rotterdam, for pathological and virological investigations. These birds were found by citizen scientists; nine birds were found dead, and two were still alive but subsequentially were euthanized after showing severe neurological signs of disease, such as torticollis and body tremors. The carcasses were refrigerated, and autopsies were performed at Bio Security Level 3 settings within 24 h after retrieval.The birds appeared freshly dead and in good state of preservation. All the birds were female based on the presence of an oviduct and ovaries in the coelomic cavity; eight were juveniles (first year) and three were adults (second year) based on their plumage. Pharyngeal and cloacal swabs were collected for virology from each bird, using sterile cotton swabs placed in 1 ml of virus transport medium. Tissue samples from nasal turbinate, air sac, trachea, lung, heart, liver, spleen, kidney, pancreas, stomach, jejunum, duodenum, colon, and brain were collected for virology and kept at -80 °C until analysis. Duplicate samples of the same tissue were collected for histopathology and immunohistochemistry and fixed in 10% neutral-buffered formalin until analysis. Blood from the heart was collected in a plain 2 ml tube, and centrifuged for 15 min at 1500 g.Serology. Sera were tested for nucleoprotein (NP)-specific antibodies with a commercial blocking enzyme-linked immunosorbent assay (bELISA) . Samples were tested according to the manufacturer’s instructions. A sample was considered NP positive when the signal-to-noise ratio  was 0.5 or lower.10. Extracted total RNA of tissue samples, pharyngeal and cloacal swabs were tested for the presence of influenza A virus matrix gene-fragment; pharyngeal and cloacal swabs were further subtyped using real-time RT-PCR targeting fragments of the H5 and N8 genes. Samples were characterized as HPAI H5 virus by detection of a multi-basic cleavage site upon Sanger sequencing of the HA gene9. Full genome sequence of the virus isolated from the pharyngeal swab of Common buzzard 9 (B9), was obtained by Sanger sequencing. In order to assess the excretion of infectious virus, pharyngeal and cloacal swabs were also tested by virus culture. Virus titers of the pharyngeal and cloacal swabs were obtained with triplicate tenfold serial dilutions in confluent layers of Madin-Darby canine kidney (MDCK) cells10.Tissue samples were first weighed and then homogenized with a FastPrep 24  in Hankʾs balanced salt solution and centrifuged briefly before dilution in lysis buffer for RNA extraction10.Tissues samples were embedded in paraffin, sectioned at 4 μm, and stained: with hematoxylin and eosin (HE) for presence of histopathological changes; with an immunohistochemical test, using monoclonal antibody against nucleoprotein of influenza A virus as a primary antibody, for detection of influenza viral antigen


With the challenges that dengue fever (DF) presents to healthcare systems and societies, public health officials must determine where best to allocate scarce resources and restricted budgets. Constrained optimization (CO) helps to address some of the acknowledged limitations of conventional health economic analyses and has typically been used to identify the optimal allocation of resources across interventions subject to a variety of constraints.Wolbachia-infected mosquitoes and paediatric vaccination) within the constraints of a fixed budget, set no higher than cost estimates of the current vector control programme, to minimize the number of dengue cases and disability-adjusted life years (DALYs) lost. Epidemiological, cost, and effectiveness data were informed by national data and the research literature. The time horizon was 10 years. Scenario analyses examined different disease management and intervention costs, budget constraints, vaccine efficacy, and optimization time horizon.A dynamic transmission model was developed to predict the number of dengue cases in Thailand at steady state. A CO was then applied to identify the optimal combination of interventions  with corresponding coverages under lower bound  and upper bound  budget ceilings. Scenario analyses indicated that the most impactful situations related to the costs of Wolbachia and paediatric vaccination with decreases/ increases in costs of interventions demonstrating a direct correlation with coverage (increases/ decreases) of the respective control strategies under examination.Under base-case budget constraints, the optimal coverage of the two interventions to minimize dengue incidence was predicted to be nearly equal  over a 10-year time horizon, within the constraints of the estimated budgets for vector control in the absence of vaccination and The online version contains supplementary material available at 10.1186/s12889-021-10747-3. Aedes Aegypti species and, to a lesser extent, Aedes Albopictus, with both prevalent in the country. All four of the dengue virus serotypes  circulate in Thailand  coverage 0–100%) is divided by 100 and then 10,000 simulations (i.e. 100 × 100) are run. The programme eliminates all combinations that exceed the pre-specified budget constraint and retains only those permutations that fall within the programme scope. The process then concludes with the presentation of the optimal combination of the two interventions that minimize the number of dengue cases and DALYs (subject to budget constraints).In the grid search, the parameter space of the respective interventions , exposed to infection (Eh), infected and infectious (Ih), and recovered (Rh) compartments. The total human population (Nh) is equal to the sum of the populations of humans in all human compartments, i.e. Nh = Sh + Eh + Ih + Rh. The life cycle of the mosquito is represented by three infection phases, susceptible mosquitoes (vectors) (Sv), exposed (incubating) mosquitoes (Ev), and infected and infectious mosquitoes (Iv). The total vector (mosquito) population is equal to Nv (i.e. Nv = Sv + Ev + Iv). The complete model without study interventions is presented in the system of equations below:In the current study, we do not model population age structure and assume only one ‘global’ dengue serotype is circulating, as the use of a single serotype/ infection model was considered sufficient to answer the research question under investigation and adhere to the principle of parsimony. The human population is divided into four compartments comprising: humans susceptible to dengue infection , exposed (Ew), and infectious (Iw) mosquitoes, where Sw + Ew + Iw = Nw. In total, the model comprises 11 compartments; four for the human population, three each for the two mosquito populations, and one for vaccination. The complete model with study interventions is presented in the system of equations below:In the presence of h) is treated as constant, i.e. births balance deaths at rate μh with no immigration of infected individuals into the human populace.The total human population  and is The population is homogeneous, which means that every individual in a compartment is homogenously mixed with the other individuals.Mosquito bites are homogeneously distributed amongst all human hosts, which means that each mosquito can bite any human host with equal probability.There is no natural protection, i.e. humans and mosquitoes are assumed to be born susceptible and losses of immunity are not considered, nor are maternally derived antibodies.The mosquito has no resistant phase due to its relatively short life expectancy.The coefficient of transmission of the disease is fixed and does not vary seasonally in the base case.Initial conditions were derived by running the model to equilibrium steady state without any control interventions. Key model assumptions are as follows:Table Similar to Knerer et al. , epidemiThe average number of reported cases was adjusted by an expansion factor of 8.5 to derive total ‘actual’ dengue cases. This is consistent with suggested expansion factors in South-East Asia for converting total reported dengue cases into estimated ‘actual’ cases, ranging from approximately 3.8 in Malaysia, to 8.5 in Thailand and 19 in East Timor . Similarβhv) and vector(βvh) were calibrated using a gradient-based optimization loop that minimized the mean-square difference between the model and recorded observations (adjusted for under-reporting). Model code was written in MATLAB and the optimization function ‘fminsearch’ was used. At steady state, the model predicted an average of approximately 697,000 dengue cases per year in Thailand for all age groups combined. This compares to the average number of reported DF/ DHF cases in Thailand for the period 2008–2012 [n = 701,256), which indicates a good fit between observed and predicted data.Model estimates were calibrated with figures reported by the National Epidemiological Surveillance in Thailand in 2008–2012 –64 multi008–2012 –64 adjus008–2012 , all ageDALY estimates were taken from Knerer et al. , which wAs with the outcomes described above, we derived disease as well as intervention costs from our earlier paper  and highi.direct medical costs for inpatient and outpatient dengue cases.Payer perspective:ii.direct medical costs for inpatient and outpatient dengue casesdirect non-medical costs for inpatient and outpatient dengue casesindirect costs for inpatient and outpatient dengue cases.Societal perspective:In brief, unit costs (per DF episode) derived from Shepard et al.  were useTotal costs are comprised of direct medical costs and intervention costs (detailed below) from the payer perspective; and direct medical costs, direct non-medical costs, and indirect costs, in addition to intervention costs, from the societal perspective.Studies with applicable unit costs , 72 and Cost inputs and other values are presented in Table Where costs were ascribed to unreported cases for type of treatment, it was assumed that any treatment costs for unreported cases were on an outpatient basis only , in line with the likely less severe nature of these cases , 69. UnrAny economic costs associated with premature mortality (i.e. productivity loss and lifetime earnings foregone) were not included in calculations due to concerns over the risk of double counting benefits associated with averted deaths , 80.Wolbachia infection [Wolbachia intervention (due to uncertainty in the costs of such an intervention): firstly, a Wolbachia cost per dengue case averted of $1  and secondly, a Wolbachia cost per person covered of $1 .Wolbachia cost of $1 per dengue case averted being used in base-case analyses and a Wolbachia cost per person covered of $1 being examined in scenario analyses.In the current study and continuing with the exploratory nature of analyses, we use similar costs to those above, with a Wolbachia programme ranging from approximately $12 to $21 per person. This includes both urban areas (~$12 per person covered) and rural areas (~$14–21 per person covered).As an additional scenario analysis, we also use a cost per person covered of $15.05  adjusteWolbachia implementation to the point where Wolbachia-infected mosquitoes had reached steady state/ fixation in the population.Costs were assigned over 4 years to simulate accelerated For vaccination, a cost of $40 per vaccination course and assumed vaccine administration costs of $4 was used .Costs were discounted at 3% per annum as suggested by Thailand’s Health Technology Assessment guidance and the WHO , 82.Scenario analyses were carried out on different features and input data of the model to test the robustness of simulated findings and identify key parameters of influence that may impact base-case findings. Analyses predominantly focused on different budget constraints, disease management costs (i.e. unit cost profile), intervention costs, vaccine efficacy, and time horizon. An additional scenario was examined in which the parameter search space for paediatric vaccination was restricted to 70–100% (rather than 0–100% in the base case). Table Wolbachia and vaccination coverage, dengue reductions, and associated costs (including disease and intervention costs) stratified by different budgetary constraints.At steady state, the simulation model predicted approximately 7 million symptomatic dengue cases (7.175 million) in Thailand for all age groups combined over a 10-year period. The estimated total DALYs lost in this period were approximately 67,831 with cumulative disease costs of $338 million from the payer perspective. In the following sections, we detail Wolbachia and paediatric vaccination (to minimize dengue incidence) comprised 100% coverage of each intervention. In this situation, a reduction of approximately 6 million dengue cases and 58,000 DALYs with an associated budget of $679 million, was forecast over 10 years  although with different constituent costs . Corresponding intervention coverages estimated under lower and upper bound budgetary limits were Wolbachia 54% and paediatric vaccination 10% for the lower and Wolbachia 67% and paediatric vaccination 100% for the upper budget ceilings respectively. When resources become limited under the lowest budget constraints (≤ $251 million), Wolbachia has more impact on the population level of disease (as it becomes more affordable relative to vaccination cost) and vaccination is effectively reduced to a targeted hotspot control strategy.In the unconstrained case, i.e., absence of budget restrictions or limits on investment  in blue to the absence of any budget constraint in red.Figure Wolbachia and vaccination coverage that are feasible without exceeding upper limit budget constraints (as an example). In practical terms, this could take the form of either, for example, 100% Wolbachia coverage combined with approximately 74% paediatric vaccination coverage or approximately 67% Wolbachia coverage combined with 100% paediatric vaccination coverage. In effect, this suggests that one or other intervention can have very high coverage (i.e. 100%), but not both interventions without exceeding the budget ceiling. Similarly, from a more restricted budgetary standpoint , but high levels of coverage for both interventions together are not compatible within budget constraints. Depending on the public health goal, trade-offs may need to be determined to fulfil the desired objective. These trade-offs become less urgent as the budget available to fund interventions expands (as observed in the current study when the budget constraint is loosened). Additional file Wolbachia and vaccination coverage.The heatmap illustrates, for example, the limits of intervention combinations by budgetary ceiling; the chart showing the highest possible combinations of Wolbachia and paediatric vaccination with decreases and/ or increases in costs of interventions demonstrating a direct correlation with the coverage (increases and/ or decreases) of the interventions under examination. For example, a reduction in vaccine acquisition costs results in a corresponding increase in paediatric vaccination coverage (100%) and a smaller reduction in Wolbachia coverage (i.e. more resources are directed to the lower-cost vaccination programme). Similarly, a decrease in Wolbachia costs gives rise to greater coverage  as well as an increase in vaccination coverage (i.e. more resources are directed to vaccination). Conversely, as intervention costs increase, more investment flows to the less costly option and coverage increases as a result. When substituting a different and, in this case, lower unit cost profile [Wolbachia, reflecting the influence of unit costs in this regard. An increase in vaccine efficacy, from 73% to 80% representing the best case, results in greater resources being targeted towards vaccination and less to Wolbachia . A decrease in vaccine efficacy, from 73% to 50% representing the worst case, results in the converse with resources directed more to Wolbachia and away from vaccination. When paediatric vaccination is restricted to the range 70–100% (in the grid search), left over investment above the minimum vaccination coverage of 70%, flows to Wolbachia (22%) and away from vaccination in order to optimally maximise public health outcomes at the lowest cost.When assessing the impact of alternative situations as part of wider scenario analyses Table , approxi profile , more fuWolbachia infection (wMel strain) and vaccination on the dengue disease burden in Thailand was investigated as part of a constrained optimization problem. The primary goal of the exercise was to identify the best combination of vaccination and Wolbachia to minimize the number of dengue cases (and DALYs lost) subject to explicit budgetary constraints. We used a case study of Thailand for the analysis and set the budget constraint to be equal to the estimated current per capita spend on vector control in Thailand [This study aimed to provide further insights into the prioritization and combination of dengue control strategies. The impact of Thailand .The paper acts as a complement to a CEA conducted by the same authors , which iThe base epidemiological model underpinning the optimization analyses was shown to calibrate well at steady state with average reported symptomatic DF cases in Thailand for the years 2008–2012 –64, adjuWolbachia and vaccination (paediatric) can produce analogous reductions in the incidence of dengue cases yet have different budget impacts (comprising disease and intervention costs) to achieve the respective coverages. In the base case, the optimal mix between the two study interventions was shown to be approximately equivalent. Conversely, when an alternative (lower) unit cost profile [Wolbachia, reflecting the influence of unit costs in this regard. A priori hypotheses in relation to the optimal mix and cost of interventions were also borne out. For example, reduced Wolbachia costs would lead one to surmise a congruent increase in Wolbachia coverage (and decrease in vaccination coverage) whereas an increase in Wolbachia costs would lead to the opposite. Similarly, reductions/ increases in both vaccine acquisition costs, and efficacy would have parallel effects.Our results suggest that several different combinations of  profile  was subsWolbachia-infected vector mortality rate  was appropriate, citing evidence that an increased rate would result in the Wolbachia-infected mosquito population dying out and the non-Wolbachia infected population dominating the environment. Geographical specificity/ heterogeneity was also not considered in the analyses, but would perhaps be of value to help to characterize the optimal split between the two study interventions (to minimize dengue infection) at a finer spatial resolution, for example, north versus south, urban hotspots versus rural locations, etc. Wolbachia coverage will have additional benefits to the human population (beyond the dengue mitigation included in the current model), for example, in those areas of Thailand where there is a preponderance of chikungunya and/ or Zika virus. Whilst dengue is prevalent throughout Thailand, research on the long-term circulation of Zika virus indicates elevated risks of the disease (relative to the country as a whole) in the northeast and east of Thailand and reduced risks in the south of the country [This study is subject to a number of important limitations. Similar to Knerer et al. , the tra country . Convers country . Histori country , reachin country . NotwithWolbachia-based dengue control strategy could be implemented. These include, for example, the optimal choice of Wolbachia strain, appropriate surveillance, and monitoring of environmental and evolutionary changes, as well as community ‘buy-in’ and acceptance [Wolbachia-infected mosquitoes have arrived at equilibrium/ steady state fixation in areas where they have been released. Although coverage is likely to be limited initially, such analyses provide insights into the human population impact of a potential Wolbachia programme on a large, countrywide scale, both separately and in combination with other control strategies.As previously highlighted in Knerer et al. , it is aceptance , 89. CerWolbachia and paediatric vaccination) that minimize dengue burden at the lowest budget. If proposals/ suggestions are usefully to be put forward in relation to broader vaccine and/ or Wolbachia introduction for dengue control, policy and decision makers will likely need to determine which dengue interventions to prioritize to optimize the health status of the population, which may necessitate trade-offs depending on the public health goal. As alluded to above, practical operational realities may conceivably be more complicated than the somewhat simplified analyses presented here; in particular, the source of funding budgets for vaccination and/ or Wolbachia may be quite distinct, and thus not reflect the trade-offs discussed in this study. Notwithstanding this, commentators suggest that long-term dengue control necessitates increasing investment, complementary control strategies, and intervention programmes across a broad geographic area to minimize cross-border infection [Our model provides a tool for developing estimates of optimal coverage of combined dengue control strategies  by levels of Wolbachia and vaccination coverage


Viruses are the smallest living things known to science, yet they hold the entire planet in their way.A Planet of Viruses, Carl Zimmerfrom The corona virus disease 2019 (COVID-19) pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has become a big threat to human health. Studies have shown that SARS-CoV-2 can also invade the central nervous system (CNS) [Acta Epileptologica is an international academic journal that publishes advances in epileptic research. Viruses are an important cause for seizures. To highlight the important roles of viruses in epilepsy, especially under the current COVID-19 pandemic, Acta Epileptologica publishes this special issue on “Virus and Epilepsy”, focusing on four viruses: arbovirus, human immunodeficiency virus (HIV), picornavirus, and human herpes virus 6 (HHV6), from perspectives of pathogenesis, pathogenicity, clinical characteristics, treatment, and prognosis of seizure induction or enhancement after viral infection. Most importantly, the mechanisms underlying viral association with epileptogenesis are also discussed.Arbovirus and seizures”, Mingrui Zheng et al. summarized the most common arboviruses associated with epidemic viral encephalitis. Viral encephalitis affects both children and adults, usually leading to severe neurological sequelae. Arboviruses of the genus Flavivirus are usually transmitted by mosquitoes and other host animals. Among various arboviruses, the Japanese encephalitis virus, West Nile virus, Zika virus, Dengue virus and Chikungunya virus can induce seizures. Seizures may not be the major manifestation, but may predict a poor prognosis. The occurrence of seizure is mainly caused by direct cell damage by the virus, secondary glial and immune responses producing inflammatory cytokines, and damage to the blood-brain barrier. Different virus infections may lead to mild or severe symptoms, with varied occurrence of epilepsy. The seizure type can usually be focal or generalized, or even status epilepticus. The generalized tonic-clonic seizures are the most common type. EEG recordings have consistently found a pattern of diffuse slow activity. During occurrence of seizure, patients typically manifest with abnormalities including theta and delta coma, burst suppression, an isoelectric pattern, or occasionally alpha coma. Effective treatment of associated epileptic seizures, mostly symptomatic support and anti-virus therapy, enables good supportive care and optimal control of CNS-related comorbidities. However, usually there is a lack of effective treatment methods..In the paper entitled “The role of picornavirus infection in epileptogenesis”, Runxuan Zhang et al. summarized the clinical characteristics of picornavirus infection, and the pathogenesis of Theiler’s murine encephalomyelitis virus (TMEV)-induced epilepsy. Picornaviruses are a family of small positive-strand RNA viruses and transmitted via the respiratory or fecal-oral route. The neurotropic picornaviruses can induce acute or late recurrent seizures following CNS infection, by infecting the peripheral nerve, crossing the blood-brain barrier and migrating in the Trojan-horse approach. TMEV, as a member of the Picornavirus family, can cause encephalitis, leading to chronic spontaneous seizures. The TMEV-infected animal model has been used to study the mechanisms of epileptogenesis and to evaluate drug efficacy. Several immune components are involved in TMEV-induced epileptogenesis. Minocycline (MIN) treatment can positively affect the long-term epileptogenic process. Valproic acid has a neuroprotection effect through inhibition of histone deacetylase activity, thereby reducing seizures. Conversely, Carbamazepine may exacerbate TMEV infection, making animals more susceptible to the virus. Wogonin can decrease epileptic episodes by inhibiting the activated macrophages targeted by the IL-6 producing cells in mice. .In the paper entitled “Temporal lobe epilepsy associated with human herpes virus 6”, Jiaqi Wang et al. summarized the close relationship between HHV-6B and temporal lobe epilepsy (TLE). The special target of HHV-6 is in the hippocampus and amygdala. Recently, viral genomic DNA of HHV-6B has been detected in surgically removed brain tissues of intractable epilepsy patients, suggesting the involvement of HHV-6B in the pathogenesis of epilepsy. TLE patients with HHV-6B in their brains suffer from reiterative attacks of febrile seizures and hippocampal sclerosis. In this article, the potential involvement of cytokines in the pathological process of TLE is reviewed in detail. Anti-inflammatory or immunomodulatory therapies may show prospects for TLE treatment. .HHV-6 is a ubiquitous and most common pathogen that affects humans. In the paper entitled “Current epidemiological and etiological characteristics and treatment of seizures or epilepsy in patients with HIV infection” by Changhao Yu et al. carefully reviewed the role of HIV infection in epilepsy. Opportunistic infections are a stereotypical predisposing factor for seizures in HIV patients. There is a prevalence of 2–19.8% and an incidence of 1.8–19.8% for seizures in the HIV-infected population, which are mostly of the generalized type. HIV patients with seizures need to take both antiviral and antiepileptic drugs, which increases the risk of drug-drug interactions and the occurrence of side effects. Antiepileptic drugs should be carefully selected to avoid the CYP450-induced drug-drug interactions. This review further gives a perspective on future therapeutic studies in HIV patients with seizures. .The article entitled “


In recent years, the Fibrinogen to pre-albumin ratio (FPR) has been reported in many studies to be significantly associated with the prognosis of various cancers. This systematic review and meta-analysis aimed to investigate the prognostic value of FPR in malignant tumors of the digestive system based on available evidence.The relevant articles published before July 1, 2021, were systematically retrieved from electronic databases to evaluate the effect of Fibrinogen to pre-albumin ratio (FPR) on the prognosis of patients with malignant digestive system tumors and calculate the hazard ratio (HR) and the corresponding 95% confidence interval (CI).Thirteen articles, all from China, including 15 cohort studies and a total of 5116 cases, were included in this study. A high FPR was associated with poor overall survival , recurrence-free survival , progression-free survival , complications , disease-free survival  was significantly associated with cancer-specific survival . Even though intergroup differences were present, FPR was strongly associated with overall and relapse-free survival, and sensitivity analysis suggested that our results were stable.FPR can be used as a valuable indicator to predict the prognosis of patients with malignant digestive system tumors. Malignant tumors of the digestive system are among the most common malignancies globally, including colorectal carcinoma, carcinoma of the stomach, hepatic carcinoma, and esophageal carcinoma .Most patFibrinogen is a 340 KDa hepatocyte-produced glycoprotein that can be converted to fibrin by activated thrombin. It modulates the coagulation and thrombosis process and plays an important role in hemostasis, cell attachment, and systemic inflammatory reactions .High FibEven though pre-albumin and Fibrinogen are all widely acknowledged to be associated with tumor prognosis, the Fibrinogen to pre-albumin ratio (FPR) has rarely been reported to evaluate the prognosis of patients with digestive tumors. At present, only one meta-analysis mentioned the correlation between FPR and OS of cancer patients, which proved that high FPR was associated with poor OS in cancer patients, but there was no independent report on the relationship between FPR and malignant digestive system tumors, and there was no report on the relationship between FPR and recurrence-free survival . TherefoThis systematic review followed the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines .In this The eligibility criteria were defined according to the Population, Intervention, Comparison, Outcome and Study Design (PICOS) strategy. Patients with digestive tumors were identified as "Population" at the time of retrieval; high FPR and low FPR represented "Intervention" and "Comparison" respectively; overall survival (OS), recurrence-free survival (RFS), cancer-specific survival (CSS), disease-free survival (DFS), progression-free survival (PFS) and complication were the "Outcome" of this study; "Study design" choice was retrospective and prospective research, and case reports, letters, reviews, editorials and comments were ruled out. If there were duplicates, the most recently published record would be included.Duplicates were removed using EndNote (version X8), and eligible studies were entered into a database built by EpiData (version 3.0). Two reviewers independently extracted the required data from the included studies using a special Excel spreadsheet, including author name, publication date, study location, study design (prospective or retrospective research), type of patient, sample size, age composition, gender, primary treatment , cutoff value, methods for choosing FPR cutoff value, outcome, data source (crude data or fitted curve) and duration of follow-up. Outcome indicators included primary (OS and RFS) and secondary outcome indicators . The inclusion of relevant literature and the processing of all data were performed by two independent reviewers (BL and HD). Points of disagreement were reconciled by a discussion with a third reviewer.2 statistics were used to quantify the heterogeneity between the included studies and calculated the proportion of variation due to heterogeneity rather than due to chance; I2 values ranged from 0 to 100%, with higher values indicating greater heterogeneity. If I2 > 50%, the random-effects model was chosen; otherwise, the fixed-effects model was selected. Moreover, subgroup analysis was conducted to explore whether publishing time, methods for choosing FPR cutoff value (ROC or X-tile), cutoff value, types of cancer , study design (prospective or retrospective), treatment option  and sample capacity had any influence on the results. In addition, sensitivity analysis was used to evaluate the reliability and stability of the results, and Begger's and Egger's tests were used to test whether there was a potential publication bias in the study. P > 0.05 is considered to be free of publication bias, otherwise the trim-and-fill method will be used for reevaluation. All tests were bilateral tests, and P < 0.05 was considered to be statistically significant.All statistical analyses were performed using STATA (version 12). The extracted data was used for calculating hazard ratios (HRs) and 95% CI and used to evaluate the prognostic value of FPR in digestive system malignant tumors. IThe flowchart documenting retrieval of studies from electronic databases is illustrated in Fig. 2 = 50.8%, p = 0.032), we used the random effect model . Due to the significant heterogeneity , we used a fixed-effect model . In the absence of heterogeneity  The combined results showed that PFS was significantly shorter in patients with high FPR than in patients with low FPR . A study involving 584 patients showed that high FPR was an independent risk factor for complications and disease-free survival   and fibrinogen regulate the occurrence and development of various tumors, and their levels in peripheral blood are closely related to the patient's survival and sensitivity to therapy , 32.As aSingle markers of fibrinogen and pre-albumin are limited and unstable in predicting the prognosis of digestive system tumors . Hailun The reason why FPR is related to the prognosis of digestive tract tumors is not clear. However, there are several possible mechanisms. Fibrinogen and pre-albumin are liver acute phase positive and negative proteins, respectively . Kris A Herein, we included 13 studies involving 5116 patients with malignant tumors of the digestive system. Existing evidence indicates that FPR is a sensitive indicator for predicting the prognosis of patients in this population, and patients with a high FPR exhibited worse OS than those with a low FPR. In the meantime, subgroup analysis was also conducted to explore the influence of various factors on our final findings. Although publication time, sample size, cutoff value, source of cutoff value, cancer site, treatment option and type of study varied in different groups, a high FPR was still significantly associated with poor prognosis. We further verified the stability of the meta-analysis by conducting sensitivity analysis and adjusting for censoring. In addition, we further discussed the relationship between FPR and recurrence-free survival in patients. The comprehensive results showed that FPR was an independent predictor of RFS in patients with malignant digestive system tumors. At the same time, subgroup analysis showed that although there were differences in publication time, sample size, research type and cancer system among different subgroups, high FPR still correlated significantly with poor RFS. Sensitivity analysis indicated that our conclusions on RFS were robust, and the funnel chart exhibited no potential publication bias. In addition, we also discussed the relationship between FPR and other prognostic indicators of malignant tumors of the digestive system. A high FPR was associated with adverse clinical outcomes in terms of CSS, DFS, PFS, and complications. In all events, FPR can be considered an important and practical clinical indicator to predict patient prognosis.To the best of our knowledge, this is the first meta-analysis to comprehensively examine the prognostic value of FPR in patients with malignant tumors of the digestive system. Based on the available evidence, FPR was associated with significant outcomes (OS and RFS) in the present study and correlated with adverse clinical outcome indicators . We also performed a rigorous subgroup analysis and sensitivity analysis to further demonstrate the prognostic significance of FPR in these patients. In addition, the cut-off values of FPR in most of the included studies are mainly concentrated in the range of 18 to 23.1, which provides a certain reference value for clinically determining the critical value of FPR.However, it should be noted that there are some limitations in our meta-analysis. First of all, all the included studies were conducted in China; accordingly, the drawn conclusions mainly indicate that FPR has certain clinical value in the treatment of patients with malignant tumors of the digestive system in China; accordingly, more large scale studies worldwide are needed to provide sufficient evidence to substantiate the veracity of our findings. Moreover, significant heterogeneity was present in our meta-analysis, which may have been caused by the small number of included studies and samples. Finally, due to the limited number of included studies, the cutoff value of FPR in CSS, DFS, PFS and complications remains to be explored.According to this meta-analysis, high FPR is significantly correlated with poor prognosis, which can be harnessed clinically as an indicator to predict the prognosis of patients with malignant tumors of the digestive system. However, large multicenter studies worldwide are still needed to substantiate our findings.


Virus Evolution, Volume 7, Issue 1, January 2021, veab005 https://doi.org/10.1093/ve/veab005Published: 4 February 2021Matonaviridae is incorrectly referred to as a “single-strand, negative-sense RNA virus” when the text should read “single-strand, positive-sense RNA virus”.In the Abstract of this paper a member of the


The hatching rate and pathology of IBV-infected chicken embryos were greatly improved in the presence of CNGs. CNGs with distinctive virus-neutralizing activities show great potential as a virostatic agent to prevent the spread of avian viruses and to alleviate the pathology of infected avian species.In this study, we demonstrate the synthesis of carbonized nanogels (CNGs) from an amino acid (lysine hydrochloride) using a simple pyrolysis method, resulting in effective viral inhibition properties against infectious bronchitis virus (IBV). The viral inhibition of CNGs was studied using both in vitro  and pseudorabies virus (PRV)) and in ovo (IBV) models, which indicated that the CNGs were able to prevent virus attachment on the cell membrane and penetration into the cell. A very low concentration of 30 μg mL Gammacoronavirus genus within the Coronaviridae family, with a non-segmented, positive-sense single-stranded RNA genome and an envelope /(TCID50 of control group) ×100%.Antiviral effect (%) = [ to remove the serum and then diluted with sterile isotonic physiological buffer (ca. 4.0 vol% blood cells). For the analysis, 200 µL of the RBC stock suspension was incubated with aliquots of CNGs-270 dispersions (1–100 μg mL–1) in 1.5-mL vials at 37 °C for 1 h, followed by centrifugation at an RCF of 1000 g for 10 min. Hemolysis was measured based on the absorption of hemoglobin at 576 nm (OD576) in the supernatant (150 µL). Hemolysis in sterile isotonic physiological buffer and ultrapure water was used as negative and positive control, respectively, and the hemolysis activity was calculated as:Blood samples for the hemolysis assay were collected from female 2-month-old Sprague-Dawley rats in tubes containing ethylenediaminetetraacetic acid (EDTA) and immediately (within 30 min of the collection) centrifuged (RCF 3000× 6.25 CEID50 mL–1) was incubated with the CNG samples  in phosphate-buffered saline  at room temperature for 1 h. The virus-positive control was incubated with the PBS at the same time. The virus-CNG inoculum was injected into the 13-day-old SPF chicken embryos. The embryo pathological effects were determined after 7 days of incubation . The 50% chicken embryo infectious dose (CEID50) results were determined using the Reed–Muench method. The antiviral effects of CNGs were calculated as per the formula:The animal experiments were approved by the Institutional Animal Care and Use Committee of National Pingtung University of Science and Technology. The project identification codes are NPUST-108-039 and NPUST-110-044, date of approval: 25 June 2019. The IBV virus (10


Prasiola japonica possesses several biological activities. However, reports on the anti-inflammatory activities and molecular mechanisms of its different solvent fractions remain limited. In this study, we investigated the potential anti-inflammatory activities of P. japonica ethanol extract (Pj-EE) and four solvent fractions of Pj-EE made with hexane (Pj-EE-HF), chloroform (Pj-EE-CF), butanol (Pj-EE-BF), or water (Pj-EE-WF) in both in vitro (LPS-induced macrophage-like RAW264.7 cells) and in vivo (carrageenan-induced acute paw edema mouse models) experiments. The most active solvent fraction was selected for further analysis. Various in vitro and in vivo assessments, including nitric oxide (NO), cytokines, luciferase assays, real-time polymerase chain reactions, and immunoblotting analyses were performed to evaluate the underlying mechanisms. In addition, the phytochemical constituents were characterized by Liquid chromatography-tandem mass spectrometry. In in vitro studies, the highest inhibition of NO production was observed in Pj-EE-CF. Further examination revealed that Pj-EE-CF decreased the expression of inflammation-related cytokines in LPS-induced RAW264.7 cells and suppressed subsequent AP-1-luciferase activity by inhibition of phosphorylation events in the AP-1 signaling pathway. Pj-EE-CF treatment also demonstrated the strongest reduction in thickness and volume of carrageenan-induced paw edema, while Pj-EE-BF showed the lowest activity. Furthermore, Pj-EE-CF also reduced gene expression and cytokines production in tissue lysates of carrageenan-induced paw edema. These findings support and validate the evidence that Pj-EE, and especially Pj-EE-CF, could be a good natural source for an anti-inflammatory agent that targets the AP1 pathway. Inflammation is the protective response of the immune system against various pathogens and cellular danger signals, mediated primarily by immune cells such as macrophages. Acute inflammation is a process that involves the activation of complex enzymes, secretion of free radicals, and release of several inflammatory and pro-inflammatory mediators, which are commonly characterized by redness, swelling, pain, heat, and loss of tissue function ,2,3. TheChlorophyta) are considered a representative natural source for pharmaceutical, nutraceutical, and cosmeceutical products [Prasiola, have a diverse distribution. At least 14 of 36 species are freshwater organisms, and other species grow in terrestrial habitats. Prasiola japonica belongs to the family Prasiolaceae and is found growing in freshwater ecosystems [Prasiola japonica has been reported in east Asia and especially in Japan and in the Republic of Korea [P. japonica has been shown to possess medicinal benefits including antioxidant, antiapoptotic, anti-melanogenic, and anti-inflammatory effects. However, most of those studies were limited to identifying such effects of the crude ethanolic extract of P. japonica in the NF-κB pathway and mainly focused on the skin cell lines [P. japonica ethanol extract (Pj-EE) and four of its solvent fractions—hexane (Pj-EE-HF), chloroform (Pj-EE-CF), butanol (Pj-EE-BF), and water (Pj-EE-WF)—in in vitro (LPS-induced macrophage-like RAW264.7 cells) studies. The most active fraction, Pj-EE-CF, is selected to determine additional activities. In in vivo studies in carrageenan-induced acute paw edema mouse models, the molecular targets and phytochemical constituents of Pj-EE-CF are also evaluated.Some green algae  the secretion of NO in macrophage-like RAW264.7 cells. Pj-EE-CF (100 μg/mL) had the strongest effect (p = 0.0071) on the secretion of NO and up to 80% inhibition on LPS-induced RAW264.7 cells. In contrast, treatment with Pj-EE, Pj-EE-HF, Pj-EE-BF, and Pj-EE-WF did not exert significant inhibition. Furthermore, using a conventional 3--2,5-diphenyltetrazolium bromide (MTT) assay, we checked the cell viability on macrophage-like RAW264.7 cells upon treatment with Pj-EE and the four solvent fractions. However, treatment with Pj-EE-CF at a concentration that significantly reduced NO production indicated an interference of cell viability by up to 25%  . As demonstrated in p to 25% b. Conseqctively) d. These p < 0.001), and treatment with Pj-EE-CF downregulated the expression of pro-inflammatory genes. In line with the result of the NO assay, LPS-stimulated RAW264.7 cells pretreated with Pj-EE-CF at concentrations of 25 and 50 μg/mL significantly inhibited in a concentration-dependent manner the mRNA levels of iNOS , the enzyme that is responsible for catalyzing the secretion of NO . We initially determined that Pj-EE-CF treatment on LPS-stimulated RAW264.7 cells can reduce inflammatory mediators such as NO. To further our study, we examined whether Pj-EE-CF also can regulate the expression of pro-inflammatory mediators at the transcriptional level. Using quantitative real-time polymerase chain reaction (qPCR), we assessed the mRNA expression of iNOS, COX-2, and other pro-inflammatory cytokines, such as TNF-α, IL-1β, IL-6, and IL-10. As shown in on of NO a. Moreovp = 0.0078 and p = 0.0035, respectively). However, treatment with up to 50 μg/mL of Pj-EE-CF concentration-dependently suppressed AP-1 luciferase activity induced by either TRIF or MyD88 . However, Pj-EE-CF had the lowest measurement both of thickness and volume compared to 100 mg/kg of Pj-EE or the other solvent fraction groups, which suggested that it had more potent inhibitory effects, followed by Pj-EE, Pj-EE-HF, and Pj-EE-WF. Additionally, Pj-EE-BF did not show inhibitory symptoms on the carrageenan-induced paw edema mouse model.To determine the anti-inflammatory efficacy of Pj-EE and its solvent fractions in vivo, carrageenan-induced acute paw edema mouse models were used. Inflammation in both hind paws of each mouse was triggered by intraplantar injection of 1% newly prepared solution of carrageenan in PBS after pretreatment with 100 mg/kg Pj-EE and the four different solvent fractions once a day for 10 days, as illustrated in To evaluate the effects of Pj-EE and its solvent fractions at the molecular level, the level of inflammatory genes in the tissue lysate of paw samples was examined by qPCR a–g. In aP. japonica, has been described to possess pharmacological benefits. Last year our research group reported that the ethanol extract of P. japonica exerts antiapoptotic, anti-melanogenic, and antioxidant effects on skin cell lines [Medicinal plants with natural sources still offer promising options and are recognized as therapeutic agents. In addition, understanding the mechanisms of therapeutic action could lead to the discovery of new drugs from natural sources with reduced or no side effects ,22,23. Tll lines ,19. We all lines . HoweverIn early characterizations of NO, it was described as playing an important role in biological activities such as neurotransmission and immune defenses. However, research showed that while the production of NO is uncontrolled in the cells, it acts as one of the pro-inflammatory mediators and a key molecule during inflammatory responses . In thisIn addition, to precisely identify the molecular target and mechanism of Pj-EE-CF, other experimental approaches, including luciferase assays and immunoblot analyses, were performed. The results of the luciferase reporter genes indicated that Pj-EE-CF regulates the mRNA level of cytokines by moderating the transcriptional activity of AP-1. Strengthening these findings, immunoblot analyses of the level of phosphorylation of AP-1 subunits, c-jun and c-Fos, revealed that Pj-EE-CF modulates the transcriptional activation of AP-1. Previous studies reported the essential role and mechanism of MAPKs in activating AP-1 pathways ,23,25. CSeveral studies on the discovery and development of anti-inflammatory agents were based on the use of carrageenan-induced inflammation. Carrageenan-induced paw edema has been established as an experimental model of acute inflammatory diseases and has been widely used for studying novel analgesic and anti-inflammatory agents ,26,27,28Phytochemical screening, especially pertaining to the flavonoids, for the possible presence of Pj-EE-CF was characterized using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Numerous flavonoid molecules have been described to obtain anti-inflammatory activity through various mechanisms, including inhibition of the expression of inflammation-related enzymes via suppression of transcription factors activation, including AP-1 ,30,31. AMacrophage-like RAW264.7 cells (ATCC number TIB-71) and HEK293 cells (ATCC number CRL-1573) were obtained from the American Type Culture Collection (ATCC) . TRIzol reagent was purchased from MRCgene . Carrageenan, dimethyl sulfoxide (DMSO), carboxymethylcellulose (CMC), lipopolysaccharide , polyethylenimine (PEI), sodium dodecyl sulfate, and 3--2,5-diphenyltetrazolium bromide (MTT) were purchased from Sigma-Aldrich Co. . Fetal bovine serum was acquired from Biotechnics Research, Inc. . RPMI 1640, DMEM, trypsin, PBS, and penicillin-streptomycin were obtained from HyClone . Primers used for qPCR were obtained from Macrogen Inc. . Phospho-specific or total-protein antibodies against c-Fos, c-Jun, JNK, ERK, p38, and β-actin were purchased from Cell Signaling Technology  and Santa Cruz Biotechnology . A CATX124 balance was purchased from CAS Co. , a Mitutoyo thickness gauge 547 was received from Mitutoyo , and a plethysmometer 37,140 was obtained from UgoBasile . P. japonica was acquired from the Prasiola Japonica Research Center  and was extracted as in previous studies [w/v). Subsequently, the extract was filtered using 120 nm filter paper , concentrated using a vacuum concentrator , and then dried using a vacuum freeze dryer . The yield of the dried samples was measured. The final weight of the extract was 29.974 g  with a yield of 14.24%. In addition, the ethanolic extract of P. japonica (crude extract) was then fractionated by various polarity solvents including n-hexane (Pj-EE-HF), chloroform (Pj-EE-CF), n-butanol (Pj-EE-BF), and water (Pj-EE-WF) as illustrated in  studies ,19,20. BThe murine macrophage cell line (RAW264.7) and the human embryonic kidney cell line (HEK293) were cultured as previously reported . For a p6 cells/mL) were seeded in a 96-well plate and then pretreated with the specified concentrations of Pj-EE and its solvent fractions for 30 min. LPS (1 μg/mL) was then treated for the next 24 h. The suppression effect of Pj-EE and its solvent fractions on the secretion of NO was analyzed using Griess reagents, as previously described [RAW264.7 cells  that were pretreated with Pj-EE-CF  for 30 min followed by induction with LPS (1 μg/mL) for 6 h. Paw tissues of mice were ground, and then stored at −70 °C until use. Total RNA was prepared using TRIzol per the manufacturer’s instructions, and then 1 μg of total RNA was immediately made for cDNA synthesis using a cDNA synthesis kit  per the manufacturer’s instructions. The qPCR was performed as previously reported [Total RNA was obtained from macrophage-like RAW264.7 cells (1 × 10reported . The pri5 cells/mL) were seeded in 24-well plates and preincubated overnight before transfection of plasmids encoding AP-1-Luc under cotransfection conditions with MyD88 and TRIF, by PEI methods as previously reported [HEK293 cells (2 × 10reported . After 26 cells/mL) were seeded in a 6-well plate. After pretreatment with different concentrations of PJ-EE-CF for 30 min, LPS was treated and then incubated for the next 24 h. After harvesting and washing with PBS, RAW264.7 cells were lysed in lysis buffer as previously described [RAW264.7 cells (1 × 10escribed . Approxiescribed .n = 6 per group) had water and a pellet diet  ad libitum in separate cages under a 12 h light–dark cycle. The in vivo experiments were conducted in agreement with the guidelines of the Institutional Animal Care and Use Committee, Sungkyunkwan University .ICR mice  were purchased from Daehan Biolink . The mice , paw edema was stimulated by a 100 μL subplantar injection of 1% newly made solution of carrageenan in PBS into both hind paws, as in the previously published report . ICR miceviously .p-value of <0.05 was considered to be statistically significant. All data in this study represent the mean ± standard deviation of four samples (in vitro experiments). Similar experimental data were obtained from additional independent experiments performed under the same conditions with six mice per group (in vivo experiments). Statistical analyses was performed using the computer program SPSS . A comparison of statistical differences of all measured data was subjected to one-way ANOVA followed by the Holm–Sidak test or the Kruskal–Wallis/Mann–Whitney test. A The present study, using in vitro and in vivo experiments, clearly indicated and supported the evidence of the anti-inflammatory effects of Pj-EE. Pj-EE-CF showed a particularly potent inhibition of inflammatory responses by targeting ERK and p38, thereby suppressing the activity of AP-1 that could result in attenuation of various inflammatory mediators and cytokines, as summarized in